Patent Application
20080095764
All patents, patent applications and publications cited herein are hereby incorporated by reference in their entirety. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.
Neoplasia is a disease characterized by an abnormal proliferation of cells, or neoplasm. In neoplasia, cells divide and grow uncontrollably, they invade and disrupt other tissues, and often spread to other areas of the body (metastasis). Neoplasms may manifest as a blood disorder (e.g. leukemia) or a tumor, and may be benign or malignant (cancer). Successful management of neoplasms depends, in part, upon early detection of tumors. A correlation generally exists between the tumor burden in a patient with a neoplasm, and the patient's chances of survival. Thus, the mortality from cancer can be reduced if tumors are found and treated at an early stage.
The major therapies for treating neoplasia are surgery and radiotherapy (for local and local/regional neoplasms) and chemotherapy (for systemic sites) (Beers and Berkow, Eds., The Merck Manual of Diagnosis and Therapy, 17th ed. (Whitehouse Station, N.J.: Merck Research Laboratories, 1999) 973-74, 976, 986, 988, 991). While treatment-related improvements in survival may have resulted in a decline in cancer mortality, current treatment protocols can be invasive (surgery) or can produce deleterious side effects (chemotherapy and radiation).
By way of example, breast cancer is the most common malignancy among women, and remains the leading cause of cancer-related death in women aged 20-59 (Greenlee et al., Cancer Statistics 2000. A Cancer Journal for Clinicians, 50:7-33, 2000). Breast examination, mammography, xerography, and termography are established methods of detecting malignant breast masses. These methods, in conjunction with personal history, may strongly suggest breast cancer, however, a true diagnosis can only be made by microscopic examination of tissue removed by excisional biopsy or aspiration. There are a number of methods currently used to treat breast cancer, including surgery, radiotherapy, hormone therapy, and chemotherapy. Because breast tumors may be cured with combined modality therapy, each of these methods may be used alone, or in addition to one or more other therapies. Thus, local and regional therapy, surgery, or radiotherapy is often integrated with systemic therapy (e.g. chemotherapy).
Despite the various methods for detecting, diagnosing, and treating neoplasias, including breast neoplasms, cancer remains prevalent in all segments of society, and is often fatal. Clearly, alternative strategies for detection (including the development of markers that can identify neoplasias at an early stage) and for treatment are needed to improve survival in cancer patients. In particular, a better understanding of tumor suppressors, and tumor-suppression pathways, would provide a basis from which novel detection, diagnostic, and treatment regimens may be developed.
Cadherins are functionally-related integral membrane glycoproteins that play an important role in calcium-dependent cell-cell adhesion. Cadherins promote cell adhesion via a homophilic mechanism, and play a role in the construction of tissues and of the animal body as a whole. There are three types of cadherin, each of which is distinct in its immunological specificity and tissue distribution: N cadherin is found in neural tissue, E cadherin (also known as uvomorulin or L CAM) is found in epithelial tissue, and P cadherin is found in placental tissue. Each cadherin protein has a 600-amino-acid extracellular domain, containing four repeats (believed to contain the calcium-binding sites), a transmembrane domain, and a one 50-amino-acid intracellular domain. Cadherin expression appears to be tightly regulated during development, with each tissue or cell type showing a characteristic pattern of cadherin molecules. Inappropriate regulation of cadherin expression levels or functionality has been observed in human malignancies, and has been shown to lead to aggravated cancer-cell invasion and metastasis.
There are at least six subfamilies in the cadherin superfamily: classical or type-I cadherins, atypical or type-II cadherins, desmocollins, desmogleins, protocadherins, and Flamingo cadherins. These subfamilies are distinguished on the basis of protein domain composition, genomic structure, and phylogenetic analysis of protein sequence. The protocadherins constitute a large subfamily of the cadherin superfamily, and function in a variety of tissues and multicellular organisms. Protocadherins are usually localized at synapses in the central nervous system (CNS), and their expression is spatiotemporally regulated.
A highly conserved protocadherin (PCDH) cluster exists at human chromosome 13q. This cluster contains PCDH8, PCDH9, PCDH17, and PCDH20. These protocadherins are single-pass transmembrane proteins that share greater similarity with each other than with other members of the protocadherin family. Like other protocadherins, the PCDHs in the 13q cluster have six cadherin repeats in the extracellular domain, a single transmembrane domain, a signal peptide sequence, and an intracellular domain; however, they have unique cytoplasmic tails. Interestingly, while the extracellular domain of PCDH8 is most similar to that of PCDH17, the intracellular domain of PCDH8 is most similar to that of PCDH9.
Protocadherin 8 and protocadherin 9 constitute a linkage group on human chromosome 13 and mouse chromosome 14. Like other protocadherins, PCDH8 and PCDH9 are predominantly expressed in the brain, and exhibit expression patterns that are developmentally regulated; PCDH9 is also expressed in a broader variety of tissues. In contrast to a classical or a desmosomal cadherin—which generally consists of 15-17 exons, and shares a remarkable degree of conservation in intron positions—PCDH8 has only three exons. The first exon encodes the extracellular domain, the transmembrane region, and part of the cytoplasmic tail; the second and third exons encode the remainder of the cytoplasmic region, including the 3′ untranslated region. Protocadherin 17, also known as PCDH68, encodes a protein which contains six extracellular cadherin domains, a transmembrane domain, and a cytoplasmic tail that differs from those of the classical cadherins.
Many protocadherins are believed to play in important role in the organization; of the CNS. Prior to the present invention, however, it was not known that PCDHs 8, 9, and 17 are also markers for, and form a basis for designing therapeutics to treat, neoplasia.
