The present invention relates to methods for screening for tumour sensitivity to anti-mitotic agents based on the characterisation of β-tubulin expression by the tumour, methods for modulating the expression of β-tubulin, and in particular II-, III-, or IVb-β-tubulin, in tumour cells. The present invention also relates to molecules and methods for enhancing the sensitivity of tumour cells to tubulin-binding agents, such as vinca alkaloids, taxanes and epothilones and to other anti-cancer agents. The present invention also relates to methods for modulating the expression of tubulin in tumour cells to reduce tumorigenicity, for example to reduce the incidence of rate of growth of a tumour in a subject.
Microtubules are required for cell division in eukaryotic cells, functioning as part of the spindle to ensure chromosome separation and segregation. The principal components of microtubules are α- and β-tubulin, which form heterodimers. At least seven different β-tubulin isotypes have been identified in humans to date. These isotypes are classified according to the sequence of carboxy terminal domains as follows (with the Roman numeral class numbers referring to the protein isotype and the human gene classification in parentheses): class I (HM40), class II (Hβ9), class III (Hβ4), class IVa (H5β), class IVb (Hβ2), class V and class VI (Hβ1). Different isotypes exhibit distinct patterns of tissue expression. For example, class III β-tubulin is normally expressed only in neuronal cells.
Tubulin-binding agents are an important component in the treatment of many human cancers, including ovarian cancer, breast cancer, non small cell lung cancer, prostate cancer, advanced stage neuroblastoma and various lymphomas. These clinically important agents include taxanes, such as paclitaxel (Taxol), epothilones, and vinca alkaloids such as vinblastine, which bind to β-tubulin from α/β-tubulin and disrupt microtubule dynamics and thereby induce mitotic arrest and apoptosis.
Upon binding to tubulin, taxanes and epothilones are known to enhance microtubule assembly, resulting in the stabilization of polymerized microtubules, abnormal spindle esters and cell cycle arrest in mitosis. In contrast, vinca alkaloids are a second class of tubulin-binding agents which induce the destabilization of polymerized tubulin by blocking the region involved in tubulin dimer attachment, thereby preventing microtubule assembly. Platinum-based DNA-damaging agents are a large class of anti-cancer drugs which bind and modify DNA, which are effective in the treatment of a variety of different cancers, and which are commonly administered in combination therapies with tubulin-binding agents.
The development of resistance by tumour cells to microtubule-binding agents such as taxanes, epothilones and vinca alkaloids is, however, a significant obstacle to the success of a range of chemotherapeutic regimens.
Lung cancer is the most common cancer in the world with over one million cases diagnosed every year and remains the leading cause of cancer death in both men and women. Advanced non-small cell lung carcinoma (NSCLC) accounts for more than 80% of these cases. More than half of these subjects have developed metastasis at the time of diagnosis, and so chemotherapy remains the most effective treatment option. Over the last decade, the use in phase 2 studies of chemotherapeutic agents such as paclitaxel and vinorelbine, either as a single agent or as part of a combination chemotherapeutic regimen, has shown promising activity and prolonged survival rates at one year. However, in general the prognosis for subjects remains poor due to the emergence of drug-resistant tumour cells that significantly limits the clinical utility of these drugs in the treatment of lung cancer. Accordingly, there is a clear need for methods and approaches to combat and overcome this resistance to ensure the continued efficacy of anti-mitotic agents in the treatment of cancer.
On the basis of differential tissue expression and highly conserved sequences across all species, it has been suggested that each β-tubulin isotype may provide unique characteristics which impart functional differences in microtubules. Although alterations in tubulin isotype expression have been demonstrated in cell lines selected for resistance to antimicrotubule agents, including paclitaxel, docetaxel and estramustine, the available data focuses on the role of βIII tubulin in paclitaxel resistance. The relevance of βIII tubulin in the response of cells to other types of tubulin-binding agents, or the relevance of non-βIII-tubulin isotypes in responses to tubulin-binding agents is poorly understood. Class II β-tubulin, for example, was found in both normal and tumour breast tissues, suggesting that it may not be a good biomarker for these tumours. Further, despite the common use of DNA-damaging agents in combination therapy with vinca alkaloids, the effects of βIII-tubulin expression on the efficacy of these agents has not been addressed.
One particular difficulty with the studies of drug resistant cells is to ascertain whether changes in β-tubulin expression which may be observed in resistant cells contributes to the resistant phenotype or arise as a secondary mechanism. It is not unusual for multidrug resistant cells to undergo multiple cellular changes and they hence may incorporate more than one mechanism of resistance. Many factors which are associated with poor prognosis, such as the diverse factors which cause or promote tumorigenesis in tumour cells, are not well understood. Accordingly, there is a clear need for methods and approaches which offer alternatives or adjuncts to treatment with conventional anti-mitotic agents in the treatment of cancer.
In view of the above, for therapeutic applications, there is a need for agents which enhance tumour sensitivity to tubulin-binding agents and/or other anti-mitotic agents or DNA-damaging agents. In addition, it would be desirable to be able to screen a tumour to predict its sensitivity or potential sensitivity to a range of tubulin-binding agents and/or other anti-mitotic agents, both before commencing treatment with such agents and during the course of treatment. Furthermore, many factors which are associated with poor prognosis, such as the diverse factors which cause or promote tumorigenesis in tumour cells, are not well understood. Accordingly, there is a clear need for methods and approaches which offer alternatives or adjuncts to treatment with conventional anti-mitotic agents in the treatment of cancer.
The present inventors have now demonstrated an association between the expression of class II, class III or class IVb β-tubulin in NSCLC cells and leukemic cells with the response of these cells to various tubulin-binding agents. The inventors have now also identified that suppression of class III β-tubulin expression in class III β-tubulin expressing tumour cells results in a reduction in the tumorigenic phenotype in the tumour cells.
Accordingly, in a first aspect there is provided a method of screening a tumour cell for resistance to or potential resistance to a tubulin-binding agent, the method comprising detecting the expression of any one or more of class II, class III and class IVb β-tubulin by the tumour cell, wherein the expression of any one or more of class II, class III and class IVb β-tubulin indicates that the tumour cell has resistance or potential resistance to the tubulin-binding agent.
In one embodiment, the tubulin-binding agent is a microtubule destabilizing agent. In one embodiment, the tubulin-binding agent is a microtubule stabilizing agent.
In one embodiment the method comprises detecting the expression of any two of class II, class III and class IVb β-tubulin by the tumour cell. In another embodiment, the method comprises detecting the expression of class II, class III and class IVb β-tubulin by the tumour cell.
In certain embodiments, the step of detecting the expression of one or more of class II, class III and class IVb β-tubulin by the tumour cell comprises detecting the expression of one or more of class II, class III and class IVb β-tubulin protein.
In certain embodiments the step of detecting the expression of one or more of class II, class III and class IVb β-tubulin by the tumour cell comprises detecting the expression of one or more of class II, class III and class IVb β-tubulin mRNA.
The step of detecting the expression of one or more of class II, class III and class IVb β-tubulin by the tumour cell may comprise quantifying the level of expression of the tubulin.
The step of detecting the expression of one or more of class II, class III and class IVb β-tubulin by the tumour cell may comprise comparing the expression of one or more of class II, class III and class IVb β-tubulin by the tumour cell with the expression of one or more of class II, class III and class IVb β-tubulin by a control cell. The control cell may be tumour cell or a non-tumour cell, for example a cell taken from tissue surrounding the tumour. The control cell may be a tumour cell which is resistant to the tubulin-binding agent. The control cell may be a tumour cell which is sensitive to the tubulin-binding agent. The control cell may be a tumour cell in which the expression of any one or more of class II, class III and class IVb β-tubulin has been reduced.
Also provided is a method of assessing the resistance of a tumour cell to a tubulin-binding agent. The method includes detecting an amount of a class II, class III and class IVb β-tubulin protein or a class II, class III and class IVb β-tubulin mRNA by the tumour cell. A subject tumour cell tubulin amount is thereby obtained. The subject tumour cell tubulin amount is compared to a control cell tubulin amount thereby determining a tubulin amount difference. The control cell tubulin amount is a respective amount of a class II, class III or class IVb β-tubulin protein or any one or more of class II, class III and class IVb β-tubulin mRNA in a control cell. The resistance of the tumour cell to the tubulin-binding agent is assessed based on the tubulin amount difference.
The control cell of the above methods may be a control non tumour cell. The control cell of the above methods may be a control tumour cell. The control tumour cell may be a tumour cell which is resistant to the tubulin-binding agent, or a tumour cell which is sensitive to the tubulin-binding agent.
In certain embodiments the detecting of an amount of a class II, class III and class IVb β-tubulin protein is detecting an amount of class II, class III and class IVb β-tubulin protein. In a particular embodiment, the detecting of an amount of any one or more of class II, class III and class IVb β-tubulin protein is detecting an amount of class III β-tubulin protein only.
In other embodiments, the detecting of an amount of a class II, class III and class IVb β-tubulin mRNA is detecting an amount of class II, class III and class IVb β-tubulin mRNA. In a particular embodiment the detecting of an amount of any one or more of class II, class III and class IVb β-tubulin mRNA is detecting an amount of class III β-tubulin mRNA only. Thus, where “a” class II, class III or class IVb β-tubulin protein or mRNA is detected, any one or more of the class II, class III or class IVb β-tubulin protein or mRNA is detected.
In another aspect there is provided a method for enhancing the sensitivity of a tumour cell to at least one tubulin-binding agent, the method comprising introducing into the tumour cell an effective amount of at least one nucleic acid construct comprising a nucleotide sequence specific to at least a portion of the class II, class III or class IVb β-tubulin gene product, wherein the construct decreases the expression of class II, class III or class IVb β-tubulin in the tumour cell.
In another aspect, there is provided a method for treating a tumour in a subject, comprising administering to the subject an effective amount of at least one nucleic acid construct comprising a nucleotide sequence specific to at least a portion of the class II, class III or class IVb β-tubulin gene product and administering to the subject at least one tubulin-binding agent, wherein the nucleic acid construct decreases the expression of class II, class III or class IVb β-tubulin in the tumour and thereby increases the sensitivity of the tumour to the at least one tubulin-binding agent.
In one embodiment, the nucleic acid construct and the at least one tubulin-binding agent are administered simultaneously. In one embodiment, the nucleic acid construct and the at least one tubulin-binding agent are administered concurrently. In another embodiment, the nucleic acid construct is administered before the at least one tubulin-binding agent.
In one embodiment of the above aspects, the nucleotide sequence is an antisense sequence. In another embodiment, the nucleotide sequence is an siRNA sequence. In another embodiment, the nucleotide sequence is a short hairpin RNA. In yet another embodiment, the nucleotide sequence is a ribozyme sequence.
In a particular embodiment of any of the above aspects, the nucleotide sequence is specific to at least a portion of the class IVb β-tubulin gene product. In another particular embodiment, the nucleotide sequence is specific to at least a portion of the class II β-tubulin gene product. In a particular embodiment, the nucleotide sequence is specific to at least a portion of the class III β-tubulin gene product.
In a particular embodiment of any of the above aspects, the at least one tubulin-binding agent may be at least one microtubule destabilizing agent. The at least one microtubule destabilizing agent may be selected from the group consisting of any one or more of vinca alkaloid agents, dolostatins, colchicines, cryptophycins, curacin A, 2-methoxyestradiol and derivatives, analogues or prodrugs thereof. In a particular embodiment, the microtubule destabilizing agent is a vinca alkaloid agent. In one embodiment, the vinca alkaloid agent is selected from the group consisting of any one or more of vincristine, vinblastine, vinflunine, vindesine, and vinorelbine, or a derivative, analogue or prodrug thereof.
In a particular embodiment of any of the above aspects, the at least one tubulin-binding agent may be at least one microtubule stabilizing agent. The at least one microtubule stabilizing agent may be selected from the group consisting of any one or more of a taxane and an epothilone. The taxane may be paclitaxel or docetaxel. The epothilone may be epothilone A, epothilone B or epothilone D. In a particular embodiment of the above aspects, the tubulin-binding agent is a vinca alkaloid agents, a dolostatin, colchicine, a cryptophycin, curacin A, 2-methoxyestradiol or an epothilone.
In a particular embodiment, the tumour cells are non-small cell lung carcinoma cells (NSCLCs).
In a particular embodiment, the subject is a human.
In another aspect there is provided the use of at least one nucleic acid construct comprising a nucleotide sequence specific to at least a portion of the class II, class III or class IVb β-tubulin gene, wherein the construct is able to decrease the expression of class II, class III or class IVb β-tubulin in a tumour cell, in the preparation of a medicament for increasing the sensitivity of a tumour cell to at least one tubulin-binding agent.
Also provided is a pharmaceutical composition for increasing the sensitivity of a tumour to at least one tubulin-binding agent, the pharmaceutical composition comprising at least one nucleic acid construct comprising a nucleotide sequence specific to at least a portion of the class II, class III or class IVb β-tubulin gene, wherein the construct is able to decrease the expression of class II, class III or class IVb β-tubulin in a tumour, together with a pharmaceutically acceptable carrier, diluent or excipient.
In another aspect there is provided a kit when used for assessing the sensitivity of a tumour to a tubulin-binding agent, the kit comprising at least one nucleic acid construct comprising a nucleotide sequence specific to at least a portion of the class II, class III or class IVb β-tubulin gene product, wherein the construct is used to detect the expression of class II, class III or class IVb β-tubulin in the tumour. In certain embodiments, the kit further comprises one or more tubulin-binding agents. In particular embodiments, the diagnosing the sensitivity of a tumour to a tubulin-binding agent is an in vitro diagnosis. In another aspect there is provided a kit when used for assessing the sensitivity of a tumour to a tubulin-binding agent, the kit comprising at least one antibody which is specific to any one of class II, class III or class IV β-tubulin polypeptide, wherein the antibody is used to detect the expression of class II, class III or class IV β-tubulin in the tumour.
In another aspect there is provided a kit when used for treating a tumour, the kit comprising (i) a pharmaceutical composition comprising at least one nucleic acid construct comprising a nucleotide sequence specific to at least a portion of the class II, class III or class IVb β-tubulin gene, together with a pharmaceutically acceptable carrier, diluent or excipient, and optionally (ii) one or more tubulin-binding agents.
Also provided is a veterinary composition for increasing the sensitivity of a tumour to one or more tubulin-binding agents, the veterinary composition comprising at least one nucleic acid construct comprising a nucleotide sequence specific to at least a portion of the class II, class III or class IVb β-tubulin gene, together with a acceptable carrier, diluent or excipient.
In another aspect there is provided a method of screening for agents which disrupt microtubule dynamics associated with class II, class III and/or class IVb β-tubulin, the method comprising exposing a candidate agent which is suspected of disrupting microtubule dynamics associated with at least one of class II, class III or class IVb β-tubulin to a first cell which expresses at least one of class II, class III or class IVb β-tubulin and detecting the response of the cell to the candidate agent, exposing the candidate agent to a second cell of the same type in which the expression of at least one of class II, class III or class IVb β-tubulin in the cell has been decreased and detecting the response of the second cell to the candidate agent, and comparing the responses of the first and second cells to the candidate agent, wherein an altered response of the second cell to the candidate agent compared to the response of the first cell signifies an activity for the candidate agent in disrupting microtubule dynamics associated with class II, class III or class IVb β-tubulin. In particular embodiments the response of the cell is an increase or decrease in the rate of apoptosis. In particular embodiments the response of the cell is a change in the rate of cell division.
The inventors have now also identified that suppression of class III β-tubulin expression in class III β-tubulin expressing tumour cells results in a reduction in the tumorigenic phenotype in the tumour cells. Accordingly, provided herein is a method of reducing the tumorigenicity of a class III β-tubulin expressing tumour cell in a subject, the method comprising administering to the subject a nucleic acid molecule comprising a polynucleotide sequence which is specific to at least a portion of the class III β-tubulin gene, wherein the polynucleotide sequence is able to decrease the expression of class III β-tubulin in the tumour cell and thereby reduce the tumorigenicity of the tumour cell.
Also provided is a pharmaceutical composition for reducing the tumorigenicity of a class III β-tubulin expressing tumour cell in a subject, the pharmaceutical composition comprising a nucleic acid molecule which comprises a polynucleotide sequence which is specific to at least a portion of the class III β-tubulin gene, together with a pharmaceutically acceptable carrier, diluent or excipient, and wherein the polynucleotide is able to decrease the expression of class III β-tubulin in the tumour cell.
In a particular embodiment the pharmaceutical composition is formulated for systemic administration. In yet another embodiment the pharmaceutical composition is formulated for parenteral administration. In yet another embodiment the pharmaceutical composition is formulated for topical administration.
Also provided is the use of a nucleic acid molecule comprising a polynucleotide sequence which is specific to at least a portion of the class III β-tubulin gene, wherein the polynucleotide sequence is able to decrease the expression of class III β-tubulin in a class III β-tubulin expressing tumour cell, in the preparation of a medicament for reducing the tumorigenicity of a tumour cell in a subject.
In one embodiment of the above aspects, the polynucleotide sequence is an antisense sequence. In another embodiment, the polynucleotide sequence is an siRNA sequence. In yet another embodiment, the polynucleotide sequence is a ribozyme sequence. In yet another embodiment, the polynucleotide sequence is a shRNA sequence. In yet another embodiment the nucleic acid molecule consists of an antisense sequence. In yet another embodiment, the nucleic acid molecule consists of an siRNA sequence.