The present invention is based upon the surprising discovery that human protocadherins on chromosome 13q 14-21 are activated or inactivated, as the case may be, in an oncogenic switch. For example, the inventors have discovered that protocadherin 17 is turned on, and protocadherins 8 and 9 are shut off, in cancer.
Accordingly, in one aspect, the present invention provides a method for determining whether a subject has neoplasia, by assaying a diagnostic sample of the subject for expression of at least one protocadherin selected from the group consisting of PCDH8, PCDH9, and PCDH17, wherein detection of PCDH8 expression decreased below normal is diagnostic of neoplasia in the subject, detection of PCDH9 expression decreased below normal is diagnostic of neoplasia in the subject, and detection of PCDH17 expression elevated above normal is diagnostic of neoplasia in the subject.
The present invention further provides a method for assessing the efficacy of therapy to treat neoplasia in a subject who has undergone or is undergoing treatment for neoplasia, by assaying a diagnostic sample of the subject for expression of at least one protocadherin selected from the group consisting of PCDH8, PCDH9, and PCDH17, wherein normal expression of PCDH8, PCDH9, and PCDH17 in the diagnostic sample is indicative of successful therapy to treat neoplasia, and wherein PCDH8 expression decreased below normal in the diagnostic sample and/or PCDH9 expression decreased below normal in the diagnostic sample and/or PCDH7 expression elevated above normal in the diagnostic sample is indicative of a need to continue therapy to treat neoplasia.
The present invention also provides a method for assessing the prognosis of a subject who has neoplasia, by assaying a diagnostic sample of the subject for expression of at least one protocadherin selected from the group consisting of PCDH8, PCDH9, and PCDH17, wherein the subject's prognosis improves with an increase in expression of PCDH8 in the diagnostic sample and/or an increase in expression of PCDH9 in the diagnostic sample and/or a decrease in expression of PCDH17 in the diagnostic sample, and wherein the subject's prognosis worsens with a decrease in expression of PCDH8 in the diagnostic sample and/or a decrease in expression of PCDH9 in the diagnostic sample and/or an increase in expression of PCDH17 in the diagnostic sample.
Additionally, the present invention provides a method for determining whether a subject has neoplasia, by assaying a diagnostic sample of the subject for methylation of PCDH8 promoter and/or methylation of PCDH9 promoter, wherein detection of methylation of the PCDH8 promoter and/or the PCDH9 promoter is diagnostic of neoplasia in the subject.
The present invention further provides a method for assessing the efficacy of therapy to treat neoplasia in a subject who has undergone or is undergoing treatment for; neoplasia, by assaying a diagnostic sample of the subject for methylation of PCDH8 promoter and/or methylation of the PCDH9 promoter, wherein no detection of methylation of the PCDH8 promoter and/or the PCDH9 promoter in the diagnostic sample is indicative of successful therapy to treat neoplasia, and wherein detection of methylation of the PCDH8 promoter and/or the PCDH9 promoter in the diagnostic sample is indicative of a need to continue therapy to treat neoplasia.
The present invention also provides a method for assessing the prognosis of a subject who has neoplasia, by assaying a diagnostic sample of the subject for methylation of PCDH8 promoter and/or PCDH9 promoter, wherein the subject's prognosis improves with a decrease in methylation of the PCDH8 promoter and/or the PCDH9 promoter in the diagnostic sample, and wherein the subject's prognosis worsens with an increase in methylation of the PCDH8 promoter and/or the PCDH9 promoter in the diagnostic sample.
Furthermore, the present invention provides a method for determining whether a subject has neoplasia, by assaying a diagnostic sample of the subject for at least one mutation selected from the group consisting of a PCDH8 mutation and a PCDH17 mutation.
Additionally, the present invention provides a kit for use in detecting neoplasia, including: (a) at least one agent reactive with PCDH8, PCDH9, and/or PCDH17; and (b) reagents suitable for detecting expression of at least one protocadherin selected from the group consisting of PCDH8, PCDH9, and PCDH17. Also provided is a kit for use in detecting neoplasia, including: (a) at least one nucleic acid probe that hybridizes to nucleic acid encoding PCDH8, PCDH9, and/or PCDH17; and (b) reagents suitable for detecting expression of at least one nucleic acid encoding at least one protocadherin selected from the group consisting of PCDH8, PCDH9, and PCDH17.
The present invention also provides a method for treating or preventing neoplasia in a subject, by modulating level and/or function of at least one protocadherin in the subject, wherein the at least one protocadherin is selected from the group consisting of PCDH8, PCDH9, and PCDH17.
The present invention further provides a pharmaceutical composition that includes a modulator of PCDH8 or PCDH9 expression, or a PCDH8 or PCDH9 protein, in an amount effective to treat or prevent neoplasia in a subject to whom the composition is administered, and a pharmaceutically-acceptable carrier. Also provided is a pharmaceutical composition that includes an agent reactive with PCDH17, in an amount effective to treat or prevent neoplasia in a subject to whom the composition is administered, and a pharmaceutically-acceptable carrier.
Additionally, the present invention provides a method for identifying an agent for use in treating and/or preventing neoplasia, by: (a) obtaining a collection of cells having at least one characteristic selected from the group consisting of PCDH8 expression decreased below normal, PCDH9 expression decreased below normal, and PCDH17 expression elevated above normal; (b) contacting a candidate agent with the cells; and (c) determining the effect, if any, of the candidate agent on PCDH8 expression, PCDH9 expression, and/or PCDH17 expression in the cells. Also provided are an agent identified by this method, and use of this agent in a method for treating or preventing neoplasia in a subject.
Finally, the present invention provides a method for identifying an agent for use in treating and/or preventing neoplasia, by: (a) obtaining an animal comprising cells having at least one characteristic selected from the group consisting of PCDH8 expression decreased below normal, PCDH9 expression decreased below normal, and PCDH17 expression elevated above normal; (b) administering a candidate agent to the animal; and (c) determining the effect, if any, of the candidate agent on PCDH8 expression, PCDH9 expression, and/or PCDH17 expression in the animal. Also provided are an agent identified by this method and use of this agent in a method for treating or preventing neoplasia in a subject.
Additional aspects of the present invention will be apparent in view of the description which follows.
As disclosed herein, the inventors have discovered that human protocadherins on chromosome 13q14-21 are activated or inactivated, as the case may be, in an oncogenic switch. For example, the inventors have discovered that protocadherin 17 is turned on, and protocadherins 8 and 9 are shut off, in cancer. In normal breast cells, protocadherin 17 is typically silent, while protocadherins 8 and 9 are typically expressed. Contrastingly, in breast cancer, protocadherin 17 is expressed, while protocadherins 8 and 9 are either mutated or silenced through methylation of their promoters. Altered expression of these genes is also seen in prostate cancer.
Accordingly, the present invention provides a method for determining whether a subject has neoplasia. As used herein, the “subject” is a mammal, including, without limitation, a cow, dog, human, monkey, mouse, pig, or rat. Preferably, the subject is a human. The inventors demonstrate herein that patterns of protocadherin expression are: altered in cancer, including breast cancer and prostate cancer. Thus, the method of the present invention comprises assaying a diagnostic sample of the subject for expression of a protocadherin, wherein detection of protocadherin expression elevated above normal, or decreased below normal, as the case may be, is diagnostic of neoplasia in the subject. By way of example, the method of the present invention may comprise assaying a diagnostic sample of the subject for expression of PCDH8, PCDH9, and/or PCDH17, wherein detection of PCDH8 expression decreased below normal is diagnostic of neoplasia in the subject, detection of PCDH9 expression decreased below normal is diagnostic of neoplasia in the subject, and/or detection of PCDH17 expression elevated above normal is diagnostic of neoplasia in the subject.
As used herein, “PCDH” includes both a PCDH (protocadherin) protein (e.g. PCDH8, PCDH9, PCDH17, PCDH20, etc.) and a PCDH analogue. For example, “PCDH8” includes both a PCDH8 (protocadherin 8) protein and a PCDH8, analogue; “PCDH9” includes both a PCDH9 (protocadherin 9) protein and a PCDH9 analogue; and “PCDH17” includes both a PCDH17 (protocadherin 17) protein and a PCDH17 analogue. Unless otherwise indicated, a “protein” shall include a protein, protein domain, polypeptide, or peptide. A “PCDH analogue”, as used herein, is a functional variant of the PCDH protein of interest (e.g. PCDH8, PCDH9, PCDH17, PCDH20, etc.), having PCDH biological activity, which has 60% or greater (preferably, 70% or greater) amino-acid-sequence homology with the PCDH protein of interest. A PCDH “analogue” includes a variant of the PCDH protein that has a homologous three-dimensional conformation. As further used herein, the term “PCDH biological activity” refers to the function/activity of a protein or peptide that demonstrates an ability to influence the generation, termination, and/or course of progression of a neoplasia, as described herein.
As further used herein, the PCDH8, PCDH9, and PCDH17 amino acid sequences are well known in the art. See, e.g. GenBank Accession Nos. AAH36025: NP—002581, 095206, NP—116567, AAK21986, and AAC70009, including conservative substitutions thereof, for PCDH8; see, e.g. GenBank Accession Nos. NP—982354, NP—065136, Q9HC56, and AAF89689, including conservative substitutions thereof, for PCDH9, and see, e.g. GenBank Accession Nos. NP—055274 and 014917, including conservative substitutions thereof, for PCDH17. “Conservative substitutions” are those amino acid substitutions which are functionally equivalent to the substituted amino acid residue, either because they have similar polarity or steric arrangement, or because they belong to the same class as the substituted residue (e.g. hydrophobic, acidic, or basic). The term “conservative substitutions”, as defined herein, includes substitutions having an inconsequential effect on the ability of the PCDH of interest to influence the generation, termination, and/or course of progression of a neoplasia.
PCDH proteins of interest (e.g. PCDH8, PCDH9, PCDH17, PCDH20, etc.) and PCDH analogues may be produced synthetically or recombinantly, or may be isolated from native cells. PCDHs of interest are preferably produced recombinantly, using conventional techniques and cDNA encoding the PCDH proteins (e.g. PCDH8, PCDH9, PCDH17, PCDH20, etc.).
The method of the present invention may be used to determine whether a subject has a neoplasia, thereby permitting the diagnosis of the neoplasia in the subject. As used herein, “neoplasia” refers to the uncontrolled and progressive multiplication of cells of a neoplasm (i.e., neoplastic cells, such as tumor cells), under conditions that would not elicit, or would cause cessation of, multiplication of normal cells. Neoplasia results in a “neoplasm”, which is defined herein to mean any new and abnormal growth, particularly a new growth of tissue, in which the growth of cells is uncontrolled and progressive. Thus, neoplasia includes “cancer”, which herein refers to a proliferation of neoplastic cells having the unique trait of loss of normal controls, resulting in unregulated growth, lack of differentiation, local tissue invasion, and/or metastasis.