In a particular embodiment of the above aspect, the class III β-tubulin expressing tumour cell is a non-small cell lung carcinoma cell (NSCLC). In another embodiment the class III β-tubulin expressing tumour cell is selected from an ovarian cancer cell, a breast cancer cell, a gastric cancer cell, a melanoma cell, a prostate cancer cell, or a cell from a tumour of unknown origin.
In a particular embodiment of the above aspects, the subject is a human.
In a particular embodiment of the above aspects, the reduction in tumorigenesis reduces the rate of division of the tumour cell, and thereby reduces the rate of tumour growth in the subject.
In the context of this specification, the term “specific” when used in relation to the nucleotide sequence of an antisense, ribozyme, shRNA or siRNA construct means substantially specific, but not necessarily exclusively so. That is, while being specific for class II, class III or class IVb β-tubulin gene product, the nucleotide sequence may also cross-hybridise with other β-tubulin isotype gene product sequences in an amount sufficient to also inhibit their expression. It may be preferable that the nucleotide sequence does not decrease the expression of non-class II, class III and/or class IVb β-tubulin isotypes, or that it decreases the expression of these other isotypes by a lesser proportion than it decreases the expression of class II, class III and/or class IVb isotypes. Further, for example, the nucleotide sequence of an siRNA construct may display less than 100% sequence identity with the class II, class III or class IVb β-tubulin gene and yet retain specificity thereto. In the context of the present specification, a polynucleotide which is specific, for example, to the class III β-tubulin gene may be able to hybridize to a portion of the genomic sequence found within the class III β-tubulin gene, and/or may be able to hybridize to a portion of the class III β-tubulin mRNA or a class III β-tubulin cDNA sequence in a manner which is able to reduce the expression of class III β-tubulin polypeptide in a cell.
The term “specific” when used in relation to an antibody which is specific to class II, class III or class IVb β-tubulin polypeptide is intended to encompass an antibody which is capable of distinguishing one of these tubulin polypeptide isotypes from other tubulin polypeptide isotypes, for example in an ELISA or Western blot assay. In the case of an antibody which is “specific” for class IVb β-tubulin polypeptide, the antibody will recognise polypeptides of the class IV β-tubulin isotype, while not recognising class II or class III β-tubulin isotypes. An antibody specific for class IVb β-tubulin isotype need not necessarily be able to distinguish between the class IVa and class IVb β-tubulin polypeptides.
As used herein the term “effective amount” includes within its meaning a sufficient amount of an agent or compound to provide the desired therapeutic or prophylactic effect. The exact amount required will vary from subject to subject depending on factors such as the species being treated, the age and general condition of the subject, the severity of the condition being treated, the particular agent being administered and the mode of administration and so forth. Thus, it is not possible to specify an exact “effective amount”. However, for any given case, an appropriate “effective amount” may be determined by one of ordinary skill in the art using only routine experimentation. In the case of an anti-cancer agent preferably the effective amount will be substantially non-toxic to the subject while being toxic to the tumour.
As used herein, a construct which “decreases the expression” of class II, class III and/or IVb β-tubulin in a cell is intended to encompass not only the complete inhibition of the expression of any one, any two or all three of these genes, but also diminution of tubulin gene product or polypeptide expression which is sufficient to enhance the sensitivity of the cell to one or more tubulin-binding agents. Thus a decrease in expression of class II, class III and/or IVb β-tubulin mRNA or polypeptide in a cell of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90% when compared with a cell not exposed to the construct is contemplated. It may be preferable that the decrease in expression of the class II, class III and/or IVb β-tubulin in a cell is sufficient to obtain a maximal enhancement of sensitivity of the cell to one or more microtubule desensitizing agents. It will be understood that the decrease of expression of class II, class III and/or IVb β-tubulin by the construct may be permanent, for example where the construct is stably incorporated into the genome of the somatic cell, such as the tumour cell, to which it is exposed, or may be only temporary. A “construct” is intended to encompass an antisense polynucleotide sequence, a ribozyme, an siRNA sequence, or an shRNA sequence, and also polynucleotide molecules which comprise an antisense polynucleotide sequence, a ribozyme, an siRNA sequence, or an shRNA sequence, such as vectors or expression constructs.
In the context of this specification, the term “comprising” means “including, but not necessarily solely including”. Furthermore, variations of the word “comprising” such as “comprise” and “comprises” have correspondingly varied meanings.
Throughout this specification, reference to “a” or “one” element does not exclude the plural, unless context determines otherwise. For instance, reference to “a nucleic acid construct” should not be read as excluding the possibility of multiple copies of such nucleic II) acid constructs. Similarly, reference throughout the specification to “a tumour cell” is intended to be read as including either a single tumour cell or a plurality of tumour cells, for example a collection of cells obtained from a tumour.
The term “any one or more of class II, class III and class IVb β-tubulin” is intended to encompass any one of class II β-tubulin, class III β-tubulin, class IVb β-tubulin, class II and class III β-tubulin, class II and class IVb β-tubulin, class III and class IVb β-tubulin, and class II, class III and class IVb β-tubulin.
The term “sensitivity” and corresponding terms such as “sensitive” in the context of the interaction between a tumour cell and a tubulin-binding agent or DNA damaging agent is intended to encompass the response of the tumour cell to the anti-mitotic and/or cytotoxic activities of the tubulin-binding agent or DNA damaging agent. Thus, tumour cells with an enhanced sensitivity to a tubulin-binding agent may demonstrate a greater degree of inhibition of mitosis, and/or may exhibit a greater degree of cell death on exposure to a tubulin-binding agent than tumour cells which are less sensitive.
Alternatively, or in addition, a tumour cell which has an increased or enhanced sensitivity to a tubulin-binding agent may respond to the anti-mitotic and/or cytotoxic activity of the tubulin-binding agent where previously it did not respond.
In addition or alternatively, tumour cells with an increased or enhanced sensitivity to a tubulin-binding agent may exhibit an enhanced sensitivity to ionizing radiation and/or a DNA damaging agent on exposure to the tubulin-binding agent.
Tumour cells with an increased or enhanced sensitivity to a tubulin-binding agent may also respond to the anti-mitotic and/or cytotoxic activities of a tubulin-binding agent at lower concentrations of the tubulin-binding agent than a cell which is less sensitive.
In the context of tumour cells in vitro or in situ, an enhanced sensitivity to one or more tubulin-binding agents may be expressed in terms of an enhanced decrease in the number of viable cells in culture or in situ, an enhanced decrease in the progression of tumour enlargement or an enhanced reduction in the size of the tumour on treatment with the one or more tubulin-binding agents. Conversely, the term “resistant” and similar terms such as “resistance” have a corresponding but opposite meaning to “sensitive”.
The resistance of tumour cells to a tubulin-binding agent or a DNA-damaging agent may be directly assessed by a variety of methods available in the art. Examples of such techniques include exposing tumour cells to a series of dilutions of the tubulin-binding agent or DNA damaging agent and determining the concentration at which 50% killing is observed, or the concentration at which the rate of apoptosis is increased or inhibition of clonogenic growth of the cells or the concentration at which the rate of cell division is decreased. Suitable assays are described, for example, in Verrills et al. (2003). Chemistry and Biology 10: 597-607, and in Gan et al. (2007) Cancer Research 67:(19) 9356-9363, the contents of which are incorporated herein by reference.
Similarly, where the “response” of a tumour cell to a tubulin-binding agent is described, it is contemplated that the response may include any one or both of the presence of tumour cell apoptosis or a reduction or halting of the rate of tumour cell division.
As described herein, “tumorigenesis” and associated terms such as “tumorigenicity” relate to the ability of a cancer cell to divide in a manner which results in the production of a tumour. Cancer cells which are capable of forming tumours do not respond to controls on cell division in the same manner as non-tumour cells. For example tumorigenic cancer cells may grow and continue to divide in an anchorage independent manner, that is they do not require contact with a substratum in order to continue to divide, whilst less tumorigenic cancer cells or normal cells substantially do not divide in the absence of contact with a suitable substrate.
Tumour cells that are tumorigenic have the ability to divide to form a tumour. A tumour cell with reduced tumorigenesis has a phenotype in which the ability of the cell to divide and form a tumour is reduced or is substantially inhibited when compared to a tumour cell of the same type. A cell in which tumorigenesis is reduced may, for example exhibit a reduced rate of colony formation in an in vitro assay for anchorage independent cell growth, for example in an soft agar colony formation assay as described herein in Example 18, when compared to a cell of the same type in which tumorigenesis is not reduced. A cell in which tumorigenesis is reduced may, for example, exhibit a reduced rate of tumour formation when implanted in a nude mouse when compared with a cell of the same type in which tumorigenesis is not reduced. A cell in which tumorigenesis is reduced may, for example, exhibit a increased rate of contact inhibition and thus a reduced rate of cell division in a cell culture assay when compared with a cell of the same type in which tumorigenesis is not reduced. A cell with reduced tumorigenesis is less likely to form a tumour in a subject when compared with a cell of the same type in which tumorigenesis is not reduced, and thus is less likely to adversely affect the subject.
In certain embodiments, a cell with reduced tumorigenicity may exhibit an increased expression of one or more tumour suppressors, such as any one or both of Tropomyosin 1 (TM1) and Maspin protein when compared with a cell of the same type in which tumorigenesis is not reduced.
The term “tubulin-binding binding agent” is intended to include molecules which modulate the dynamics of tubulin polymerisation and/or depolymerization within a cell, and comprises microtubule destabilizing agents and microtubule stabilizing agents.
The term “microtubule destabilizing agent” is intended to broadly encompass the class of compounds which bind to tubulin dimers and which destabilize the formation of the tubulin dimers into microtubules. Tubulin and microtubules are commonly targeted for chemotherapy, as their functions are often dysregulated in many types of cancer. Agents which target tubulin or microtubules constitute one of the most effective classes of chemotherapeutics amongst anticancer agents presently in use for the prolongation of survival of subjects with advanced disease. Typically, microtubule destabilizing agents may bind the vinca domain or the colchicine domain of tubulin. A variety of antimitotic agents which interact with tubulin including a variety of microtubule destabilizing agents are described in Hamel (1996) Med. Res. Rev. 16:207-231, the entire contents of which are incorporated herein by reference. Microtubule destabilizing agents may be vinca alkaloid agents, dolostatins, colchicines, cryptophycins, curacin A, 2-methoxyestradiol or derivatives, analogues or prodrugs thereof. The term microtubule destabilizing agent is not intended to encompass compounds which bind to the microtubule polymer and stabilize microtubules, such as the taxanes (which comprises paclitaxel and docetaxel) and the epothilones.
The term “vinca alkaloid agent” is intended to broadly encompass the class of alkaloid compounds which were originally derived from plants of the genus Catharanthus, which possess antimicrotubule activity and which act as mitotic inhibitors. Members of the class include, but are not limited to, vinblastine, vincristine, vinflunine, vindesine, and vinorelbine, and semisynthetic or synthetic analogues or derivatives thereof.
The term “microtubule stabilizing agent” is intended to encompass those compounds which bind to polymerised microtubules and which inhibit the depolymerization of the polymerized microtubules. Compounds which are microtubule stabilizing agents comprise, but are not limited to, the taxanes and the epothilones.
One or more preferred embodiments of the present invention will now be described, by way of an example only, with reference to the accompanying figures, wherein:
In one aspect, there is provided herein are methods of screening for the resistance of a tumour cell to a tubulin-binding agent. Such methods comprise detecting the expression of any one or more of class II, class III and class IVb β-tubulin by the tumour cell,
wherein expression of any one or more of class II, class III and class IVb β-tubulin by the tumour cell is predictive of the resistance to or potential resistance to the tubulin-binding agent.
Also provided is a method of assessing the resistance of a tumour cell to a tubulin-binding agent. The method includes detecting an amount of a class II, class III and class IVb β-tubulin protein or a class II, class III and class IVb β-tubulin mRNA by the tumour cell. A subject tumour cell tubulin amount is thereby obtained. The subject tumour cell tubulin amount is compared to a control cell tubulin amount thereby determining a tubulin amount difference. The control cell tubulin amount is a respective amount of a class II, class III or class IVb β-tubulin protein or any one or more of class II, class III and class IVb β-tubulin mRNA in a control cell. The resistance of the tumour cell to the tubulin-binding agent is assessed based on the tubulin amount difference.
A “subject tumour cell” is simply a tumour cell derived from a sample tumour that is the subject of the resistance assessment. And as indicated above, a subject tumour cell tubulin amount is simply the amount of any one or more of a class II, class III and class IVb β-tubulin protein or a class II, class III and class IVb β-tubulin mRNA detected in the method.
A “control cell” is a cell with a known amount of a class II, class III and/or class IVb β-tubulin protein or a class II, class III and/or class IVb β-tubulin mRNA and a known level of resistance to the tubulin binding agent. In some embodiments, the control cell is a control tumour cell, discussed below. Control cells may be obtained from organisms (or organs) or derived using methods of the present invention. The control cell may be part of a dose response curve generated from exposing cells to a plurality of difference dosages of the tubulin binding agent. Moreover, the method of assessing the resistance of a tumour cell may itself be used to generate such a dose response curve. In some embodiments, the control cell is a mammalian control cell such as a human control cell.
The control cell tubulin amount is the amount of a class II, class III and/or class IVb β-tubulin protein or a class II, class III and/or class IVb β-tubulin mRNA respective to the subject tumour cell tubulin amount. For example, where the subject tumour cell tubulin amount is an amount of class II and class III β-tubulin protein detected from the subject tumour cell, the control cell tubulin amount is the amount of class II and class III β-tubulin within the control cell.
As is apparent from the description of the method of assessing the resistance of a tumour cell above, a “tubulin amount difference” is simply the difference between the subject tumour cell tubulin amount and the control cell tubulin amount.
As is apparent from the description of the methods herein, where the subject tumour cell tubulin amount is higher than the control cell tubulin amount, in some embodiments the subject tumour cell would be assessed as having greater resistance to the tubulin-binding agent than the control cell. For example, where the subject tumour cell class III tubulin amount is higher than the control cell tubulin amount, the subject cell would be assessed as having greater resistance (and less sensitivity) to a vinca alkaloid, a taxane, an epothilone or a DNA damaging agent. For example, where the subject tumour cell class II tubulin amount is higher than the control cell tubulin amount, the subject cell would be assessed as having greater resistance (and less sensitivity) to a vinca alkaloid or 2-methoxy estradiol. For example, where the subject tumour cell class IVb tubulin amount is higher than the control cell tubulin amount, the subject cell would be assessed as having greater resistance (and less sensitivity) to a vinca alkaloid, and to have less resistance (and greater sensitivity) to an epothilone.
Conversely, where the subject tumour cell tubulin amount is lower than the control cell tubulin amount, in some embodiments the subject tumour cell may be assessed as having less resistance (and greater sensitivity) to the tubulin-binding agent than the control cell. For example, where the subject tumour cell class III tubulin amount is lower than the control cell tubulin amount, the subject cell would be assessed as having less resistance (and greater sensitivity) to a vinca alkaloid, a taxane, an epothilone or a DNA damaging agent. For example, where the subject tumour cell class II tubulin amount is lower than the control cell tubulin amount, the subject cell would be assessed as having lower resistance (and greater sensitivity) to a vinca alkaloid or 2-methoxy estradiol. For example, where the subject tumour cell class IVb tubulin amount is lower than the control cell tubulin amount, the subject cell would be assessed as having lower resistance (and greater sensitivity) to a vinca alkaloid, and to have greater resistance (and lower sensitivity) to an epothilone.
Control tumour cells of known resistance to the agent may be, for example, tumour cells which are isolated from an individual prior to successful treatment with the agent. A series of control tumour cells of a range of known resistances to the agent may be assembled for example from tumour cells which are isolated from a group of individuals prior to successful and unsuccessful treatments with the agent.
As demonstrated herein, the presence of certain classes of tubulin in a tumour cell contributes to the responsiveness of the tumour cell to the administration of anti-mitotic agents, such as tubulin-binding agents, or DNA-damaging agents. Accordingly, the assessment of expression of any one or more of class II, class III and class IVb β-tubulin by the tumour cell provides guidance as to the tubulin-dependent responsiveness of the tumour cell to different tubulin-binding agents or DNA-damaging agents, and provides a method by which agents to treat the tumour may be selected.
The resistance of a tumour cell to a tubulin-binding agent may be a complete resistance, or it may be a partial resistance. A tumour cell may be considered “resistant” to a tubulin-binding agent if it is unresponsive to the administration of a tubulin binding agent. An unresponsive tumour cell may demonstrate, for example, an unchanged rate of cell division, an unchanged rate of apoptosis or unchanged cell morphology on exposure to the tubulin binding agent at concentrations which are cytotoxic to non-tumour cells or tumour cells which are sensitive to the agent.
The tumour cell may be resistant to the administration of the tubulin-binding agent in vitro. The tumour cell may be resistant to the administration of the tubulin-binding agent in situ.
In some embodiments a tumour cell may be considered “resistant” to a tubulin-binding agent if it demonstrates a reduced response to the administration of a tubulin-binding agent when compared to control tumour cells of known resistance to the agent.