As used herein, neoplasms include, without limitation, morphological irregularities of cells in tissue of a subject, as well as pathologic proliferation of cells in tissue of a subject, as compared with normal proliferation in the same type of tissue. Additionally, neoplasms include benign tumors and malignant tumors (e.g. breast tumors) that are either invasive or noninvasive. Malignant neoplasms are distinguished from benign in that the former show a greater degree of anaplasia, or loss of differentiation and orientation of cells, and have the properties of invasion and metastasis. Examples of neoplasms or neoplasias which may be assessed, detected, diagnosed, monitored, or treated in accordance with inventions described herein include, without limitation, carcinomas, particularly those of the bladder, breast, cervix, colon, head, kidney, lung, neck, ovary, prostate, and stomach; lymphocytic leukcmias, particularly acute lymphoblastic leukemia and chronic lymphocytic leukemia; myeloid leukemias, particularly acute monocytic leukemia, acute promyelocytic leukemia, and chronic myelocytic leukemia, malignant lymphomas, particularly Burkitt's lymphoma and Non-Hodgkin's lymphoma; malignant melanomas; myeloproliferative diseases; sarcomas, particularly Ewing's sarcoma, hemangiosarcoma, Kaposi's sarcoma, liposarcoma, peripheral neuroepithelioma, and synovial sarcoma; and mixed types of neoplasias, particularly carcinosarcoma and Hodgkin's disease (Beers and Berkow (eds.), The Merck Manual of Diagnosis and Therapy, 17th ed. (Whitehouse Station, N.J.: Merck Research Laboratories, 1999) 973-74, 976, 986, 988, 991). In a preferred embodiment of the present invention, the methods and compositions of the present invention are directed to the assessment, detection, diagnosis, monitoring, and treatment of breast cancer or prostate cancer.
According to the method of the present invention, the diagnostic sample of a subject may be assayed in vitro or in vivo. Where the assay is performed in vitro, a diagnostic sample from the subject may be removed using standard procedures. The diagnostic sample may be tissue, including any bone, brain tissue, breast tissue, colon tissue, muscle tissue, nervous tissue, ovarian tissue, prostate tissue, retinal tissue, skin tissue, or soft tissue, which may be removed by standard biopsy. In addition, the diagnostic sample may be a bodily fluid, including cerebrospinal fluid, pericardial fluid, peritoneal fluid, saliva, serum, sputum, and urine, or a solid, such as feces. Furthermore, the diagnostic sample taken from the subject or patient may be, for example, any tissue known to have a neoplasm, any tissue suspected of having a neoplasm, or any tissue believed not to have a neoplasm.
Protein may be isolated and purified from the diagnostic sample of the present invention using standard methods known in the art, including, without limitation, extraction from a tissue (e.g. with a detergent that solubilizes the protein), where necessary, followed by affinity purification on a column, chromatography (e.g. FTLC and HPLC), immunoprecipitation (e.g. with an antibody to the PCDH of interest), and/or precipitation (e.g. with isopropanol and a reagent such as Trizol). Isolation and purification of the protein may also be followed by electrophoresis (e.g. on an SDS-polyacrylamide gel). Nucleic acid may be isolated from a diagnostic sample using standard techniques known to one of skill in the art.
In accordance with the method of the present invention, neoplasia in a subject may be diagnosed by assaying a diagnostic sample of the subject for expression of a protocadherin, wherein detection of protocadherin expression elevated above normal, or decreased below normal, as the case may be, is diagnostic of neoplasia in the subject. For example, neoplasia may be diagnosed in a subject by assaying a diagnostic sample of the subject for expression of PCDH8, PCDH9, and/or PCDH17, wherein detection of PCDH8 expression decreased below normal is diagnostic of neoplasia in the subject, detection of PCDH9 expression decreased below normal is diagnostic of neoplasia in the subject, and detection of PCDH17 expression elevated above normal is diagnostic of neoplasia in the subject.
As used herein, “expression” means the transcription of a gene into at least one mRNA transcript, or the translation of at least one mRNA into a protein. For example, “expression of PCDH” means the transcription of the PCDH gene of interest into at least one mRNA transcript, or the translation of at least one mRNA into a PCDH protein, as defined above. Accordingly, a diagnostic sample may be assayed for PCDH expression by assaying for PCDH protein, PCDH cDNA, and/or PCDH mRNA. The appropriate form of PCDH will be apparent based on the particular techniques discussed herein. It is also contemplated that the diagnostic sample may be assayed for expression of any or all forms of the PCDH protein of interest (including precursor, endoproteolytically-processed forms, and other forms resulting from post-translational modification) in order to determine whether a subject or patient has neoplasia.
As used herein, the term “elevated above normal” refers to detection (e.g. of expression of PCDH17, etc.) at a level that is significantly greater than the level expected for the same type of diagnostic sample taken from a non-diseased subject or patient (i.e., one who does not have neoplasia) of the same gender and of similar age. As further used herein, “significantly greater” means that the difference between the level (e.g. of expression of PCDH17, etc.) that is elevated above normal, and the expected (normal) level (e.g. of expression of PCDH17, etc.), is of statistical significance.
Preferably, PCDH expression (e.g. PCDH17 expression) elevated above normal is expression of the PCDH (e.g. PCDH17 expression) at a level that is at least 10% greater than the level of PCDH expression (e.g. PCDH17 expression) otherwise expected. Where PCDH expression (e.g. PCDH17 expression) is expected to be absent from a particular diagnostic sample taken from a particular subject or patient, the normal level of PCDH expression for that subject or patient is nil. Where a particular diagnostic sample taken from a particular subject or patient is expected to have a low level of constitutive PCDH expression, that low level is the normal level of PCDH expression for that subject or patient. As disclosed herein, PCDH17 expression is generally absent from non-neoplastic (normal) cells.
Additionally, as used herein, the term “decreased below normal” refers to detection (e.g. of expression of PCDH8, of expression of PCDH9, etc.) at a level that is significantly lower than the level expected for the same type of diagnostic sample taken from a non-diseased subject or patient (i.e., one who does not have neoplasia) of the same gender and of similar age. As further used herein, “significantly lower” means that the difference between the level (e.g. of expression of PCDH8, of expression of PCDH9, etc.) that is decreased below normal, and the expected (normal) level (e.g. of expression of PCDH8, of expression of PCDH9, etc.), is of statistical significance. Preferably, PCDH expression (e.g. expression of PCDH8, expression of PCDH9, etc.) decreased below normal is expression of the PCDH at a level that is at least 10% less than the level of PCDH expression otherwise expected.
Expected or normal levels of PCDH expression (e.g. PCDH8 expression, PCDH9 expression, PCDH17 expression, etc.) for a particular diagnostic sample taken from a subject or patient may be easily determined by assaying non-diseased subjects of a similar age and of the same gender. For example, diagnostic samples may be obtained from at least normal, healthy men between the ages of 25 and 80, to determine the normal quantity of PCDH expression (e.g. PCDH8 expression, PCDH9 expression, PCDH17 expression, etc.) in males. A similar procedure may be followed to determine the normal quantity of PCDH expression (e.g. PCDH8 expression, PCDH9 expression, PCDH17 expression, etc.) in females. Once the necessary or desired samples have been obtained, the normal quantities of PCDH expression (e.g. PCDH8 expression, PCDH9 expression, PCDH17 expression, etc.) in men and women may be determined using a standard assay for quantification, such as flow cytometry, Western-blot analysis, or an ELISA for measuring protein quantities, as described in the art. For example, an ELISA may be run on each sample in duplicate, and the means and standard deviations of the quantity of the PCDH protein of interest (e.g. PCDH8 protein, PCDH9 protein, PCDH17 protein, etc.) may be determined. If necessary, additional subjects may be recruited before the normal quantities of PCDH expression (e.g. PCDH8 expression, PCDH9 expression, PCDH17 expression, etc.) are quantified.
In accordance with the method of the present invention, a diagnostic sample of a subject may be assayed for PCDH expression (e.g. PCDH8 expression, PCDH9 expression, PCDH17 expression, etc.), and PCDH expression may be detected in a diagnostic sample, using assays and detection methods readily determined from the known art (e.g. immunological techniques, hybridization analysis, fluorescence imaging techniques, radiation detection, etc.), as well as any assays and detection methods disclosed herein (e.g. immunoprecipitation, Western-blot analysis, etc.). For example, a diagnostic sample of a subject may be assayed for PCDH expression (e.g. PCDH8 expression, PCDH9 expression, PCDH17 expression, etc.) using an agent reactive with the PCDH of interest.
As used herein, “reactive” means the agent has affinity for, binds to, or is directed against a target of interest (e.g. PCDH8, PCDH9, PCDH17, etc.). As further used herein, an “agent” shall include a protein, polypeptide, peptide, nucleic acid (including DNA or RNA), antibody, Fab fragment, F(ab′)2 fragment, molecule, compound, antibiotic, drug, and any combination(s) thereof. A Fab fragment is a univalent antigen-binding fragment of an antibody, which is produced by papain digestion. A F(ab′)2 fragment is a divalent antigen-binding fragment of an antibody, which is produced by pepsin digestion. Preferably, the agent of the present invention is labeled with a detectable marker or label.
In one embodiment of the present invention, the agent reactive with the PCDH of interest (e.g. PCDH8, PCDH9, PCDH17, etc.) is an antibody. As used herein, the antibody of the present invention may be polyclonal or monoclonal. In addition, the antibody of the present invention may be produced by techniques well known to those skilled in the art. Polyclonal antibody, for example, may be produced by immunizing a mouse, rabbit, or rat with purified protein (e.g. PCDH8, PCDH9, PCDH17, etc.). Monoclonal antibody then may be produced by removing the spleen from the immunized mouse, and fusing the spleen cells with myeloma cells to form a hybridoma which, when grown in culture, will produce a monoclonal antibody. It is expected that antibodies that specifically target protocadherin 17, in particular, will lead to tumor regression.
The antibodies used herein may be labeled with a detectable marker or label. Labeling of an antibody, or any other agent, may be accomplished using one of a variety of labeling techniques, including peroxidase, chemiluminescent labels known in the art, and radioactive labels known in the art. The detectable marker or label of the present invention may be, for example, a nonradioactive or fluorescent marker, such as biotin, fluorescein (FITC), acridine, cholesterol, or carboxy-X-rhodamine, which can be detected using fluorescence and other imaging techniques readily known in the art. Alternatively, the detectable marker or label may be a radioactive marker, including, for example, a radioisotope. The radioisotope may be any isotope that emits detectable radiation, such as 35S, 32P, 125I, 3H, or 14C. Radioactivity emitted by the radioisotope can be detected by techniques well known in the art. For example, gamma emission from the radioisotope may be detected using gamma imaging techniques, particularly scintigraphic imaging. Preferably, the agent of the present invention is a high-affinity antibody labeled with a detectable marker or label.