Control tumour cells from a single tumour with a range of resistances to a tubulin-binding agent and a range of tubulin expression may be generated for the purpose of providing control cells for comparison purposes. Cells with a range of sensitivities may be generated for example by taking control tumour cells which are known to be resistant to the agent, dividing the cells into groups, and stably modulating the expression of at least one of class II, class III and class IVb β-tubulin in each of the groups of cells, using for example the siRNA or shRNA methods described herein. By generating different groups of cells in which the expression of at least one of class II, class III and class IVb is stably reduced in a controlled fashion, as demonstrated herein it is possible to generate cells from a single tumour with a range of sensitivities to a tubulin-binding agent.
The resistance of a tumour may be assessed with reference to concentration of a tubulin-binding agent which is used therapeutically in human subjects.
In some embodiments a tumour cell may be considered “resistant” to a tubulin-binding agent if it demonstrates a response to a tubulin-binding agent which is similar to the response of cells of a control tumour of known resistance to the tubulin-binding agent. A control tumour of known resistance to a tubulin-binding agent may be isolated, for example, from a subject prior to the unsuccessful treatment with the tubulin-binding agent. Alternatively, a resistant control tumour cell may be generated from a primary tumour cell or a tumour cell line in vitro by treating a tumour primary tumour cell or tumour cell line at least once with the tubulin-binding agent at a concentration which results in greater than 90% cell death, and selecting for cells which survive the treatment.
A tumour cell may be “potentially resistant” if it is suspected of resistance to or of becoming resistant to a microtubule-binding agent.
The method comprises “detecting” the expression of any one or more of class II, class III and class IVb β-tubulin by the tumour cell. In certain embodiments the identification of the presence of a particular tubulin in tumour cells as detected by a particular technique may be sufficient to assess the tumour as being resistant to a tubulin-binding agent, and conversely the identification of the absence of a particular tubulin in tumour cells by the technique may be sufficient to assess the tumour as being sensitive to the agent. In such embodiments it may not be necessary to make a determination of the amount of tubulin expressed by the tumour cells in order to assess the resistance of the tumour cell.
In other embodiments, the detecting of the expression of any one or more of class II, class III and class IVb β-tubulin by the tumour cell will involve a comparison of the amounts of tubulin of any one or more of these classes expressed by the tumour cell with the amount of tubulin of any one or more of these classes expressed by at least one cell of known resistance.
In particular embodiments the comparison will be with the amount of tubulin expressed by a plurality of control cells of different known resistances. As demonstrated in the examples provided herein, in at least certain embodiments the degree or resistance of a tumour cell may be proportional (for example in the case of class III β-tubulin and vinca alkaloids) or inversely proportional (for example in the case of class IVb β-tubulin and epothilone) to the amount of tubulin which is expressed by the tumour cell. Accordingly, a comparison may be made against a dose-response curve generated for example from multiple control tumour cells.
Thus, the detecting of expression of any one or more of class II, class III and class IVb β-tubulin by the tumour cell may comprise detecting the presence or absence of tubulin mRNA or the presence or absence of tubulin polypeptide in the tumour cell.
Alternatively, the detecting of expression of any one or more of class II, class III and class IVb β-tubulin by the tumour cell may comprise determining the relative amount of tubulin mRNA or the relative amount of tubulin polypeptide expressed by the tumour cell.
The relative amount may be in comparison with the abundance of a tubulin mRNA or the amount of a tubulin polypeptide in cells of a known control sensitive tumour. The relative amount may be in comparison with the amount of a tubulin mRNA or the amount of a tubulin polypeptide in cells of a known resistant control tumour. The relative amount may be in comparison with the amount of a tubulin mRNA or the amount of a tubulin polypeptide in control tumour cells of a range of sensitivities. The relative amount may be in comparison with the amount of tubulin mRNA or the amount of tubulin polypeptide in non-tumour cells taken from the tissue surrounding the tumour. The relative amount may be a normalised amount as determined by comparison with the expression of a mRNA or polypeptide which is expressed at relatively constant levels, such as GAPDH.
The presence or absence or relative amount of tubulin mRNA may be detected by any one or more of RT-PCR, quantitative PCR, semi-quantitative PCR, or in-situ hybridization under stringent conditions, using one or more probes or primers which are specific for any one of class II, class III and class IVb β-tubulins. Examples of polynucleotides which may be used for RT-PCR or semi-quantitative PCR techniques are described in Kavallaris et al. (1997) J Clin Invest 100, 1282-1293, the entire contents of which are incorporated herein by reference. In a particular embodiment, the presence or absence of mRNA is detected using RT-PCR, for example using methodology and specific primers as described in Kavallaris et al. (1997) J Clin Invest 100, 1282-1293.
Where a measurement of the relative amount of a tubulin mRNA is required, known techniques such as real-time reverse transcriptase polymerase chain reaction may be employed to reverse transcribe RNA and then quantitate the resulting cDNA using real-time PCR. Quantitative techniques for assessing the expression of RNA are reviewed in Ding and Cantor (2004) J Biochem Mol Biol 37(1):1-10, and in Bustin et al., (2005) Journal of Molecular Endocrinology 34: 579-601, the entire contents of which are incorporated by reference.
The presence or absence or relative amount of tubulin polypeptide may be detected using any one or more of Western blotting, ELISA, or other standard quantitative or semi-quantitative techniques available in the art, or a combination of such techniques for identifying the presence of specific polypeptides. A range of quantitative and semi-quantitative proteomic techniques are reviewed, for example in Hirsch et al., (2004) Am J Physiol Lung Cell Mol Physiol 278: L1-L23, the entire contents of which is incorporated herein by reference. Techniques relying on antibody recognition of one or more specific tubulin isotype polypeptides are contemplated in particular. In a particular embodiment, the presence or absence or relative abundance of tubulin polypeptide may be detected with techniques which comprise semi-quantitative Western blotting, for example using the Western blotting technique described in Example 2 described herein. In other particular embodiments, the presence or absence or relative abundance of tubulin polypeptide may be detected with techniques which comprise antibody capture of tubulin polypeptides in combination with electrophoretic resolution of captured tubulin polypeptides, for example using the Isonostic™ Assay (Target Discovery, Inc.).
In particular embodiments, tumour cells which are detected as expressing β-III tubulin are predicted to be resistant to a vinca alkaloid, a taxane, an epothilone or a DNA damaging agent.
In particular embodiments, tumour cells which are detected as expressing β-II tubulin are predicted to be resistant to a vinca alkaloid or 2-methoxy estradiol.
In particular embodiments, tumour cells which are detected as expressing β-IVb tubulin are predicted to be resistant to a vinca alkaloid, and to be sensitive to an epothilone.
In certain embodiments, the tubulin-binding agent is a microtubule stabilizing agent, such as a taxane or an epothilone.
In certain embodiments, the tubulin-binding agent is a microtubule destabilizing agent, such as a vinca alkaloid or 2-methoxy estradiol.
Methods described herein of screening for or assessing the resistance of a tumour cell a tubulin-binding agent may be practiced prior to the administration of an anti cancer agent, in order to determine whether the cells of the tumour will respond to one or more tubulin-binding agents, or may be practiced during a course of treatment, to determine whether the cells of the tumour are developing resistance to the tubulin-binding agent.
In a further aspect, the invention relates to the introduction of a nucleic acid construct comprising a nucleic acid sequence specific for at least a portion of the class II, class III or IVb β-tubulin gene to a tumour, wherein the nucleic acid construct decreases the expression of class II, class III or IVb β-tubulin.
Although the exemplified nucleic acid constructs comprising a nucleotide sequence described herein are siRNA or shRNA sequences, it will be clearly understood that the targeted disruption of the expression of class II, class III and/or IVb β-tubulin genes may be achieved using any molecules which selectively target and inhibit the expression of class II, class III and/or IVb β-tubulin genes, such as antisense sequences, varieties of small interfering RNA (siRNA) sequences, shRNA sequences, ribozyme sequences, microRNA and the like. The “expression” of class II, class III and/or IVb β-tubulin genes is intended to encompass the transcription and/or translation of class II, class III and/or IVb β-tubulin sequences. Accordingly, the nucleic acid sequence which is specific for at least a portion of the class II, class III or IVb β-tubulin gene is also intended to encompass a nucleic acid sequence which is specific for at least a portion of the class II, class III or IVb β-tubulin mRNA or cDNA.
“Detecting the expression” is intended to encompass not only the detection of the presence of class II, class III or IVb β-tubulin mRNA or protein but also in certain embodiments the amount of the class II, class III or IVb β-tubulin mRNA or protein.
“Detecting the amount” of a class II, class III or IVb β-tubulin mRNA or protein is intended to include detecting the absolute levels of the mRNA or protein, or in certain embodiments the relative amounts of the mRNA or protein. The relative amounts are relative to one or more other cellular proteins or mRNAs, such as other tubulin proteins or mRNAs or a housekeeping gene protein or mRNA in the cell. Thus in some embodiments the amounts are normalised relative to other cellular proteins or mRNAs.
Methods for the design, synthesis, and delivery of antisense nucleic acids are well known in the art. The antisense molecules may be DNA or RNA, or partial or complete synthetic analogues thereof. Antisense constructs may be generated which are at least substantially complementary along their length to the region of the gene in question. Binding of an antisense construct to its complementary cellular sequence may interfere with transcription, RNA processing, transport, translation and/or mRNA stability.
Suitable antisense oligonucleotides may be prepared by methods well known to those of skill in the art. Typically antisense oligonucleotides will be synthesized on automated synthesizers. Suitable antisense oligonucleotides may include modifications designed to improve their delivery into cells, their stability once inside a cell, and/or their binding to the appropriate target. For example, the antisense oligonucleotide may be modified by the addition of one or more phosphorothioate linkages, or the inclusion of one or more morpholine rings into the backbone. The antisense oligonucleotide may be 10-30 base pairs in length and will be target regions of the class II, class III and/or IVb β-tubulin gene. In one embodiment, the antisense oligonucleotide sequences will have no more than 90% sequence identity with other known tubulin isotypes.
As a practical matter, whether any particular nucleic acid molecule is no more than 90% identical to, for instance, the nucleotide sequence of other known tubulin isotypes can be determined conventionally using known computer programs such as the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis. 53711). Bestfit uses the local homology algorithm of Smith and Waterman to find the best segment of homology between two sequences (Advances in Applied Mathematics 2:482-489 (1981)). When using Bestfit or any other sequence alignment program to determine whether a particular sequence is, for instance, 90% identical to a reference sequence, the parameters are set such that the percentage of identity is calculated over the full length of the reference nucleotide sequence and that gaps in homology of up to 5% of the total number of nucleotides in the reference sequence are allowed. A preferred method for determining the best overall match between a query sequence and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag and colleagues (Comp. App. Biosci. 6:237-245 (1990)). In a sequence alignment the query and subject sequences are both DNA sequences. An RNA sequence can be compared by converting U's to T's. The result of said global sequence alignment is in percent identity. Preferred parameters used in a FASTDB alignment of DNA sequences to calculate percent identity are: Matrix=Unitary, k-tuple=4, Mismatch Penalty=1, Joining Penalty=30, Randomization Group Length=0, Cutoff Score=1, Gap Penalty=5, Gap Size Penalty 0.05, Window Size=500 or the length of the subject nucleotide sequence, whichever is shorter.
If the subject sequence is shorter than the query sequence because of 5′ or 3′ deletions, not because of internal deletions, a manual correction must be made to the results. This is because the FASTDB program does not account for 5′ and 3′ truncations of the subject sequence when calculating percent identity. For subject sequences truncated at the 5′ or 3′ ends, relative to the query sequence, the percent identity is corrected by calculating the number of bases of the query sequence that are 5′ and 3′ of the subject sequence, which are not matched/aligned, as a percent of the total bases of the query sequence. Whether a nucleotide is matched/aligned is determined by results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score. Only bases outside the 5′ and 3′ bases of the subject sequence, as displayed by the FASTDB alignment, which are not matched/aligned with the query sequence, are calculated for the purposes of manually adjusting the percent identity score.
It may be advantageous if the specific sequences to be targeted are unique to either class II, class III and/or IVb β-tubulin gene sequences. These amino acid sequences of human β-tubulin isotypes are described in Verdier-Pinard et al., (2005) Biochemistry 44:15858-15870, the entire contents of which are incorporated herein by reference. The amino acid sequence of human class III β-tubulin isotype is described in UniProtKB/Swiss-Prot Accession Number Q13509 (SEQ ID NO:26). Corresponding cDNA and/or genomic DNA sequences are also known. The complete mRNA sequence of human class III β-tubulin is provided, for example, in GenBank accession number U47634 (SEQ ID NO:27). The genomic sequence of Human class III β-tubulin is provided, for example, in Ensembl accession number ENSG00000198211 (chromosome: NCBI36:16:88512568:88530606:1)(SEQ ID NO:28). The skilled addressee will be able to readily determine whether any given sequence is specific to either class II, III and/or IVb β-tubulin gene sequences.
Small interfering RNA (siRNA) sequences are small, usually double-stranded RNA oligonucleotides, for example at 19, 21, 27 or 29 bases in length with or without overhangs, which specifically hybridise with RNA sequences of interest and which serve as substrates for the RNA-induced silencing complex. Double-stranded RNA molecules may be synthesised in which one strand is identical to a specific region of the mRNA transcript to be silenced, and this double stranded RNA may be introduced directly. Alternatively, corresponding dsDNA can be employed, which, once presented intracellularly is converted into dsRNA. Methods for the synthesis of suitable siRNA molecules for use in RNA interference (RNAi) and for achieving post-transcriptional gene silencing are known to those of skill in the art, and there are now commercial services for designing and producing siRNAs. For example, rules for the rational design of siRNA are available online in “Rules of siRNA design for RNA interference (RNAi)”. These rational design principals include are described in Elbashir S M et al. (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature. 411:494-498; Elbahir S M et al. (2001). Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate. EMBO J. 20:6877-6888; Elbashir S M et al. (2002). Analysis of gene function in somatic mammalian cells using small interfering RNAs. Methods. 26:199-213; Reynolds A, Leake D, Boese Q, Scaringe S, Marshall W S, Khvorova A. Rational siRNA design for RNA interference. Nat Biotechnol. 2004 March; 22(3):326-30. The entire contents of each of these publications is incorporated herein by reference. Examples of two siRNA design tools which implement the rational siRNA principals discussed above are design algorithms offered by Dharmacon, Inc and a downloadable Microsoft Excel™ template written by Maurice Ho.
The skilled addressee will appreciate that a range of suitable siRNA constructs capable of inhibiting the expression of the class II, class III and/or class IVb β-tubulin can be identified and generated based on knowledge of the sequence of the genes in question using routine procedures known to those skilled in the art without undue experimentation. As demonstrated herein, a variety of siRNA or shRNA sequences directed to different non-overlapping regions of a specific β-tubulin are able to influence the expression of that tubulin in a cell. Those skilled in the art will appreciate that there need not necessarily be 100% nucleotide sequence match between the target sequence and the siRNA sequence. The capacity for mismatch is dependent largely on the location of the mismatch within the sequences. In some instances, mismatches of 2 or 3 nucleotides may be acceptable but in other instances a single nucleotide mismatch is enough to negate the effectiveness of the siRNA. The suitability of a particular siRNA molecule may be determined using routine procedures known to those skilled in the art without undue experimentation.
Although the maximal effects of antisense nucleic acids and siRNA on the specific inhibition of RNA or protein expression are comparable, siRNA generally produces a longer-lasting effect. siRNAs may be introduced into a cell by way of a vector, for example via a viral-mediated delivery mechanism such as an adeno-associated virus vector, or delivered exogenously, for example when delivered as part of a liposome complex. Techniques for the non-viral delivery of synthetic siRNAs in vivo are reviewed in Akhtar and Benter (2007) J. Clin. Invest 117:3623-3632, the entire contents of which is incorporated by reference. Techniques for the administration of siRNA sequences locally or systemically to mice (Yano et al, (2004) Clinical Cancer Research 10: 7721-7726), to primates (Zimmermann et al, (2006) Nature 441(7089):111-114) and to humans (Nogawa et al. (2006) J Clin Invest 115:978-985) have been described (see for example the review of Akhtar and Benter (2007) J Clin Invest 117: 3623-3632), and these have demonstrated that this class of molecules can be used to reduce the expression of target genes in vivo. The entire contents of each of these citations is incorporated herein by reference. The use of siRNA delivery systems such as cholesterol-siRNA conjugates, cationic delivery systems, including cationic nanoparticles or cationic liposomes or cationic polymer or peptide delivery systems, or chitosan-siRNA conjugates is contemplated.
The systemic administration of siRNA has been demonstrated to result in an accumulation of the siRNA in the reticuloendothelial system, including the liver and lungs, and accordingly this may be advantageous when administering an siRNA for the treatment of lung tumours.
Ribozymes, such as hammerhead or hairpin ribozymes, are capable of the targeted catalytic cleavage and splicing of specific RNA sequences, including mRNA and genomic RNA sequences. The design and methods for the delivery of ribozymes are reviewed, for example, in Vaish, Kore and Eckstein (1998) Nucleic Acids Research 26:5237-5242; in Lieber and Strauss (1995) Mol. Cell. Biol. 15:540-551; and in Usman and Blatt (2000) J Clin Invest 106:1197-1202, the entire contents of each of which are incorporated herein by reference. The use of ribozymes to target and regress specific tumours is described in the art, for example Zhang et al. (2000) Gene Ther 7:2041-2050.