Where the agent of the present invention is an antibody reactive with a PCDH of interest (e.g. PCDH8, PCDH9, PCDH17, etc.), a diagnostic sample taken from the subject may be purified by passage through an affinity column which contains anti-PCDH antibody as a ligand attached to a solid support, such as an insoluble organic polymer in the form of a bead, gel, or plate. The antibody attached to the solid support may be used in the form of a column. Examples of suitable solid supports include, without limitation, agarose, cellulose, dextran, polyacrylamide, polystyrene, sepharose, or other insoluble organic polymers. The anti-PCDH antibody may be further attached to the solid support through a spacer molecule, if desired. Appropriate binding conditions (e.g. temperature, pH, and salt concentration) for ensuring binding of the agent and the antibody may be readily determined by the skilled artisan. In a preferred embodiment, the anti-PCDH antibody is attached to a sepharose column, such as Sepharose 4B.
Where the agent is an antibody, a diagnostic sample of the subject may be assayed for PCDH expression (e.g. PCDH8 expression, PCDH9 expression, PCDH17 expression, etc.) using binding studies that utilize one or more antibodies immunoreactive with a PCDH protein of interest (e.g. PCDH8, PCDH9, PCDH17, etc.), along with standard immunological detection techniques. For example, the PCDH protein eluted from the affinity column may be subjected to an ELISA assay, Western-blot analysis, flow cytometry, or any other immunostaining method employing an antigen-antibody interaction. Preferably, the diagnostic sample is assayed for PCDH expression (e.g. PCDH8 expression, PCDH9 expression, PCDH17 expression, etc.) using Western blotting.
Alternatively, a diagnostic sample of a subject may be assayed for PCDH expression (e.g. PCDH8 expression, PCDH9 expression, PCDH17 expression, etc.) using hybridization analysis of nucleic acid extracted from the diagnostic sample taken from the subject. According to this method of the present invention, the hybridization analysis may be conducted using Northern-blot analysis of mRNA. This method also may be conducted by performing a Southern-blot analysis of DNA using one or more nucleic acid probes, which hybridize to nucleic acid encoding a PCDH of interest (e.g. PCDH8, PCDH9, PCDH17, etc.). The nucleic acid probes may be prepared by a variety of techniques known to those skilled in the art, including, without limitation, the following: restriction enzyme digestion of PCDH nucleic acid; and automated synthesis of oligonucleotides having sequences which correspond to selected portions of the nucleotide sequence of the PCDH nucleic acid of interest, using commercially-available oligonucleotide synthesizers, such as the Applied Biosystems Model 392 DNA/RNA synthesizer.
The nucleic acid probes used in the present invention may be DNA or RNA, and may vary in length from about 8 nucleotides to the entire length of the PCDH-encoding (e.g. PCDH8-encoding, PCDH9-encoding, PCDH17-encoding, etc.) nucleic acid. The nucleic acid used in the probes may be derived from any mammal, including a human. The nucleotide sequences for human PCDH proteins, including PCDH8, PCDH9, and PCDH17, are known in the art. See, e.g. GenBank Accession Nos. BC036025, NM—002590, NM—032949, AY413-493, AY013873 and AF061573, for PCDH8, see, e.g. GenBank Accession Nos. NM—020403, NM—203487, AK057768, and AF169692, for PCDH9; and see, e.g. GenBank Accession Nos. NT—024524 and NM—014459, for PCDH17. Using one of these PCDH sequences as a probe, for example, the skilled artisan could readily clone corresponding PCDH cDNA from other species. In addition, the nucleic acid probes of the present invention may be labeled with one or more detectable markers or labels. Labeling of the nucleic acid probes may be accomplished using one of a number of methods known in the art—e.g. nick translation, end labeling, fill-in end labeling, polynucleotide kinase exchange reaction, random priming, or SP6 polymerase (for riboprobe preparation)—along with one of a variety of labels—e.g. a radioactive label (such as 35S, 32P, or 3H), a nonradioactive label (such as biotin, fluorescein (FITC), acridine, cholesterol, or carboxy-X-rhodamine (ROX)), or any other detectable marker disclosed herein. Combinations of two or more nucleic acid probes (or primers), corresponding to different or overlapping regions of the PCDH nucleic acid of interest (e.g. nucleic acid encoding PCDH8, PCDH9, PCDH17, etc.), also may be used to assay a diagnostic sample for PCDH expression (e.g. expression of PCDH8, PCDH9, PCDH17, etc.), using, for example, PCR or RT-PCR.
The detection of PCDH expression (e.g. PCDH8 expression, PCDH9 expression, PCDH17 expression, etc.) in the method of the present invention may be followed by an assay to measure or quantify the extent of PCDH expression (e.g. PCDH8 expression, PCDH9 expression, PCDH17 expression, etc.) in a diagnostic sample of a subject. Such assays are well known to one of skill in the art, and may include immunohistochemistry/immunocytochemistry, flow cytometry, mass spectroscopy, Western-blot analysis, or an ELISA for measuring amounts of PCDH protein (e.g. PCDH8, PCDH9, PCDH17, etc.). For example, to use an immunohistochemistry assay, histological (e.g. paraffin-embedded) sections of tissue may be placed on slides, and then incubated with an antibody against the PCDH protein of interest (e.g. PCDH8, PCDH9, PCDH17, etc.). The slides then may be incubated with a second antibody (against the primary antibody), which is tagged to a dye or other colorimetric system (e.g. a fluorochrome, a radioactive agent, or an agent having high electron-scanning capacity), to permit visualization of the PCDH protein of interest (e.g. PCDH8, PCDH9, PCDH17, etc.) that is present in the sections.