Short hairpin RNA (shRNA) is a sequence of RNA with a tight hairpin turn structure which is introduced into cells as part of a vector which comprises a constitutive promoter such as a U6 promoter to allow shRNA to be constitutively expressed. The shRNA hairpin structure is cleaved by the cellular machinery into siRNA, which is then bound to the RNA-induced silencing complex (RISC). This complex binds to and cleaves mRNAs which match the siRNA that is bound to it. The vector comprising the shRNA sequence is usually passed on to daughter cells in cell division, allowing the gene silencing to be inherited. Strategies for designing shRNA sequences and nucleic acid molecules comprising shRNA for use in mammalian cells are known, for example McIntyre and Fanning, Design and cloning strategies for constructing shRNA expression vectors, BMC Biotechnol. (2006) 6:1.
In general, each of these classes of molecules appears to be well tolerated, either when administered to human or animal subjects systemically, such as via intravenous routes, as a bolus or by sustained release techniques subcutaneously, or when directly applied to tumour cells or injected into or in the vicinity of tumours.
Delivery to the lungs in a formulation which comprises an inhalable polynucleotide construct is also contemplated, in particular for tumours of the lung. The administration of siRNA as an aerosol directly to the lungs by nebulizer for the treatment of respiratory syncytial virus infection is currently in clinical trials (Alnylam ALN-RSV01, Bitco et al, (2005) Nat Med 11:50-55), and has demonstrated safety, tolerance and efficacy in antiviral activity. Accordingly, the delivery of an siRNA to the lungs to modulate specific tubulin expression in tumours of the lungs is contemplated.
Suitable polynucleotide sequences may be administered in isolation or in a nucleic acid construct. The construct may be a plasmid vector, a viral vector, or any other suitable vehicle adapted for the insertion of foreign sequences and introduction into eukaryotic cells. The vector may be an expression vector capable of directing the transcription of a DNA sequence into RNA. Viral expression vectors include, for example, epstein-barr virus-, bovine papilloma virus-, adenovirus- and adeno-associated virus-based vectors.
In one embodiment, the vector is episomal. The use of a suitable episomal vector provides a means of maintaining the polynucleotide sequence in target cells in high copy number extra-chromosomally, thereby eliminating potential effects of chromosomal integration.
In the context of the present invention, it may be preferable to administer the polynucleotide construct in a form so that it is not permanently incorporated into the genome of the somatic cell to which it is administered, to allow temporal control of the inhibition of expression of class II, class III and/or class IVb β-tubulin.
Throughout the specification, where reference is made to a polynucleotide, such as a polynucleotide construct or a nucleic acid sequence, it will be understood that it is intended to encompass non-naturally occurring polynucleotides and nucleic acids, provided that they retain the ability to interact specifically in the cell while possessing low toxicity. The use of certain non-naturally occurring nucleic acids, for example, provides increased resistance to nuclease digestion, which in turn may the increase half-life of the nucleic acid following administration. Chemical stabilisation to reduce nuclease digestion, for example, has been demonstrated in the ribozyme “ANGIOZYME” which is currently undergoing clinical trials.
Where exogenous polynucleotide constructs are to be delivered to isolated cells or to a subject, they will be formulated in an appropriate diluent, such as saline. In order to promote translocation of the nucleic acid constructs into the cell cytoplasm the constructs may be encapsulated in liposomes, and/or conjugated to ligands or targeting molecules which promote the recognition of the desired target cells and/or the penetration of the plasma membrane. The use of siRNA conjugated with cell-penetrating peptides, for example, has been demonstrated to increase cellular uptake of siRNA (Veldhoen et al. (2006) Nucleic Acids Res 34: 6561-6573). Other techniques which are known in the art for increasing the transfection efficiency of an applied nucleic acid include biochemical methods, such as the use of DEAE-dextran, the conjugation of the siRNA to nanoparticles, calcium phosphate transfection methods and/or physical transfection methods such as direct micro-injection, electroporation or biolistic particle delivery.
The introduction of a polynucleotide as described herein encompasses the use of polycationic agents, which are compounds that can be used to delivery siRNA to cancer cells. As disclosed herein, the polycationic agent polyethylenimine (PEI), which is a low molecular weight compound, may be used as a delivery vehicle for the delivery of the nucleic acid construct. PEI has the ability to deliver siRNA, for example, to cells by firstly, condensing siRNA into positively charged particles which are capable of interacting with anionic proteoglycans at the cell surface and promoting the entry into cells by endocytosis. Secondly, the noncovalent complexation of siRNA with PEI efficiently stabilises the siRNAs and provides greater efficiency of action for a given concentration.
Delivery of RNA silencing agents using nanotransporters, such as nanoparticles or nanotubes, which have the potential to increase cell target specificity is also contemplated. Suitable nanotransporters are described, for example in International patent publication No. WO 2007/089607 “RNA Silencing agents for use in therapy and nanotransporters for efficient delivery of same”, the entire content of which is incorporated herein by reference.
As demonstrated herein, specific β-tubulin isotypes confer altered sensitivity to tubulin-binding agents and DNA-damaging agents. This information may be used to exploit natural differences in isotype composition among various cell types to target specific drugs to specific tumours. The hypersensitivity of a cell may result both from enhanced apoptotic pathways and from defective mitosis due to modifications in microtubules and their associated protein, thus enhancing the sensitivity of these cells to tubulin-binding agents.
Although the use of a nucleic acid construct comprising a polynucleotide sequence which is specific to class II and/or class IVb β-tubulin gene to enhance the sensitivity of certain NSCLC cell lines and leukaemia cell lines to tubulin-binding agents is described herein, due to the specificity of the mode of action of the nucleic acid construct it is anticipated that any other tumour cell types which express the class II, class III and/or class IVb β-tubulin genes will also demonstrate an enhanced sensitivity to at least one microtubule destabilizing agent when treated in a similar fashion. For example, ovarian tumours resistant to paclitaxel may express increased levels of class II and/or class IVb β-tubulin, neuroblastoma cells may express high levels of class II, and leukaemia cells and breast cancer cells may also express class II and/or class IVb β-tubulin. As used herein a tumour cell is a neoplastic cell, and is intended to encompass not only cells found in solid tumours but also isolated neoplastic cells or circulating neoplasms such as leukemic cells.
Numerous methods are available in the art to determine whether any particular tumour cell expresses the class II, Class III and/or class IVb β-tubulin gene, including the use of commercially available tubulin isotype-specific antibodies and/or isotype-specific nucleic acid primers and/or probes.
The tumour cell when exposed to the nucleic acid construct may be in vitro, in situ in a tumour removed from a subject, or in vivo. The tumour cell will be of mammalian origin, and in one embodiment of human origin.
In one embodiment the nucleic acid construct sequences are introduced before commencement of treatment with the one or more microtubule destabilizing agents. The timing of the introduction of the nucleic acid construct sequences will depend on the route of administration of the construct and the kinetics of inhibition of the class II, class III and/or class IVb β-tubulin gene of the relevant target. The level of class II, class III and/or class IVb β-tubulin gene expression may be monitored for example using tissue biopsy specimens and real-time PCR specific for the class II, class III and/or class IVb β-tubulin gene expression. For siRNA administration to a cell, maximum gene suppression occurs at approximately 72 h after the siRNA has entered the cell, and this is the point that the cells are at maximum sensitivity to the one or more microtubule destabilizing agents. Accordingly preferably the administration of the microtubule destabilizing agent may be commenced at any time whereby the agent is present while the class II and/or class IVb β-tubulin gene expression of tumour cells is maximally suppressed.
The present invention contemplates methods of treatment which involve the use of compositions comprising the nucleic acid constructs described herein and pharmaceutical compositions comprising the same.
In general, suitable compositions for use in accordance with the methods of the present invention may be prepared according to methods and procedures that are known to those of ordinary skill in the art and accordingly may include a pharmaceutically acceptable carrier, diluent and/or adjuvant.
Compositions may be administered by standard routes. In general, the compositions may be administered by the parenteral (e.g., intravenous, intraspinal, subcutaneous or intramuscular), oral or topical route. Administration may be systemic, regional or local. The particular route of administration to be used in any given circumstance will depend on a number of factors, including the nature of the condition to be treated, the severity and extent of the condition, the required dosage of the particular compound to be delivered and the potential side-effects of the compound.
In general, suitable compositions may be prepared according to methods which are known to those of ordinary skill in the art and may include a pharmaceutically acceptable diluent, adjuvant and/or excipient. The diluents, adjuvants and excipients must be “acceptable” in terms of being compatible with the other ingredients of the composition, and not deleterious to the recipient thereof.
In addition to the aforementioned agents which may increase the efficiency of transfection, examples of pharmaceutically acceptable carriers or diluents are demineralised or distilled water; saline solution; vegetable based oils such as peanut oil, safflower oil, olive oil, cottonseed oil, maize oil, sesame oils such as peanut oil, safflower oil, olive oil, cottonseed oil, maize oil, sesame oil, arachis oil or coconut oil; silicone oils, including polysiloxanes, such as methyl polysiloxane, phenyl polysiloxane and methylphenyl polysolpoxane; volatile silicones; mineral oils such as liquid paraffin, soft paraffin or squalane; cellulose derivatives such as methyl cellulose, ethyl cellulose, carboxymethylcellulose, sodium carboxymethylcellulose or hydroxypropylmethyl-cellulose; lower alkanols, for example ethanol or iso-propanol; lower aralkanols; lower polyalkylene glycols or lower alkylene glycols, for example polyethylene glycol, polypropylene glycol, ethylene glycol, propylene glycol, 1,3-butylene glycol or glycerin; fatty acid esters such as isopropyl palmitate, isopropyl myristate or ethyl oleate; polyvinylpyrridone; agar; carrageenan; gum tragacanth or gum acacia, and petroleum jelly. Typically, the carrier or carriers will form from 10% to 99.9% by weight of the compositions.
The compositions of the invention may be in a form suitable for administration by injection, in the form of a formulation suitable for oral ingestion (such as capsules, tablets, caplets, elixirs, for example), in the form of an ointment, cream or lotion suitable for topical administration, in a form suitable for delivery as an eye drop, in an aerosol form suitable for administration by inhalation, such as by intranasal inhalation or oral inhalation, in a form suitable for parenteral administration, that is, subcutaneous, intramuscular or intravenous injection.
For administration as an injectable solution or suspension, non-toxic parenterally acceptable diluents or carriers can include, Ringer's solution, isotonic saline, phosphate buffered saline, ethanol and 1,2 propylene glycol.
Some examples of suitable carriers, diluents, excipients and adjuvants for oral use include peanut oil, liquid paraffin, sodium carboxymethylcellulose, methylcellulose, sodium alginate, gum acacia, gum tragacanth, dextrose, sucrose, sorbitol, mannitol, gelatine and lecithin. In addition these oral formulations may contain suitable flavouring and colourings agents. When used in capsule form the capsules may be coated with compounds such as glyceryl monostearate or glyceryl distearate which delay disintegration.
Adjuvants typically include emollients, emulsifiers, thickening agents, preservatives, bactericides and buffering agents.
Solid forms for oral administration may contain binders acceptable in human and veterinary pharmaceutical practice, sweeteners, disintegrating agents, diluents, flavourings, coating agents, preservatives, lubricants and/or time delay agents. Suitable binders include gum acacia, gelatine, corn starch, gum tragacanth, sodium alginate, carboxymethylcellulose or polyethylene glycol. Suitable sweeteners include sucrose, lactose, glucose, aspartame or saccharine. Suitable disintegrating agents include corn starch, methylcellulose, polyvinylpyrrolidone, guar gum, xanthan gum, bentonite, alginic acid or agar. Suitable diluents include lactose, sorbitol, mannitol, dextrose, kaolin, cellulose, calcium carbonate, calcium silicate or dicalcium phosphate. Suitable flavouring agents include peppermint oil, oil of wintergreen, cherry, orange or raspberry flavouring. Suitable coating agents include polymers or copolymers of acrylic acid and/or methacrylic acid and/or their esters, waxes, fatty alcohols, zein, shellac or gluten. Suitable preservatives include sodium benzoate, vitamin E, alpha-tocopherol, ascorbic acid, methyl paraben, propyl paraben or sodium bisulphite. Suitable lubricants include magnesium stearate, stearic acid, sodium oleate, sodium chloride or talc. Suitable time delay agents include glyceryl monostearate or glyceryl distearate.
Liquid forms for oral administration may contain, in addition to the above agents, a liquid carrier. Suitable liquid carriers include water, oils such as olive oil, peanut oil, sesame oil, sunflower oil, safflower oil, arachis oil, coconut oil, liquid paraffin, ethylene glycol, propylene glycol, polyethylene glycol, ethanol, propanol, isopropanol, glycerol, fatty alcohols, triglycerides or mixtures thereof.
Suspensions for oral administration may further comprise dispersing agents and/or suspending agents. Suitable suspending agents include sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethyl-cellulose, poly-vinyl-pyrrolidone, sodium alginate or acetyl alcohol. Suitable dispersing agents include lecithin, polyoxyethylene esters of fatty acids such as stearic acid, polyoxyethylene sorbitol mono- or di-oleate, -stearate or -laurate, polyoxyethylene sorbitan mono- or di-oleate, -stearate or -laurate and the like.
The emulsions for oral administration may further comprise one or more emulsifying agents. Suitable emulsifying agents include dispersing agents as exemplified above or natural gums such as guar gum, gum acacia or gum tragacanth.
Methods for preparing parenterally administrable compositions are apparent to those skilled in the art, and are described in more detail in, for example, Remington's Pharmaceutical Science, 15th ed., Mack Publishing Company, Easton, Pa., hereby incorporated by reference herein.
The topical formulations of the present invention, comprise an active ingredient together with one or more acceptable carriers, and optionally any other therapeutic ingredients. Formulations suitable for topical administration include liquid or semi-liquid preparations suitable for penetration through the skin to the site of where treatment is required, such as liniments, lotions, creams, ointments or pastes, and drops suitable for administration to the eye, ear or nose.
The use of antisense oligonucleotide compositions for administration to the lungs, and compositions suitable for this use, are described for example in U.S. Pat. No. 6,825,174, the entire contents of which are incorporated herein by reference. The methods described therein may be used to prepare suitable compositions for inhalable or intranasal delivery.
Drops according to the present invention may comprise sterile aqueous or oily solutions or suspensions. These may be prepared by dissolving the active ingredient in an aqueous solution of a bactericidal and/or fungicidal agent and/or any other suitable preservative, and optionally including a surface active agent. The resulting solution may then be clarified by filtration, transferred to a suitable container and sterilised. Sterilisation may be achieved by filtration, followed by transfer to a container by an aseptic technique. Examples of bactericidal and fungicidal agents suitable for inclusion in the drops are phenylmercuric nitrate or acetate (0.002%), benzalkonium chloride (0.01%) and chlorhexidine acetate (0.01%). Suitable solvents for the preparation of an oily solution include glycerol, diluted alcohol and propylene glycol.
Lotions according to the present invention include those suitable for application to the skin or eye. An eye lotion may comprise a sterile aqueous solution optionally containing a bactericide and may be prepared by methods similar to those described above in relation to the preparation of drops. Lotions or liniments for application to the skin may also include an II) agent to hasten drying and to cool the skin, such as an alcohol or acetone, and/or a moisturiser such as glycerol, or oil such as castor oil or arachis oil.
Creams, ointments or pastes according to the present invention are semi-solid formulations of the active ingredient for external application. They may be made by mixing the active ingredient in finely-divided or powdered form, alone or in solution or suspension in an aqueous or non-aqueous fluid, with a greasy or non-greasy basis. The basis may comprise hydrocarbons such as hard, soft or liquid paraffin, glycerol, beeswax, a metallic soap; a mucilage; an oil of natural origin such as almond, corn, arachis, castor or olive oil; wool fat or its derivatives, or a fatty acid such as stearic or oleic acid together with an alcohol such as propylene glycol or macrogols.
The composition may incorporate any suitable surfactant such as an anionic, cationic or non-ionic surfactant such as sorbitan esters or polyoxyethylene derivatives thereof. Suspending agents such as natural gums, cellulose derivatives or inorganic materials such as silicaceous silicas, and other ingredients such as lanolin, may also be included.
The composition may also be administered or delivered to target cells in the form of liposomes. Liposomes are generally derived from phospholipids or other lipid substances, and are formed by mono- or multi-lamellar hydrated liquid crystals that are dispersed in an aqueous medium. Specific examples of liposomes used in administering or delivering a composition to target cells are synthetic cholesterol (Sigma), the phospholipid 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC; Avanti Polar Lipids), the PEG lipid 3-N-[(-methoxy poly(ethylene glycol)2000)carbamoyl]-1,2-dimyrestyloxy-propylamine (PEG-cDMA), and the cationic lipid 1,2-di-o-octadecenyl-3-(N,N-dimethyl)aminopropane (DODMA) or 1,2-dilinoleyloxy-3-(N,N-dimethyl)aminopropane (DLinDMA) in the molar ratios 55:20:10:15 or 48:20:2:30, respectively, PEG-cDMA, DODMA and DLinDMA. Any non-toxic, physiologically acceptable and metabolisable lipid capable of forming liposomes can be used. The compositions in liposome form may contain stabilisers, preservatives, excipients and the like. The preferred lipids are the phospholipids and the phosphatidyl cholines (lecithins), both natural and synthetic. Methods to form liposomes are known in the art, and in relation to this specific reference is made to: Prescott, Ed., Methods in Cell Biology, Volume XIV, Academic Press, New York, N.Y. (1976), p. 33 et seq., the contents of which is incorporated herein by reference.