It is contemplated that the diagnostic sample in the present invention frequently will be assayed for PCDH expression (e.g. PCDH8 expression, PCDH9 expression, PCDH17 expression, etc.) not by the subject or patient, nor by his/her consulting physician, but by a laboratory technician or other clinician. Accordingly, the method of the present invention further comprises providing to a subjects or patient's consulting physician a report of the results obtained upon assaying a diagnostic sample of the subject or patient for PCDH expression (e.g. PCDH8 expression, PCDH9 expression, PCDH17 expression, etc.).
Additionally, the present invention provides a method for determining whether a subject has neoplasia, by assaying a diagnostic sample of the subject for methylation of a protocadherin promoter (e.g. methylation of a CpG island within a PCDH promoter), wherein detection of methylation of the PCDH promoter is diagnostic of neoplasia in the subject. By way of example, the method for determining whether a subject has neoplasia may comprise assaying a diagnostic sample of the subject for methylation of the PCDH8 promoter and/or methylation of the PCDH9 promoter, wherein detection of methylation of the PCDH8 promoter and/or the PCDH9 promoter is diagnostic of neoplasia in the subject.
In terms of epigenetic changes, it is known that methylation at CpG sites on DNA may affect gene expression. For example, hypomethylation in the promoter regions of proto-oncogenes is seen in many cancers. Additionally, hypermethylation is seen in the promoter regions of tumor-suppressor genes, and is known to give rise to reduced expression. Methylation-sensitive enzymes (Southern blotting/PCR), methylation-specific PCR, immunohistochemical stains, bisulfite sequencing (e.g. combined with cloning), denaturing HPLC, MethyLight assay (real-time quantitative PCR), single nucleotide primer extension (SNuPE), and methylation analysis using restriction-enzyme digestion (combined bisulfite restriction assay, or COBRA), are among the many techniques used to detect methylation changes in DNA.
By way of example, a diagnostic sample of a subject may be assayed for methylation of the PCDH8 promoter and/or methylation of the PCDH9 promoter, in accordance with the method of the present invention, wherein detection of methylation of the PCDH8 promoter and/or the PCDH9 promoter is diagnostic of neoplasia in the subject. The present invention may be used to detect any neoplasia, including any of those described above. In a preferred embodiment, the neoplasia is breast cancer or prostate cancer. In accordance with the method of the present invention, a diagnostic sample may be assayed for methylation of a protocadherin promoter (e.g. the PCDH8 promoter and/or the PCDH9 promoter) in accordance with assays and detection methods readily determined from the known art and/or disclosed herein, including, without limitation, methylation-sensitive enzymes (Southern blotting/PCR), methylation-specific PCR, immunohistochemical stains, bisulfite sequencing (e.g. combined with cloning), denaturing HPLC, MethyLight assay, SNuPE, and COBRA, as described above.
The present invention further provides a method for assessing the efficacy of therapy to treat neoplasia in a subject or patient who has undergone or is undergoing treatment for neoplasia. The method of the present invention comprises assaying a diagnostic sample of the subject or patient for expression of at least one protocadherin (e.g. PCDH8, PCDH9, PCDH17, etc.). By way of example, a diagnostic sample of a subject may be assayed for expression of PCDH8, PCDH9, and/or PCDH17. In accordance with this method of the present invention, normal expression of PCDHg, PCDH9, and PCDH17 in the diagnostic sample is indicative of successful therapy to treat neoplasia. Contrastingly, PCDH8 expression decreased below normal in the diagnostic sample, and/or PCDH9 expression decreased below normal in the diagnostic sample, and/or PCDH7 expression elevated above normal in the diagnostic sample, is indicative of a need to continue therapy to treat neoplasia.
The method of the present invention may be used to assess the efficacy of therapy to treat any neoplasia, including breast cancer, prostate cancer, and all of those described above. The diagnostic sample may be a tissue or a bodily fluid or solid, as described above, and may be assayed for PCDH expression (e.g. PCDH8 expression, PCDH9 expression, PCDH17 expression, etc.) in vitro or in vivo. In addition, the diagnostic sample may be assayed for PCDH expression (e.g. PCDH8 expression, PCDH9 expression, PCDH17 expression, etc.) using all of the various assays and methods of detection and quantification described above. This method of the present invention provides a means for monitoring the effectiveness of therapy to treat neoplasia by permitting the periodic assessment of levels of PCDH expression (e.g. PCDH8 expression, PCDH9 expression, PCDH17 expression, etc.) in a diagnostic sample taken from a subject or patient.
In accordance with the method of the present invention, a diagnostic sample of a subject or patient may be assayed, and levels of PCDH expression (e.g. PCDH8 expression, PCDH9 expression, PCDH17 expression, etc.) may be assessed, at any time following the initiation of therapy to treat neoplasia. For example, levels of PCDH expression (e.g. PCDH8 expression, PCDH9 expression, PCDH17 expression, etc.) may be assessed while the subject or patient is still undergoing treatment for neoplasia.
Where levels of PCDH8 expression and/or PCDH9 expression detected in an assayed diagnostic sample of the subject or patient continue to remain decreased below normal, and/or where levels of PCDH17 expression detected in an assayed diagnostic sample of the subject or patient continue to remain elevated above normal, a physician may choose to continue with the subject's or patient's treatment for the neoplasia. Similarly, where levels of PCDH8 expression and/or PCDH9 expression in an assayed diagnostic sample of the subject or patient do not noticeably increase through successive assessments, and/or levels of PCDH17 expression in an assayed diagnostic sample of the subject or patient do not noticeably decrease through successive assessments, it may be an indication that the treatment for neoplasia is not working, and that treatment doses could be increased or otherwise altered.