The use of DOTAP and/or other cationic lipid-mediated nucleic acid delivery systems is also contemplated. DOTAP has been used for the systemic delivery of a siRNA for gene slicing, for example (Sorenson et al., (2003) J. Mol. Biol. 327:761-766, the entire contents of which are incorporated herein by reference).
The composition may be conjugated to polycationic agents for the delivery of the composition into a cell. Polycationic agents can be used as delivery vehicles for compositions. One example of a polycationic agent is poly(ethyleneimine), or PEI. PEI is a low molecular weight compound which condenses short nucleic acid strands such as siRNA into positively charged particles which interaction with the anionic surface of a cell. PEI can be used alone or further conjugated to PEG. Another example of a polycationic agent is poly-L-lysine (PLL). Use of polycationic agents for the delivery of a composition to a cell is known in the art, and in relation to this specific reference is made to Judge et al. (2005). Nature 25: 457-462, the contents of which is incorporated herein by reference.
The compositions may also be administered in the form of microparticles. Biodegradable microparticles formed from polylactide (PLA), polylactide-co-glycolide (PLGA), and epsilon-caprolactone (g-caprolactone) have been extensively used as drug carriers to increase plasma half life and thereby prolong efficacy (Kumar, M., 2000, J Pharm Pharmaceut Sci. 3(2) 234-258). Microparticles have been formulated for the delivery of a range of drug candidates including vaccines, antibiotics, and DNA. Moreover, these formulations have been developed for various delivery routes including parenteral subcutaneous injection, intravenous injection and inhalation.
The compositions may comprise nanoparticles. Nanoparticles such as PEG-containing nanoparticles, have been demonstrated to readily taken up into cells because of their size. Where the compositions described herein are associated with nanoparticles, for example by complementary charges on the composition and the surface of the nanoparticles, the delivery of the composition into cells may be enhanced by the cell delivery properties provided by the nanoparticles.
The compositions may incorporate a controlled release matrix that is composed of sucrose acetate isobutyrate (SAIB) and organic solvent or organic solvents mixture. Polymer additives may be added to the vehicle as a release modifier to further increase the viscosity and slow down the release rate. SAIB is a well known food additive. It is a very hydrophobic, fully esterified sucrose derivative, at a nominal ratio of six isobutyrate to two acetate groups. As a mixed ester, SAIB does not crystallize but exists as a clear viscous liquid. Mixing SAIB with a pharmaceutically accepted organic solvent such as ethanol or benzyl alcohol decreases the viscosity of the mixture sufficiently to allow for injection. An active pharmaceutical ingredient may be added to the SAIB delivery vehicle to form SAIB solution or suspension formulations. When the formulation is injected subcutaneously, the solvent diffuses from the matrix allowing the SAIB-drug or SAIB-drug-polymer mixtures to set up as an in situ forming depot.
For the purposes of the present invention molecules and agents may be administered to subjects as compositions either therapeutically or preventively. In a therapeutic application, compositions are administered to a patient already suffering from a disease, in an amount sufficient to cure or at least partially arrest the disease and its complications. The composition should provide a quantity of the molecule or agent sufficient to effectively treat the patient.
The therapeutically effective dose level for any particular patient will depend upon a variety of factors including: the disorder being treated and the severity of the disorder; activity of the molecule or agent employed; the composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration; the route of administration; the rate of sequestration of the molecule or agent; the duration of the treatment; drugs used in combination or coincidental with the treatment, together with other related factors well known in medicine.
One skilled in the art would be able, by routine experimentation, to determine an effective, non-toxic amount of agent or compound which would be required to treat applicable diseases and conditions.
It will be understood that methods to determine whether the sensitivity of a tumour cell to a microtubule destabilizing agent has been enhanced may include the examination of any one or more of apoptotic markers, the degree of tumour shrinkage and the change in metabolic activity of tumour cells on exposure to a microtubule destabilizing agent. The presence of markers of apoptosis, including positive TUNEL labelling or the presence of DNA laddering may be used to determine whether cells in a tumour have undergone apoptosis.
The determination of changes in tumour size may be made using techniques readily available to the clinician, such as digital radiography, x-ray techniques including x-ray mammography, magnetic resonance imaging, computed tomography, positron emission tomography, gamma camera imaging, ultrasound imaging, endoscopic imaging and clinical assessment. The presence or level of tumour specific markers in the circulation or in a tissue biopsy may also be assessed using tumour specific antibodies or by in situ hybridization with tumour specific probes. Positron Emission Tomography may be used, for example, to measure the metabolic activity of tumours in situ.
The examples are intended to serve to illustrate this invention and should not be construed as limiting the general nature of the disclosure of the description throughout this specification.
Statistical analysis was done with GraphPad Prism program. Where statistical analysis is presented, results are expressed as means of at least three independent experiments±SEM. A two-tailed Student's t test was used to determine the statistical differences between various experimental and control groups, with P<0.05 considered statistically significant.
Human NSCLC cell lines Calu-6 and H460 (ATCC: Calu6 catalogue number HTB-56 and NCI-H460 catalogue number HTB-177) were maintained as monolayer cultures in Dulbecco's Modified Eagle Medium (DMEM) and RPMI, respectively, supplemented with 10% foetal calf serum (FCS) and 2 mM L-glutamine. The cells were grown at 37° C. in a humidified atmosphere with 5% CO2.
A variety of siRNA or shRNA were used in the examples described herein.
For siRNA transfection, 3×104-5×104 cells/well were plated in 24-well plates and transfected with class II β-tubulin siRNA ON-Target plus SMARTpool reagent at up to 100 nM siRNA (Dharmacon, Chicago, Ill.).
Non-silencing control siRNA, with no sequence homology to any known human gene sequence, was used as a negative control in all experiments (Qiagen, Valencia, Calif.), with the exception of 27-mer βIII siRNA experiments. Cells were transfected with βIII siRNA at a final concentration of 25 nM (Calu-6) and 100 nM (H460).
Transfection was carried out using Lipofectamine 2000 (Invitrogen, Carlsbad, Calif.) according to the manufacturer's instructions. Control experiments were done in parallel by transfecting the cells with a non-silencing control siRNA with no sequence homology to any known human gene sequence (Qiagen, Valencia, Calif.) at equivalent concentrations as the target siRNA. Cells were harvested for experiments after 48-72 h transfection, unless otherwise stated. All experiments involved the use of a Lipofectamine only control (mock-transfected) and a non-silencing control siRNA (negative control).
The effect of siRNA transcription on the expression of β-tubulin isotypes was assessed using reverse transcription-PCR (RT-PCR) analysis of β-tubulin isotypes and by western blotting. For reverse transcription analysis, total RNA was isolated using Trizol reagent (Invitrogen), according to the manufacturer's instructions. RNA samples were DNAse-treated, and reverse transcribed for RT-PCR analysis using methodology and specific primers as described in Kavallaris et al. (1997) J Clin Invest 100, 1282-1293, the entire contents of which are incorporated herein by reference. For Hβ9 (class II), H5β (class IVa) and Hβ2 (class IVb) genes, semi-quantitative PCR-based assays involved setting up two separate PCR tubes for target (β-tubulin) and control (β2-microglobulin) gene sequences. Amplified products were resolved on a 12.5% polyacrylamide gel and visualized by ethidium bromide staining using the Gel Doc 1000 imaging system with data analysis using QuantityOne software version 4.0 (Bio-Rad, Hercules, Calif.). PCR amplifications were performed in triplicate using two independent cDNA preparations.
For western blotting, total cellular proteins from transfected cells (10 μg) were resolved on 12% SDS-PAGE and electrotransferred to nitrocellulose membrane using standard methods. Immunoblotting was performed using monoclonal antibodies to class I β-tubulin (1:5,000; kindly provided by Dr. R. Luduena, University of Texas, San Antonio, Tex.) or alternatively Anti β-I tubulin (AbCam), class II β-tubulin (1:1,000, Covance, Richmond, Calif.), class III β-tubulin (1:1,000, Covance), class IV β-tubulin (1:500, Sigma Chemical Co.-Aldrich, St Louis, Mo.) and total β-tubulin (1:500, Sigma) as described previously (Don et al. (2004) Mol Cancer Ther 3, 1137-1146) with minor modifications. Enhanced chemiluminescence (GE Healthcare, Uppsala, Sweden) was used for detection. The blots were scanned using the typhoon scanner and quantified using ImageQuant software version 5.2 (Molecular Dynamics Inc, Sunnyvale, Calif.). The experiments were repeated in triplicate with proteins isolated from three independent extractions.
As shown in
Cells were transfected for 24 h and approximately 600 cells (for Calu-6) or 150 cells (for H460) were seeded into each well of a 6-well plate and allowed to attach for 4-6 h. The cell lines used in this study were not subject to prior drug selection, and are likely to represent intrinsic drug resistance observed in lung carcinoma.
Cells were then treated with increasing concentrations of various anti-mitotic drugs. Following a 3-day incubation at 37° C., the drug-containing medium was removed and replaced with fresh complete medium. Medium was changed at 3-4 day intervals for 7 to 10 days until visible colonies formed. Control cells were treated identically, with comparable media changes. Surviving colonies were simultaneously fixed and stained with 0.5% crystal violet in methanol, rinsed with water to remove excess stain and air-dried overnight.
The colonies in each well were manually counted, and the results were presented as surviving fraction according to the formula: surviving fraction was calculated as follows: colony number/(number of cells seeded×plating efficiency), where plating efficiency is equivalent to the colony number divided by the number of cells seeded in the drug-free medium. Dose-response curves were plotted for each drug-cell line combination and inhibitory dose (ID50) values were extrapolated from this curve using GraphPad Prism program (GraphPad Software, version 4, San Diego, Calif.). ID50 was defined as the concentration of the drug required to reduce the number of colonies in drug-treated wells to 50% of that of the relevant untreated control wells. The ID50 values were the means of at least four independent experiments.
The results of this assay demonstrated that the knockdown of βII and βIVb tubulin significantly enhanced the cytotoxicity of vinca alkaloids but not of taxanes. As shown in
To rule out the possibility that these effects might be due to some unique interaction of paclitaxel with specific β-tubulin isotypes, an additional microtubule-stabilizing agent, epothilone B was included in the analysis. Treatment with epothilone B led to no significant change in drug sensitivity in H460 cells transfected with βII siRNA (
Although it was possible to specifically distinguish class IVa (H5β) and class IVb (Hβ2) expression at the gene expression (mRNA) level, there are currently no commercially available antibodies which differentiate between class IVa and class IVb protein, and so the differential expression of these polypeptides was not examined in this study. A combination of affinity capture with class IV β-tubulin specific antibodies and high resolution electrophoresis techniques, such as capillary zone electrophoretic techniques is expected to resolve these two proteins and allow their differential quantification.
A reduction of approximately 40-50% H5P gene expression in Calu-6 cells transfected with class IVb siRNA was observed (data not shown) but no significant changes were observed in H460 class IVb transfectants. Since both cell lines showed identical clonogenic results, the drug response obtained was attributed to class IVb suppression rather than class IVa.
Inhibition of either βII or βIVb in NSCLC cells did not significantly potentiate the cytotoxicity of paclitaxel. This result is consistent with the observation that transfection of CHO cells with β-tubulin classes I, II or IVb did not confer resistance to paclitaxel.
The siRNA used in this study resulted in the specific downregulation of expression of each targeted β-tubulin isotype, and no change was observed in the levels of the other β-tubulin isotypes. These results highlight the differing contribution of each individual β-tubulin isotype in determining the chemosensitivity of a cell.
Based on these results, we propose that each β-tubulin isotype is unique in terms of drug interaction, and that the isotype composition of a cell affects its response to antimicrotubule agents. The dramatic effects on vincristine sensitivity observed in IVb transfectants is of interest, since this isotype is constitutively expressed in all tissues.
It is conceivable that differences in β-tubulin composition may influence the interactions of microtubules with other elements of the cytoskeleton network and hence dictate tumour cell behaviour, including the cellular response to chemotherapeutic agents. Another possible explanation for the mechanism of sensitivity is alterations in drug binding affinity; however, since vinca alkaloids (including vincristine, vinblastine and vinorelbine) all bind to β-tubulin isotypes with similar affinity, it is unlikely that alterations in drug binding affinity due to the different isotype composition is the underlying mechanism.
Transfected Calu-6 cells were grown on sterile chamber slides until 60-70% confluent. Cells were then treated with paclitaxel and vincristine for 1 h at 10 nM. Cells were fixed in methanol and processed for staining as previously described (Don et al. (2004) supra) with minor modifications. Fixed cells were stained with α-tubulin (1:400 in 5% FCS/PBS, Sigma) for 30 min followed by Cy2 anti-mouse fluorescent tagged antibody (1:1,000 in 5% FCS/PBS, GE Healthcare). Slides were mounted on a coverslip using DAPI II Counterstain (Vysis Inc., Downers Grove, Ill.). Slides were viewed using a Zeiss Axioplan 2 Immunofluorescence Microscope (Mannheim, Germany) with Image-Pro Plus 4.1 software (Media Cybernetics, L.P., Silver Spring, Md.).
As shown in
The effects of paclitaxel treatment on βII transfectants was also examined. βII siRNA-transfected cells were stained with α-tubulin after treated with 10 nM of paclitaxel for 1 h. Consistent with the clonogenic data, microtubules of βII siRNA-treated cells did not appear significantly different from those treated control cells (
To determine whether changes in drug accumulation were contributing to drug hypersensitivity, the accumulation of [3H]-vincristine was measured in βIVb siRNA-transfected cells and MRP1-positive MCF7-VP16 cells as a positive control.
Intracellular vincristine accumulation was quantitated using radiolabelled drug as previously described (Verrills, N. M., Flemming, C. L., Liu, M., Ivery, M. T., Cobon, G. S., Norris, M. D., Haber, M., and Kavallaris, M. (2003) Chem Biol 10, 597-607) with minor modifications. Briefly, transfected cells were incubated at 37° C. for 2 h in the presence of [3H]-vincristine (5.20 Ci/mmol; final concentration, 50 nmol) (Moravek Biochemicals Inc, California). Cells were washed, hydrolyzed and the [3H] radioactivity counted as previously described. An aliquot of cell lysate was used in parallel to determine the cellular protein concentration using the BCA assay kit (Pierce, Rockford, Ill.). Intracellular vincristine accumulation was determined for duplicate samples and expressed as pmoles of vincristine/mg of protein. MCF7-VP16 cells (Schneider et al. Cancer Res. (1994) 54(1):152-8) were included as a positive control, as these cells had previously been shown to overexpress multidrug-resistance associated protein-1 (MRP1).
MCF7-VP16 cells showed a significant decrease in the accumulation of [3H]-vincristine (data not shown). There was no significant difference in intracellular vincristine levels between IVb knockdown and controls in both Calu-6 and H460 (
The distribution of DNA content in βII or βIVb tubulin siRNA-transfected H460 cells was determined by flow cytometry. After 72 h transfection, transfected cells were exposed to vincristine at 5 nM or 40 nM concentrations for 24 h. On the day of analysis, both adherent and floating cells were harvested, washed with PBS and then stained with a solution containing 0.4% Triton X-100 (Sigma), 50 μg/ml propidium iodide (Sigma), and 2 μg/ml DNase-free RNase (Roche, Indianapolis, Ind.) in the dark for 15 min at 37° C. DNA content was measured by a FACSCalibur flow cytometer (Becton Dickinson, San Diego, Calif.). The flow rate was <200 nuclei per second and 10,000 cells from each sample were analysed. Measurements were performed under the same instrumental settings. The CellQuest program was used to quantitate the distribution of cells in each cell cycle phase: sub-G1 (apoptotic cells), G1, S and G2/M.
As shown in
Class IVb transfectants, on the other hand, failed to undergo G2/M arrest and demonstrated a concomitant increase in the G0-G1 population when treated with equivalent concentrations of vincristine with (
The examples that follow describe studies in which the role of βIII tubulin knockdown on the sensitivity of tumour cells was investigated.
Paclitaxel (Calbiochem, Merck Biosciences, Nottingham, U.K.) was prepared at a stock concentration of 2 mM in dimethyl sulfoxide (DMSO). Vincristine (Sigma-Aldrich, St Louis, Mo.) was prepared at a stock concentration of 2 mM in saline (0.9% wt/vol NaCl); vinorelbine (kindly provided by Dr B Hill, Pierre Fabre, France) was solubilized in water at a stock concentration of 2 mM. Doxorubucin (doxorubicin hydrochloride; Pfizer, Sydney, Australia) was prepared at a stock concentration of 3.45 mM in saline. Etoposide (VP-16; SigmaAldrich) was prepared at a stock concentration of 68 mM in DMSO and cisplatin (Pharmacia, Rydalmere, Australia) was prepared at a stock concentration of 3.3 mM in saline.
The effects of βIII-tubulin siRNA transfection on the gene and protein expression of the isotype, were assessed by RT-PCR and western blotting. For RT-PCR, total RNA was extracted from the transfected cells using the Trizol reagent (Invitrogen), according to the manufacturer's instructions. RNA samples were DNAse-treated, reverse transcribed for RT-PCR analysis using methodology and specific primers as described in detail previously (Kavallaris et al. (1997) J Clin Invest 100, 1282-1293). Beta-2-microglobulin (β2M) was used as an internal mRNA control. Preparation of protein lysates and Western blot analysis were performed as follows. Briefly, total cellular proteins were separated on 4% to 15% SDS-PAGE and electrotransferred to nitrocellulose membranes using standard methods. Immunoblotting was carried out using monoclonal antibodies directed against PI-tubulin (clone SAP 4G5, Abcam Ltd., Cambridgeshire, UK), βII-tubulin (clone 7B9, Chemicon, Temecula, Calif.), βIII-tubulin (clone TUJ1, Chemicon), and 131V-tubulin (clone ONS 1A6, Sigma-Aldrich), GAPDH (Abcam Ltd.). Primary antibodies were detected using horseradish peroxidaselinked secondary antibody (Amersham Pharmacia Biotech, Uppsala, Sweden) and enhanced chemiluminescence (GE Healthcare, Uppsala, Sweden) was used for detection. The blots were scanned using the Typhoon® scanner and quantified using ImageQuant software version 5.2 (Molecular Dynamics Inc, Sunnyvale, Calif.).
Treatment of H460 and Calu-6 cells with βIII-tubulin siRNA resulted in significant knockdown of βIII-tubulin mRNA levels compared to the mock- and control siRNA-transfected cells (
To assess the effects of downregulation on microtubule organization, immunofluorescence staining was performed on transfected Calu-6 cells as follows. Briefly, siRNA transfected cells were plated in glass chamber slides and allowed to reach 70% confluence. Cells were then treated with 10 nM of paclitaxel or vincristine for 1 hour. Drug was then removed by washing cells with PBS and blocked in 10% FCS/PBS. For dual staining, cells were first labelled with an antibody to class III β-tubulin which was exposed to cells incubated at 37° C. for 30 minutes in a humidified chamber. Following washes in 0.1% Tween 20/PBS at room temperature, the slides were incubated with Cy3 anti-mouse fluorescent-tagged antibody (GE Healthcare) for 40 minutes in a humidified chamber in the dark at room temperature. Following incubation, the slides were washed again in 0.1% Tween 20/PBS. This was then followed by staining with α-tubulin and Cy2 anti-mouse fluorescent tagged antibody (GE Healthcare). Slides were mounted on a coverslip using DAPI II Counterstain (Vysis Inc., Downers Grove, Ill.). Immunofluorescence microscopy was performed and images were captured using a Sensicam Charged Coupled Device camera (PCO Imaging, Kelheim, Bavaria, Germany) and the Image-Pro Plus 4.1 software (Media Cybernetics, L.P., Silver Spring, Md.).
The βIII siRNA-transfected cells showed no observable changes to microtubule morphology. Consistent with the Western blot data, a clear decrease in class III β-tubulin immunofluorescence intensity was observed in the βIII-tubulin siRNA treated cells compared to both the control siRNA- and mock-transfected cells when imaged under identical conditions. Control cells (mock- and control siRNA-transfected cells) expressed similar levels of class III β-tubulin as compared to βIII knockdown where βIII-tubulin expression levels were almost undetectable by fluorescence microscopy.
To examine the effect of tubulin-binding agents on βIII-tubulin siRNA treated cells, cells were exposed for 1 h to either 10 nM paclitaxel or vincristine and cellular morphology was examined. As shown in
The data in the preceding example suggested that silencing βIII-tubulin expression may have increased the sensitivity of tumour cells to both paclitaxel and vincristine. To quantitate any change in drug sensitivity, drug treated clonogenic assays were performed. Twenty-four hours after siRNA transfection, cells were harvested and plated into 6-well plates for 6 h, prior to the addition of various drugs as indicated in the figure legends. After 72 h incubation, the drug-containing media was removed and replaced with complete growth media. Medium was changed every 3 days for 7 to 10 days until visible colonies formed. Colonies were simultaneously fixed and stained with 0.5% crystal violet in methanol, and manually counted. Individual stained colonies in each well were counted and the surviving fraction was calculated as follows: colony number/(number of cells seeded×plating efficiency), where plating efficiency equals the colony number divided by the number of cells seeded in drug-free media.
Cellular uptake and retention of tritiated substrate [3H]-paclitaxel and [3H]-vincristine were measured as previously described in Verrills, et al. J Natl Cancer Inst 2006; 98:1363-74. Briefly, cells were transfected in 12-well plates for 48 h. Drug uptake was monitored by adding [3H]-paclitaxel (14.7 Ci/mmol; final concentration, 50 nM; Moravek Biochemicals Inc, Brea, Calif.) or [3H]-vincristine (7.1 Ci/mmol; final concentration of 12.5 nM; GE Healthcare) to the transfected cells for 2 h at 37° C. Cells were washed, hydrolyzed and counted as described in previous examples. The amount of tritiated drug accumulated in the cells was determined for duplicate samples and expressed as pmoles of the drug/mg protein. At least three independent experiments were performed. Relevant positive controls were included, including vincristine resistant neuroblastoma (BE/VCR10) and VP-16 resistant breast cancer cells (MCF7-VP16), which are known to overexpress multidrug resistance 1 (MDR1) and multidrug resistance associated protein 1 (MRP1) respectively.
Consistent with the immunofluorescence observations, βIII-tubulin silencing resulted in a significant increase in sensitivity to paclitaxel and vincristine (
Drug treated clonogenic assays were also performed using DNA damaging agents, VP-16, cisplatin and doxorubicin. Interestingly, βIII-tubulin silencing resulted in increased sensitivity to all three DNA damaging agents tested in H460 cells (
To determine whether βIII-tubulin silencing affects the cell cycle profiles, cell cycle analysis using flow cytometry was performed. Cell cycle analysis was determined by transfecting H460 cells with siRNA for 72 h and harvesting (adherent and suspension) cells 24 h after drug treatment. DNA content was stained for 15 min at 37° C. with a solution containing 0.4% Triton X-100 (Sigma-Aldrich), 50 μg/ml propidium iodide (Sigma-Aldrich), and 2 μg/ml DNAse-free RNAse (Roche, Indianapolis, Ind.). The cells were then analyzed for cell cycle perturbation using a FACSCalibur (Becton-Dickinson, Franklin Lakes, N.J.). The CellQuest program was used to quantitate the distribution of cells in each cell cycle phase: sub-G1 (apoptotic cells), G1, S and G2/M.
The cell cycle profiles of H460 cells were not affected by βIII-tubulin silencing. To determine whether the tubulin-binding agents affected the cell cycle profiles of βIII-tubulin siRNA transfectants, cells were treated with either paclitaxel or vincristine. Following 24 h incubation with 5 nM paclitaxel, the βIII-tubulin silenced cells had a higher sub-G1 (apoptotic cells) content compared to the control siRNA treated cells (p<0.05) (
Annexin V-FITC staining followed by flow cytometry analysis was performed to address whether the increase in the sub-G1 population following exposure to tubulin-binding agents in the βIII-tubulin siRNA treated cells was related to an increase in apoptosis induction. Apoptosis induction was determined by transfecting H460 cells with siRNA for 72 h and harvesting (adherent and suspension) cells 48 h after drug treatment, as previously described in Pasquier et al., Mol Cancer Ther 2004; 3:1301-10. Briefly, 1×105 cells were incubated with Annexin V-FITC and propidium iodide for 15 min in the dark (Becton-Dickinson), immediately followed by flow cytometry using a FACSCalibur (Becton-Dickinson). Cytogram analysis was performed using Cell Quest software.
Incubation of H460 cells with paclitaxel for 48 h, induced apoptosis from 1 nM in βIII-tubulin siRNA treated cells and from 5 nM in control siRNA treated cells (
Similarly, in βIII-tubulin siRNA treated cells exposed to cisplatin for 48 h, there was a significant increase in the number of apoptotic cells in the 0.4 or 1 μM cisplatin treated cells compared to controls (
Class III β-tubulin knockdown of H460 and Calu-6 cells was carried out as described in previous examples, and drug treated clonogenic assays were performed using the tubulin stabilizing agent epothilone B. The results of these experiments are illustrated in
Knockdown of class III β-tubulin in both cell lines increased the sensitivity of these cells to epothilone B.
In order to investigate whether knockdown of tubulin expression by other siRNA was able to induce equivalent levels of class III β-tubulin silencing changes to responses to tubulin-binding agents, class III β-tubulin expression in H460 cells was knocked down using 27mer-siRNA reagents designated sequence 8 (SEQ ID NOS: 11 and 12) or sequence 11 (SEQ ID NOS: 13 and 14). These siRNAs recognise different regions of the class III β-tubulin mRNA sequence.
The results of these experiments are illustrated in
A short hairpin RNA construct (SEQ ID NO:15) was used in an attempt to stably knock down the expression of class III β-tubulin in H460 cells.
Three clones, designated clone 4, clone 59 and clone 60, were identified in which stable class III β-tubulin knock down was observed. As illustrated in
The tubulin-binding agent 2-methoxyestradiol (2ME2) binds to β-tubulin near the colchicine-binding site, inhibits microtubule polymerization, and induces mitotic arrest. CCRF-CEM Leukaemia cells that display increasing resistance to 2ME2 were selected, and four of the highly 2ME2 resistant leukaemia sublines were chosen for detailed analysis. The 2ME2 cells selected in 3.6-28.8 μM 2ME2 were found to be 11- to 107-fold more resistant to 2ME2 than cells of the parent line.
The 2ME2-resistant cells were hypersensitive to epothilone B and only the lowest resistant subline, CEM/2ME2-3.6R, was cross-resistance to colchicine and vincristine. The 2ME2-resistant cells did not exhibit G2/M cell cycle arrest as demonstrated in the parental cells and demonstrated higher levels of polymerized tubulin than the parent cells. Four class I β-tubulin mutations, S25N, D197N, A248T and L350N, were detected in the 2ME2-resistant cells. The S25N mutation is within the paclitaxel-binding site, whereas A248T and L350N are within colchicine-binding site on β-tubulin, yet the resistant cells were not cross resistant to paclitaxel or colchicine.
This suggests that the mutations may have induced conformational change to the binding sites. In addition, 2ME2 resistant sublines were demonstrated to have upregulated their expression of class II β-tubulin (
To determine whether the levels of class II β-tubulin could predict sensitivity and be contributing to resistance, siRNA mediated knockdown of class II β-tubulin in drug sensitive H460 (NSCLC) cells was performed. Knockdown of class II β-tubulin significantly increased sensitivity of H460 cells to 2ME2 and colchicine.
A subject diagnosed with or suspected of having a NSCLC tumour undergoes a tumour biopsy. Biopsy tissue is probed with specific commercially available monoclonal antibodies or nucleic acids by RT-PCR as described herein or as previously described (Kavallaris et al. (1997) J Clin Invest 100:1282) to determine whether the tumour expresses III, βIII or IVb β-tubulin mRNA or protein. If the tumour expresses III, βIII or IVb β-tubulin the subject is treated according to the method of the invention.
One or more siRNA constructs directed to class II, class III or class IVb β-tubulin, either encapsulated in a cationic liposome as described previously (Nogawa et al. (2005) supra), conjugated with a nanoparticle or suspended in saline, and optionally synthesised using nuclease-resistant chemical modifications, is injected directly into the tumour at a dose of 2×109 constructs/injection, and the injections repeated every second day 3 or 5 times. Alternatively the construct conjugated to a nanoparticle is administered systemically, where the ability of the nanoparticle to localise the construct is the lungs is utilised.
In order to monitor the inhibition of class II, class III or class IVb β-tubulin expression in the tumour, a further biopsy may be taken at approximately 72 hours and the tissue examined for gene expression as described above.
After substantial inhibition of expression of class II, class III or class IVb β-tubulin in the tumour, the subject is administered the standard dose of tubule-binding agent for the tumour type in question using the standard rate of administration.
*ID50=concentration of drug that killed 50% of the cells (ID50);Student's t test (two-sided).
P value was determined by comparing the ID50 of the mock-transfected cells with the ID50 of the siRNA-transfected cells.
Drug treated clonogenic assay on H460 cells following βIII silencing using 27-mer βIII siRNA.
Drug treated clonogenic assays of H460 βIII-shRNA stable cells 1 Drug concentration required to inhibit 50% of colony formation.2 Relative sensitivity (RS) is the fold sensitivity of the siRNA treated cells compared to ctrl clone 1. RS was determined by dividing the ID50 of ctrl clone 1 by the ID50 of the βIII stable clones or ctrl clone 2. NS—not significant
Materials and Methods
Cisplatin was obtained from Pfizer, West Ryde, New South Wales, Australia.
Reagents for tissue culture were purchased as follows: Dulbecco's Modified Eagle Medium (DMEM), Roswell Park Memorial Institute (RPMI), trypsin, L glutamine, foetal calf serum (FCS), trypan blue and G418 from Invitrogen Life Technologies (Carlsbad, Calif.). Phosphate buffered saline (PBS) and puromycin were purchased from Sigma. 24-well, 12-well plates for transfection were purchased from Corning Life Sciences (Acton, Mass.) and 6-well plates for clonogenic assay were obtained from Techno Plastic Products (Trasadingen, Switzerland); tissue culture flasks were purchased from Greiner Bio-One (Frickenhausen, Germany).
Reagents for siRNA transfection were obtained as follows: Lipofectamine 2000 and Opti-MEM I reduced serum medium were obtained from Invitrogen. The βII-tubulin ON Target plus SMARTpool siRNA reagents, βIII-tubulin SMARTpool siRNA reagent and βIVb tubulin siRNA were obtained from Dharmacon Research Inc. (Chicago, Ill.). Non-silencing control siRNA were from Qiagen (Valencia, Calif.). TAMRA-labelled βIII tubulin SMARTpool siRNA reagent and rhodamine-labelled non-silencing control siRNA were from Dharmacon and Qiagen, respectively. The 27-mer 13111-tubulin siRNAs were purchased from Integrated DNA Technologies (Coralville, Iowa). The pRS vector containing the βIII-tubulin shRNA expression cassettes (pRS-shβIII), along with two negative control shRNA plasmids including the original pRS vector (empty vector) and the pRS vector containing a non-effective shRNA cassette against GFP (pRS-shctrl), were purchased from OriGene Technologies, Inc. (Rockville, Md.).
Reagents for RNA isolation were purchased as follows: Trizol reagent from Invitrogen, buffer-saturated phenol from Progen (Toowong, Queensland, Australia), nuclease-free water from Promega (Madison, Wis.); chloroform from Lab-Scan (Dublin, UK); isopropanol and ethanol from Sigma, DNAse treatment kit was obtained from Promega.
Reagents for cDNA synthesis and PCR reactions were obtained from various suppliers: moloney murine leukaemia virus (MMLV) reverse transcriptase, 5× first strand buffer and 0.1M dithiothreitol (DTT) from Invitrogen; RNAsin ribonucleotide inhibitor from Promega; deoxynucleoside triphosphates (dNTPs) and random hexanucleotide primers from GE Healthcare Biosciences (Piscataway, N.J.), and AmpliTaq Gold DNA polymerase, Amplitaq Gold PCR buffer II (without MgCl2), MgCl2 solution from PerkinElmer (Waltham, Mass.). Primers were obtained from Sigma Genosys (Sydney, New South Wales, Australia).
Reagents for protein isolation, western blot analyses and tubulin polymerisation assay were purchased as follows: 4-15% Tris-HCl gradient polyacrylamide gels and dual colour precision plus protein standards from Bio-Rad (Hercules, Calif.). Ponceau S sodium salt, ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), protease inhibitor cocktail, phenylmethylsulfonyl fluoride (PMSF), Tween-20 and Trizma base (tris) from Sigma. Sodium dodecyl sulphate (SDS) from ICN Biomedicals (Aurora, Ohio); Noniodet P-40 from Fluka (Buchs, Switzerland); glycerol from Fronine (Riverstone, New South Wales, Australia); Hybond C extra nitrocellulose membranes and ECL plus Western Blotting Detection System from GE Healthcare; bicinchoninic acid (BCA) protein assay kit from Pierce (Rockford, Ill.); 3MM blotting paper from Whatman (Maidstone, UK).
Primary antibodies were obtained from various suppliers: anti-α-tubulin (clone DM1A), anti total β-tubulin (cone TUB 2.1), anti-class II β-tubulin (βII-tubulin; clone JDR.3B8) and anti-class IV β-tubulin (βIV-tubulin; clone ONS.1A6) from Sigma; anti-class II β tubulin (βII-tubulin; clone 7B9) and anti-class III β-tubulin tubulin; clone TUJ1) from Covance (Richmond, Calif.); anti class I β-tubulin tubulin; clone SAP 4G5) from Abcam (Cambridge, Mass.). Anti-stathmin (clone 53) and anti MAP4 (clone 18) antibodies from BD (Franklin Lakes, N.J.). Anti glyceraldehyde-3-phosphate dehydrogenase (GAPDH; clone 6C5) antibody was purchased from AbCam. Secondary antibody, mouse IgG, HRP-linked whole antibody from sheep was obtained from GE Healthcare.
Reagents for electrophoresis were purchased as follows: boric acid and bromophenol blue from Sigma; glycine from ICN Biomedicals Inc.; Ultrapure Acrylagel and Ultra bis-acrylagel from National Diagnostics (Atlanta, Ga.); ammonium persulfate, ethidium bromide and N,N,N′,N′-tetramethylethylenediamine (TEMED) from Sigma, 1 kb DNA ladder from Invitrogen.
35 mm sterile tissue culture dishes were obtained from BD, SeaPlaque® agarose from Lonza Australia Pty Ltd (Mt Waverley, Victoria, Australia).
Reagents for transformation of plasmid DNA into competent cells were purchased as follows: LB broth, LB agar, ampicillin sodium salt and SOC medium from Sigma; Kanamycin from Invitrogen, competent E. coli cells (JM109) from Promega and 17×100 mm, 14 ml polypropylene tube from BD; Reagents for preparation of plasmid DNA were obtained as follows: QIAfilter™ Plasmid Maxi kit for DNA purification (Qiagen). TE buffer used to resuspend purified DNA pellet was purchased from Promega.
Monoclonal rabbit antibody against βIII-tubulin was obtained from Covance. Monoclonal mouse antibody against Ki67 was obtained from Dako (Sydney, New South Wales, Australia). Biotinylated anti-rabbit IgG, biotinlyated anti-mouse IgG, M.O.M. (Mouse-on-Mouse) blocking reagent, normal goat serum and Vectastain Elite ABC (avidin-biotin-peroxidase complex) kit were purchased from Abacus ALS (Brisbane, Queensland, Australia). Haematoxylin and diaminobenzidine (DAB) liquid from Dako.
For in vivo tumorigenicity studies female 5-6 week nude mice of BALB/c background (bred by the Biological Resources Centre (BRC), University of New South Wales, Sydney, NSW, Australia) were used. Twenty animals, in groups of four, were placed in four Ventiracks cages for each experiment. The animals were allowed to acclimatise for a week at the animal facility prior to experiments.
O.C.T. freezing compound, tissue-tek cryomold standard and 4% paraformaldehyde were purchased from ProSciTech (Thuringowa, Queensland, Australia). RNA later stabilization reagent was from Qiagen.
Maintenance of Cell Lines
Human NSCLC cell lines NCI-H460 (H460) and Calu-6 (both from ATCC) were maintained in RPMI and DMEM, respectively, supplemented with 10% FCS and 2 mM L-glutamine. Both cell lines are large cell carcinoma and exhibit anchorage-independent growth in soft agar and high tumorigenicity in nude mice. H460 cells express wild-type p53, whereas Calu-6 cells harbor mutant p53. For microtubule dynamic measurements, H460 cells stably expressing GFP tubulin were generated and were cultured in RPMI growth media. The cells were grown at 37° C. in a humidified atmosphere with 5% CO2. All cultures were routinely screened for Mycoplasma spp. and found to be free of contamination.
siRNA Transfection
Table 4 shows the target sequence of each siRNA. SMARTpool siRNA reagent is a pool of four siRNA duplexes targeting separate regions of a single gene to suppress expression. Gene knockdown was also performed on these individual siRNA duplexes whenever possible to confirm the gene silencing effect. Rhodamine-conjugated GFP siRNA was used as a control siRNA initially to optimise transfection conditions. The non-silencing control siRNA, that has no sequence homology to any known human genome, was subsequently used as a negative control for non-specific silencing effects in all experiments. All siRNA sequences underwent a basic local alignment search tool (BLAST) search to avoid off-target gene silencing. Cells were transfected immediately after passage using Lipofectamine 2000 reagent as per the manufacturer's instructions. This method has been shown to improve the siRNA silencing effect for adherent cell lines (Amarzguioui, 2004). The optimal amount of siRNA used for transfection was determined by screening cell lines against a range of final concentrations of 25-200 nM of the target siRNAs.
Western blot analysis was performed 72 h post-siRNA transfection to determine the level of knockdown in target protein expression. The lowest siRNA concentration that gave effective silencing of the targeted protein and caused minimal stress to the cells was used in all subsequent experiments. Control experiments were done in parallel by transfecting the cells with a non silencing control siRNA at equivalent concentrations as the target siRNA. All experiments involved the use of a Lipofectamine only control (mock-transfected) and a non-silencing control siRNA (negative control). For βIII tubulin siRNA experiments, SMARTpool siRNAs and its associated 21-mer non-silencing control siRNA were used throughout this thesis unless otherwise stated. In microtubule dynamic experiments, the tubulin siRNAs were labeled with TAMRA and the negative control siRNA was labeled with rhodamine to determine transfection efficiency or to ensure siRNA uptake into the cells prior image acquisition.
It has recently been shown that 27-mer siRNAs can be up to 100-fold more potent than traditional 21-mer siRNAs. To validate results obtained using conventional 21-mer siRNAs (ie. SMARTpool siRNAs); four 27-mer siRNA duplexes specifically targeting βIII-tubulin were tested. For consistency, a corresponding control siRNA at a duplex length of 27 base pair (control-1) was used in the experiments involving 27 mer siRNAs at equivalent concentrations as the target siRNA. The location of this control sequence overlapped with the 21-mer non silencing control sequence with four sequences randomly inserted at the end of the 21-mer sequence. This new 27-mer control sequence was blast searched to ensure it has no sequence homology to any known human or mouse genome.
Generation of βIII-Tubulin shRNA Stable Line
The sequence of the βIII-tubulin specific 29-mer shRNA is shown in Table 5. H460 cells were transfected with the pRS vector containing the βIII-tubulin shRNA constructs (pRS-shβIII) or non-effective GFP shRNA (pRS-shctrl) as a negative control. Cells were also transfected with the pRS vector plasmid only as an empty vector control. The pRS vector expresses the puromycin resistance gene for selection of stable transfected cell lines. The minimum concentration of puromycin that would kill untransfected H460 cells was determined by the following method. Cells (2.0×105 per well) were seeded in 6-well plates and exposed to increasing concentrations of puromycin (from 0.5 to 2.5 μg/ml), in addition to a drug-free control. The cells were incubated for 10-14 days, with the selective medium being replaced every 3 days and examined for evidence of cell death. The lowest concentration of puromycin that gave significant cell death within 3-5 days and killed all the cells within the 14 days was determined to be optimal for selecting stable transfectants. From this procedure, the optimal puromycin concentration used for the selection of stable transfectants for H460 cells was 1.5 μg/ml.
The pRS shRNA expression vector contains both 5′ and 3′murine retroviral long terminal repeats (LTRs) that flank the puromycin selection marker and the U6 small nuclear RNA gene promoter. The U6 promoter is followed immediately downstream by a 29 bp target gene specific sequence (βIII-tubulin), cloned into the BamH I/Hind III cloning sites of the pRS vector, a 7 bp loop, the 29 bp sequence in reverse complement, followed by a stretch of six thymidine nucleotides (TTTTTT) termination sequence. Upon introduction of the plasmid into mammalian cells, the U6 promoter expresses the insert sequence, resulting in the formation of shRNA, which is then processed by cellular machinery to inhibit the expression of the target gene.
To prepare plasmid DNA for use in the transfection experiments, the four pRS-shrβIII constructs, along with the original pRS vector and the pRS-shctrl (all were supplied in the form of pure plasmid DNA) were first amplified using the heat shock method. Aliquots (100 μl) of JM109 competent cells were thawed on ice and transferred to prechilled 17×100 mm polypropylene tubes. Each expression plasmid was then added gently to an aliquot of competent cells before incubation on ice for 30 min. The mixture of DNA and cells were heat shocked at 42° C. for exactly 45 sec without shaking, and placed immediately on ice for 2 min. Recovery medium (such as SOC) was added to the cells and the sample was incubated at 37° C. for 1 h with agitation using Certomat® lab shaker (D & A laboratory services; Baulkham Hills, New South Wales, Australia). The transformation mixture was then streaked onto ampicillin-containing agar plates (LB amp) and incubated at 37° C. overnight. The following day, a single bacterial colony was carefully picked and inoculated into a sterile 50 ml falcon tube containing 5 ml of LB broth with 100 μg/ml ampicillin. Cultures were allowed to grow for 8 h with vigorous agitation prior to large scale DNA isolations.
Plasmid DNA isolation for large cultures was performed using the QIAfilter™ Plasmid Maxi kit. The initial 5 ml culture described in the previous section was diluted 1:500 into a large 150 ml culture containing 100 μg/ml of ampicillin and similarly grown at 37° C. with vigorous agitation for approximately 16 h. The remaining steps were performed according to the manufacturer's instructions. Following such preparation, the pRS, pRS-shctrl and each of the four pRS-shrβIII constructs were precipitated with isopropanol, washed in 70% ethanol, and resuspended in a small volume of TE buffer (10 mM Tris Cl, pH 8; 1 mM EDTA), prior to transfection. The concentration of plasmid DNA was determined using spectrophotometric absorbance measured at 260 nm (UltraSpec III, Pharmacia LKB, Uppsala, Sweden), with 1 A260 OD unit equivalent to 50 μg/ml DNA. The final product was stored at 4° C.
Selection of Stable Transfectants
Each of the four pRS-shrβIII constructs and control plasmid DNA were transfected separately into H460 cells. Briefly, 2×105 cells were plated in 6-well plates a day before transfection. Following an overnight culture, cells in each well were transfected with 4 μl Lipofectamine 2000 and 2 or 4 μg of the indicated constructs in 100 μl Opti MEM I. Cells were harvested after 72 h transfection for protein analysis to determine the efficacy of the constructs at being able to silence βIII-tubulin protein expression. Alternatively, transfected cells were passaged after 24 h transfection and cultured in the growth medium containing puromycin (1.5 μg/ml) for 1-2 weeks to enrich the population of cells that had retained the expression plasmid. Stable transfectants were selected two weeks later by transferring a well-isolated single clump of cells into a well of a 24-well plate and expanded for characterization studies. Approximately 6-60 clonal populations were isolated for each construct. After clonal expansion, cells from each individual colony were examined for βIII-tubulin expression by western blotting analysis and immunofluorescence staining. Clones expressing the desirable levels of βIII-tubulin were cryopreserved for additional experiments. Selected clones were grown continuously in the presence of puromycin, however, prior to experimental assays; the cells were grown in the puromycin-free medium for 7 days.
RT-PCR and Western Blotting
Total RNA was isolated using Trizol reagent, according to the manufacturer's instructions. RNA was resuspended in sterile nuclease-free water and stored at −80° C. The concentration of RNA was determined using spectrophotometric absorbance measured at 260 nm (UltraSpec III), with 1 A260 OD unit equivalent to 40 μg/ml RNA. The A260/A280 ratio was used to assess RNA purity, where a ratio of 1.8-2.1 is desirable. In addition, background correction was also performed by performing readings at 320 nm. Since the tubulin family of genes consists of several pseudogenes, RNA samples were DNAse-treated to remove contaminating genomic DNA using the DNAse treatment kit as per the manufacturer's instructions. RNA was precipitated with 100% ethanol at −20° C. overnight and pelleted by high-speed centrifugation (14,300×g for 15 min at 4° C.). RNA pellet was rinsed in chilled 70% ethanol, recentrifuged, and air-dried for 10 min. RNA was resuspended in sterile nuclease-free water and concentration was determined as described above. cDNA was prepared from 1 μg total RNA in a 10 μl reaction mixture containing 5× first strand buffer (50 mM Tris-HCl pH 8.3, 75 mM KCl, 3 mM MgCl2), 500 μM of each dNTP, 10 mM DTT, 10 mM RNasin ribonucleotide inhibitor, 100 ng random hexanucleotide primers, and 100 units MMLV reverse transcriptase. To verify complete removal of genomic DNA, each RNA sample was subjected to a mock reverse transcription by omitting the reverse transcriptase from the reaction mix. In addition, each sample was also subjected to a PCR amplification using the GAPDH gene, which spans the intronic sequence and detects contaminating DNA. The reaction mix was incubated at 37° C. for 1 h, diluted 1:5 with sterile water and stored at −20° C. until required.
Reverse transcriptase (RT)-PCR was performed by using 1 μl of cDNA and specific primers for Hβ4, H5β, Hβ2 and β2-microglobulin (β2M) as the control gene. The gene-specific oligonucleotide primers are listed in Table 3 along with the size of the PCR products. The 25 μl PCR reaction mix contained the following: lx PCR Gold buffer, 2.5 mM MgCl2, 250 μM of each dNTP, forward and reverse primers (each at a final concentration of 1.43 μg/μl), 0.025 U/μl Gold Taq Polymerase and sterile nuclease-free water. For the amplification of H5β, 1.5 mM MgCl2 and 1.43 μg/μl oligonucleotide primers were used instead. In contrast, 1.5 mM MgCl2 and 1.67 μg/μl oligonucleotide primers were used for the amplification of Hβ2.
PCR reaction was set up by preparing a master mix that composed of all listed reagents, except the cDNA template. The reaction mix was dispensed in aliquots (24 μl) into individual PCR tubes to which the cDNA was added separately. The reaction mix was subjected to PCR amplification for 30 cycles (for amplification of H134) or 35 cycles (for amplification of H5β and H2β genes) in a programmable thermal cycler (Gene AMP® PCR System 9700 thermocycler), supplied by PerkinElmer. Following an initial period of denaturation for 10 min at 94° C., each PCR cycle consisted of a 30 sec denaturation at 94° C., 30 sec primer annealing at 55° C. and 90 sec extension at 72° C. A final extension at 72° C. for 10 min was performed before completion of the reaction at 4° C. for storage. Amplified products were resolved on a 12.5% polyacrylamide gel together with a 1 kb DNA ladder.
The gel was visualised by ethidium bromide staining using the Gel Doc 1000 imaging system with data analysis using QuantityOne software version 4.0 (Bio-Rad).
Hβ4 gene expression was determined by co-amplifying the target gene with a constitutively expressed control gene, β2-microglobulin β2M). Primers for both the target and control genes were included in the reaction mix resulting in concurrent amplification of the target and control gene sequences in the same PCR tube. For H5β and Hβ2 genes, semi-quantitative PCR based assay involved setting up two separate PCR tubes for target and control gene sequences. In addition, PCR amplification was initially performed using GAPDH gene primers, which span the intronic sequence and detect contaminating DNA. For each PCR reaction, a water control was always included, in which amplification was performed in the absence of cDNA template, to exclude the possibility of contamination of reagents and primers used in the reaction. Where available, a positive control, such as plasmid DNA of the target gene or cell line known to express the gene of interest, was also included in the analysis. The levels of target gene expression were determined by dividing the ratio of the band intensity of the target gene by that of the internal control β2M gene and the results were expressed as a normalized ratio to the mock-transfected cells. PCR amplifications were performed in triplicate using two independent cDNA preparations.
For Western blotting, whole cellular protein was extracted by resuspending cell pellets at a density of 5×106 cells/ml in ice-cold RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate; 0.1% SDS; 50 mM Tris, pH 7.5), containing 1% protease inhibitor and incubated for 30 min on ice. Frozen mouse tumour tissues were homogenised using OMNI TH tissue homogeniser (Lomb Scientific Pty Ltd, Tarren Point, New South Wales, Australia) on ice in 0.5 ml of ice cold RIPA buffer as described for whole cell lysate. The DNA in the protein lysates was sheared by pulse sonication for 10 sec using a Microson™ Ultrasonic homogeniser (Model XL2000; Misonix Inc., Farmingale, N.Y.) on ice and then centrifuged at 14,300×g for 20 min at 4° C. to remove cellular debris. The protein in the resulting supernatant was then transferred to a new microfuge tube and stored at −80° C. Protein concentration was quantified by using the BCA protein assay kit, according to the manufacturer's instructions. Absorbance values were determined using a Benchmark Microplate reader equipped with Microplate Manager III (version 1.57) program (Bio-Rad).
Prior to electrophoresis, protein preparations were mixed with an equal volume of 2× sample buffer (0.125 M Tris, pH 6.8, 4% SDS, 20% glycerol, 0.1% bromophenol blue, 10% 2 mercaptoethanol) and incubated at 100° C. for 5 min. A total of 10 μg of protein were separated by electrophoresis using a 12% SDS-PAGE gel or pre-cast 4 15% Tris-HCl gradient gel for 2 h at 100 V in a Hoefer Mighty Small™ II system (Model SE250; Hoefer Scientific Instruments, San Francisco, Calif.) or pre-cast gel running apparatus (Model Mini-PROTEAN II Cell, Bio-Rad) respectively using a running buffer of 0.025 M Tris, 0.192 M glycine, 0.1% SDS.
The separated proteins were electrotransferred onto a nitrocellulose membrane using the Hoefer Mini Transphor unit (Model TE22; Hoefer Scientific Instruments) containing transfer buffer (0.025 M Tris/Glycine, pH 8.2; 20% methanol) overnight at 70 mA at 4° C. or 200 mA for 2 h at 4° C. Following transfer, the nitrocellulose membrane was stained with 0.5% (w/v) Ponceaus S in 1% acetic acid to confirm efficient transfer and equal protein loading. The membrane was then blocked in Tris Buffered Saline (TBS; 20 mM Tris, pH 8; 150 mM NaCl) containing 10% (w/v) skim milk (SM) for 1 h, and then probed with different primary antibodies for 1 h, as set out in Table 4.
The blot was washed three times for 10 min in TBS containing 0.05% Tween-20 (TBST) and probed with anti-mouse horseradish peroxidase-conjugated IgG secondary antibody for 1 h. All incubations were performed at room temperature unless otherwise specified. After three washes for 10 min in TBST, the blot was developed by a 5 min incubation in enhanced chemiluminescence detection kit (ECL plus Western Blotting Detection Reagents). The house-keeping protein, GAPDH was used as a loading control. Blots were scanned using the typhoon scanner (Model 9410, GE Healthcare) and quantified using ImageQuant software version 5.2 (Molecular Dynamics Inc., Sunnyvale, Calif.). The protein expression was determined by dividing the densitometric value of the target protein by that of the control protein (GAPDH). Whenever applicable, the normalized ratios were expressed against the mock transfected cells in which the protein level was arbitrarily set as 1.0. The experiments were repeated in triplicate with protein isolated from three independent extractions. When necessary the membrane was stripped and reprobed with appropriate antibodies.
Soft Agar Colony Assay (Anchorage-Independent Cell Growth Assay)
H460 cells were transfected with βIII-tubulin siRNA or control siRNA for 24 h as described above. Cells from each sample (1,000 cells per dish) were resuspended in 0.33% agar (37° C.) in RPMI containing 20% FCS, and plated onto 0.5% solidified agar/20% FCS/RPMI (37° C., bottom supportive layer) in 35 mm dishes. Triplicate plates were set up for each sample. The plates were placed at 4° C. for 10 min to solidify the top agar and then incubated at 37° C. in 5% CO2 for 12 days. After 12 days of culture, the colonies were counted and photographed using the Zeiss Axiovert 5100 inverted microscope, 5× objective and SPOT digital camera (Thornwood, N.Y.).
Tumorigenicity in Nude Mouse Xenograft Model
Female BALB/c athymic nude mice (5-6 week old) were maintained under pathogen-free conditions. Tumor cells (1×106 cells) were resuspended in 100 μl of sterile PBS and then injected subcutaneously into the right flank of mice. Samples of the injected cells were collected for determination of the knockdown level of βIII-tubulin at the time of injection by western blotting. Mice were randomized into 4 groups (five mice per group) and injected with either pRS-shctrl (clone 1 and 2) or pRS shrβIII (clone 4 and 59). The animals were monitored twice a week from the second week post-injection for tumor formation and health status. Tumor diameter was measured twice per week using a digital caliper throughout the experiment, and tumor volume in mm3 was calculated using the formula: Volume=(length×width×depth)/2. Mice were sacrificed when the tumor size reached 1 cm3 or at the end of the 120-day experimental period. Tumors were harvested, weighed and photographed. Tumors were divided into 4 parts; one part was fixed in 4% paraformaldehyde for histologic and immunohistochemistry analysis, the second part was stored in RNA later stabilization reagent according to the manufacture's instructions for RNA extraction, the third part was snap frozen in liquid nitrogen for protein isolation and the remainder embedded in O.C.T freezing compound and snap-frozen in methylbutane for preparation of frozen tissue sections. Two independent animal experiments were performed. Animal experiments were done in compliance with the Animal Care and Ethics Committee of the University of New South Wales (ACEC 07/90B). The time to tumor formation (event=1 cm3 tumor size) of each group of mice was expressed using Kaplan-Meier analysis.
Immunohistochemistry Staining
Tissues were paraffin-embedded, and stained with haematoxylin and eosin (H&E). For immunohistochemical analysis, slides were incubated at 60° C. for 30 min to ensure the sections remain attached to the slides and then deparaffinised with a total of three incubations in xylene (each for 5 min). Sections were then rehydrated through a graded alcohol series beginning with 100%, 95% and 70%, and finally with water. Slides were subjected to antigen retrieval by microwave oven treatment for 15 min in 10 mM sodium citrate buffer (pH 6.0), cooled to room temperature for 1 h, and washed extensively with water. Endogenous peroxidase activity was blocked with 1% hydrogen peroxide/methanol for 10 min. Sections were pretreated with 10% normal goat serum for 1 h in a humid chamber to block non specific binding sites and then overnight at 4° C. with βIII-tubulin antibody (1:1,000 dilution). For detection of Ki67 in tumor tissue, sections were pretreated with the vector M.O.M blocking reagent (allows for the detection of mouse antibodies in mouse tissue) for 1 h in a humid chamber to block non-specific binding sites and then for 2 h at room temperature with a monoclonal mouse Ki67 anti-human antibody (1:200 dilution). Two negative control reactions were also included to ensure that the staining was specific. One section was incubated with a rabbit IgG control antibody (for βIII-tubulin antibody) or mouse IgG control antibody (for Ki67 antibody) as well as one section being incubated with the antibody diluent alone. The IgG control antibodies were used at the same concentrations as the primary antibody. Slides were washed three times (5 min each) with PBST (PBS with 0.05% TBST) followed by one wash with PBS (5 min). The slides were then incubated with biotinylated anti rabbit secondary antibody (for βIII-tubulin) or biotinylated anti mouse secondary antibody (for Ki67) at 1:250 dilution for 10 min at room temperature. The sections were rinsed with PBST and incubated with avidin-biotin-peroxidase complex (ABC) for 5 min at room temperature. After washing two times with PBS (each for 2 min), immunoreactivity was visualised by incubating with DAB substrate at room temperature until the desired intensity developed. The slides were rinsed with tap water and counterstained with Mayer's haematoxylin (nuclear counterstain) for 1 min. Slides were then dehydrated and mounted. Staining was examined and photographed with an Olympus microscope and camera.
A two-tailed Student's t test was used for statistical analysis of comparative data using the GraphPad Prism program. Results were expressed as means of at least three independent experiments±SEM, with P<0.05 was considered statistically significant.
Functional Characterization of the Role of βIII-Tubulin in Tumorigenesis
H460 cells stably expressing βIII tubulin shRNA were generated and characterized in vitro and in vivo. H460 cells are known to display substrate-independent growth in soft agar and tumourigenicity in nude mice and hence serve as an ideal model to investigate the role of βIII-tubulin in tumourigenesis. Colony formation of H460 cells stably expressing βIII tubulin shRNA was dramatically reduced in soft agar and tumourigenicity was decreased in xenografted nude mice when compared to respective controls (
The ability of transformed cells to grow under anchorage-independent conditions is a property which is highly correlated with the malignant phenotype in vivo. A colony formation assay was performed on soft agar to determine whether siRNA-mediated knockdown of βIII tubulin expression alone would influence the tumourigenic potential of H460 cells in vitro. As shown in
This result indicates that siRNA-mediated specific knockdown of βIII-tubulin significantly inhibits anchorage-independent growth in vitro. This growth inhibition is directly attributable to the transient knockdown of βIII-tubulin expression.
To investigate the pharmacokinetics of both 21-mer and 27-mer βIII tubulin siRNAs in silencing βIII-tubulin protein expression, H460 cells were transfected with the respective siRNA and the duration of gene silencing was determined. In a subculturing time course experiment, the maximum level of siRNA-mediated knockdown of βIII tubulin protein was around 72-96 h and βIII-tubulin expression gradually increased by day 4. The only exception was sequence 11 of 27 mer siRNA where knockdown persisted till day 6 but the protein level recovered to original levels after this time.
Transfection of Class III β-Tubulin shRNA Construct into NSCLC H460 Cells and Selection of Stable Clones
Results described above demonstrated that knockdown of class III β-tubulin using either 21 mer or 27-mer class III β-tubulin siRNAs was relatively short-lived (4-6 days). In some circumstances, stable knockdown of βIII-tubulin may be advantageous.
Four class III β-tubulin shRNA constructs (pRS-shβIII) were tested initially to knockdown the expression of class III β-tubulin in H460 cells in a transient manner. Of the four constructs tested, construct 3 gave rise to over 40% knockdown of class III β-tubulin expression. Cells transfected with either the empty vector (pRS) or the control non-functional shRNA (pRS-shctrl) did not show any significant level of class III β-tubulin knockdown. The transfected cells were also subjected to short term puromycin selection (6 days) to enrich for a population of cells which expressed the pRS-shrβIII. The surviving cell populations were pooled and analyzed for class III β-tubulin expression. The transfection efficiency was increased by approximately 10% after 6 days of puromycin selection in cells transfected with construct 3. However, other pRS-shrβIII constructs did not show any significant level of βIII tubulin knockdown even after short-term puromycin selection when compared to controls. In fact, immunofluorescence staining of the surviving cell populations showed a mixed population of cells with high and low expression of class III β-tubulin (data not shown).
Methylation of promoter sequences can inactivate expression of plasmid constructs so in order to eliminate clones that were puromycin resistant but did not exhibit βIII tubulin knockdown, single cell clones were isolated from stably transfected cells to identify the clones with >50% gene silencing. Approximately six puromycin resistant clones transfected with pRS shctrl and 60 puromycin resistant clones transfected with pRS shrβIII (construct 3) were isolated at random and tested for their expression of class III β-tubulin (data not shown). Each clone was screened by both western blot analysis and immunofluorescence microscopy to ensure that results were consistent between the two methods and also to assess the percentage of cells in each population that were negative for class III β-tubulin expression.
Specific and Stable Knockdown of Class III β-Tubulin in H460 Cells
After initial screening, two clones of pRS-shctrl (referred to as control clone 1 and control clone 2) and three clones of pRS-shrβIII were selected for further characterization. The class III β-tubulin stable knockdown clones, designated clone 4, clone 59 and clone 60, were identified as cells which stably express significantly reduced levels of class III β-tubulin when compared to the control clones 1 and 2 (non-functional 29 mer shRNA) that did not display altered class III β-tubulin levels. Each of the class III β-tubulin stable knockdown clones demonstrated a different level of knockdown of the class III β-tubulin isotype (as shown by the variation in intensity of the class III β-tubulin protein detected in western blots). To determine the stability of the shRNA, stable clones were harvested at 7 day intervals for 4 weeks to examine the expression of class III β-tubulin.
Class III β-tubulin expression did not recover to control levels even after 28 days (four weeks) of culture. Further observation showed that there was no significant difference between the level of knockdown in the absence or presence of puromycin. This observation is of importance since cells were maintained in the absence of selective pressure during experiments. Thus, efficient and stable knockdown of class III β-tubulin expression was achieved in these cells.
Effect of Stable Knockdown of Class III β-Tubulin on the Expression of Other β-Tubulin Isotypes
Cellular tubulin levels are tightly regulated and stable overexpression of a specific β-tubulin isotype can result in compensatory changes in the expression of other β-tubulin isotypes. To verify the specificity of siRNA against class III β-tubulin and to evaluate protein compensatory changes, expression of other β-tubulin isotypes were analyzed in the class III β-tubulin stable knockdown clones. Results showed that specific stable knockdown of class III β-tubulin was achieved without altering the expression of other β tubulin isotypes.
Sensitivity of Class III β-Tubulin Stable Knockdown Clones to Cisplatin
As described herein, transient knockdown of class III β-tubulin using synthetic siRNA is associated with hypersensitivity to tubulin binding agents (TBAs) and DNA damaging agents. To confirm that cells stably expressing class III β-tubulin shRNA have similar phenotypic characteristics and also display similar sensitivity to TBAs as was found with siRNA, cells were exposed to various concentrations of paclitaxel. Consistent with results described herein, class III β-tubulin stable clones were found to be significantly more sensitive to paclitaxel than control clones 1 and 2. Clone 4, 59 and 60 had ID50 values of 1.36±0.09, 1.78±0.07 and 1.58±0.10 respectively, which were significantly lower than ID50 values for control clone 1 and 2 (2.76±0.13 and 2.83±0.09, respectively) (P<0.001).
Similarly, when these class III β-tubulin stable knockdown clones (clone 4, 59 and 60) were examined for sensitivity to the DNA damaging agent cisplatin, all clones were found to exhibit increased sensitivity to cisplatin. Clone 4, which has the greatest amount of knockdown, showed the greatest sensitivity to cisplatin (ID50=0.17±0.01). Similarly, clones 59 and 60 were significantly more sensitive to cisplatin (ID50 values of 0.22±0.01 and 0.24±0.01, respectively) compared to control clones 1 and 2 (ID50 values of 0.41±0.04 and 0.38±0.04, respectively) (P<0.001). These results demonstrate that consistent with transient class III β-tubulin knockdown, stable knockdown of class III β-tubulin can enhance the chemosensitivity of H460 cells to TBAs and DNA damaging agents. Therefore, the stable shRNA expressing clones maintain phenotypic features of transient knockdown of βIII-tubulin.
Stable Knockdown of Class III β-Tubulin Decreases Anchorage-Independent Growth in Soft Agar
Consistent with the data presented using synthetic class III β-tubulin siRNA (both 21-mer and 27-mer), stable knockdown of class III β-tubulin significantly reduced the number of colonies formed on soft agar in class III β-tubulin stable knockdown clones (clone 4, 59 and 60) (P<0.001) compared with control clones 1 and 2. In addition, class III β-tubulin stable knockdown clones also exhibited smaller colonies when compared to the control clones (data not shown). Thus, knockdown of class III β-tubulin impedes anchorage-independent growth of H460 cells in vitro, and might therefore contribute to decreasing tumorigenicity potential in vivo.
Stable Knockdown of Class III β-Tubulin Suppresses Tumor Development in Nude Mice
To evaluate the in vivo biological effect of silencing class III β-tubulin expression on tumorigenic potential, four groups of 5-6-week old Balb/c nude mice (five-six mice/group) were injected subcutaneously into the flank (1×106 cells) control clone 1 (group one), control clone 2 (group two), class III β-tubulin stable knockdown clone 4 (group three) and clone 59 (group four). After 9 days post injection, tumors were visible in all nude mice injected with the two control clones, whereas only two animals injected with class III β-tubulin stable knockdown clone 4 developed tumors during this time. Tumor size was evaluated twice per week using the formula: V=(length×width×depth)/2, as described above. Mice were culled when the tumor size reached 1 cm3. The survival rates of these groups of mice during the experimental period of 120 days were displayed on a Kaplan-Meier curve (
To confirm that class III β-tubulin levels were significantly reduced in subcutaneous tumors injected with class III β-tubulin stable knockdown clones, tumor tissues were harvested for class III β-tubulin protein expression by western blot and immunohistochemistry. There was significant knockdown of class III β-tubulin protein expression in tumors harvested from three independent mice injected with clone 4 when compared with the control clone 2 (
Histological examination by H&E staining did not reveal any obvious morphological changes between the tumors generated from control mice and βIII-tubulin stably knocked down clones (
Tumour cell proliferation was determined using Ki67. The staining intensity and hence expression of Ki67 was similar between the βIII-tubulin knockdown and control tumour sections, suggesting that cell proliferation rates when tumours reached 1 cm3 (time of sacrifice) did not differ between βIII-tubulin stable knockdown clones and control clones. Thus, knockdown of βIII tubulin, in the context of a highly aggressive NSCLC cell line, decreased tumourigenic growth and incidence in an in vivo NSCLC xenograft model.
Herein, stable knockdown of βIII-tubulin expression in H460 cells was found to decrease anchorage-independent growth in vitro and to suppress tumorigenicity in vivo. In vitro, stable knockdown of βIII tubulin was found to suppress anchorage-independent growth of the H460 cells compared to controls. Stable knockdown of βIII tubulin delayed tumor growth and reduced tumor incidence of subcutaneous xenografted tumors. It should be noted that despite the fact that βIII-tubulin is present in relatively small amounts in the H460 cells (7.8% of all β tubulin isotypes) knockdown of this minor isotype produces distinct effects in drug response and tumorigenesis suggesting that βIII-tubulin is functionally important in NSCLC cells. The ability of class III β-tubulin knockdown to markedly suppress the growth of the H460 cells suggests that class III β-tubulin may play an important role in lung cancer cell growth and survival.
Number | Date | Country | Kind |
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2007901131 | Mar 2007 | AU | national |
2007905307 | Sep 2007 | AU | national |
This application is a continuation of U.S. patent application Ser. No. 12/555,522 filed Sep. 8, 2009, which is a continuation-in-part of International Application No. PCT/AU2008/000298 filed Mar. 5, 2008, which claims the benefit of priority to Australian Patent Application No. 2007901131 filed Mar. 5, 2007, Australian Patent Application No. 2007905307 filed Sep. 28, 2007, and U.S. Provisional Patent Application No. 61/157,135 filed Mar. 3, 2009, the disclosures of all of which are hereby incorporated by reference in their entireties. To the extent appropriate, a claim of priority is made to each of the above-disclosed applications.
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61157135 | Mar 2009 | US |
Number | Date | Country | |
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Parent | 12555522 | Sep 2009 | US |
Child | 14951992 | US |
Number | Date | Country | |
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Parent | PCT/AU2008/000298 | Mar 2008 | US |
Child | 12555522 | US |