On the other hand, where detected levels of PCDH8 expression ad/or PCDH9 expression in an assayed diagnostic sample of the subject or patient increase through successive assessments, and/or where detected levels of PCDH17 expression in an assayed diagnostic sample of the subject or patient decrease through successive assessments, it may be an indication that the treatment for neoplasia is working, and that treatment doses could be decreased or even ceased. Where PCDH8 expression and PCDH9 expression are no longer detected in an assayed diagnostic sample of a subject or patient at levels decreased below normal, and where PCDH17 expression is no longer detected in an assayed diagnostic sample of a subject or patient at levels elevated above normal (e.g. PCDH17 expression is absent from the diagnostic sample), a physician may conclude that the treatment for neoplasia has been successful, and that such treatment may cease.
It is within the confines of the present invention to assess levels of PCDH expression (e.g. PCDH8 expression, PCDH9 expression, PCDH17 expression, etc.) following completion of a subject's or patient's treatment for neoplasia, in order to determine whether the neoplasia has recurred in the subject or patient. Accordingly, an assessment of levels of PCDH expression (e.g. PCDH8 expression, PCDH9 expression, PCDH17 expression, etc.) in an assayed diagnostic sample may provide a convenient way to conduct follow-ups of patients who have been diagnosed with neoplasias. Furthermore, it is within the confines of the present invention to use assessed levels of PCDH expression (e.g. PCDH8 expression, PCDH9 expression, PCDH17 expression, etc.) in an assayed diagnostic sample as a clinical or pathologic staging tool, as a means for determining the extent of neoplasia in the subject or patient, and as a means for ascertaining appropriate treatment options.
The present invention also provides a method for assessing the efficacy of therapy to treat neoplasia in a subject who has undergone or is undergoing treatment for neoplasia, by assaying a diagnostic sample of the subject for methylation of the PCDH8 promoter and/or methylation of the PCDH9 promoter, wherein no detection of methylation of the PCDH8 promoter and/or the PCDH9 promoter in the diagnostic sample is indicative of successful therapy to treat neoplasia, and wherein detection of methylation of the PCDH8 promoter and/or the PCDH9 promoter in the diagnostic sample is indicative of a need to continue therapy to treat neoplasia. The neoplasia may be any of those described above, including breast cancer and prostate cancer. Suitable diagnostic samples, assays, and detection and quantification methods for use in the method of the present invention have already been described.
A correlation exists, in general, between tumor burden and the survival of a patient who has cancer. Therefore, it is also contemplated in the present invention that assaying a diagnostic sample of a subject for PCDH expression (e.g. PCDH8 expression, PCDH9 expression, PCDH17 expression, etc.) may be a useful means of providing information concerning the prognosis of a subject or patient who has neoplasia. Accordingly, the present invention further provides a method for assessing the prognosis of a subject who has neoplasia, by assaying a diagnostic sample of the subject for PCDH expression (e.g. PCDH8 expression, PCDH9 expression, PCDH17 expression, etc.). By way of example, the prognosis of a subject who has neoplasia may be assessed by assaying a diagnostic sample of the subject for expression of PCDH8, PCDH9, and/or PCDH17. In accordance with this method of the present invention, the subject's prognosis improves with an increase in expression of PCDH8 in the diagnostic sample and/or an increase in expression of PCDH9 in the diagnostic sample, the subject's prognosis improves with a decrease in expression of PCDH17 in the diagnostic sample, the subject's prognosis worsens with a decrease in expression of PCDH8 in the diagnostic sample and/or a decrease in expression of PCDH9 in the diagnostic sample, and the subject's prognosis worsens with an increase in expression of PCDH17 in the diagnostic sample. Suitable diagnostic samples, assays, and detection and quantification methods for use in the method of the present invention have already been described. This method of the present invention provides a means for determining the prognosis of a subject or patient diagnosed with neoplasia based upon the level of PCDH expression (e.g. PCDH8 expression, PCDH9 expression, PCDH17 expression, etc.) in an assayed diagnostic sample of the subject or patient.
According to the method of the present invention, a diagnostic sample of a subject or patient may be assayed, and levels of PCDH expression (e.g. PCDH8 expression, PCDH9 expression, PCDH17 expression, etc.) may be assessed, at any time during or following the diagnosis of neoplasia in the subject or patient. For example, levels of PCDH expression (e.g. PCDH8 expression, PCDH9 expression, PCDH17 expression, etc.) in an assayed diagnostic sample may be assessed before the subject or patient undergoes treatment for neoplasia, in order to determine the subject's or patient's initial prognosis. Additionally, levels of PCDH expression (e.g. PCDH8 expression, PCDH9 expression, PCDH17 expression, etc.) in an assayed diagnostic sample may be assessed while the subject or patient is undergoing treatment for neoplasia, in order to determine whether the subject's or patient's prognosis has become more or less favorable through the course of treatment.
By way of example, where levels of PCDH8 expression and/or PCDH9 expression detected in an assayed diagnostic sample of the subject or patient are, or continue to remain, significantly lower than normal, a physician may conclude that the subject's or patient's prognosis is unfavorable. Similarly, where levels of PCDH17 expression detected in an assayed diagnostic sample of the subject or patient are, or continue to remain, significantly higher than normal, a physician may conclude that the subject's or patient's prognosis is unfavorable. Where PCDH8 expression and/or PCDH9 expression in an assayed diagnostic sample of the subject or patient increases through successive assessments, and/or where PCDH17 expression in an assayed diagnostic sample of the subject or patient decreases through successive assessments, it may be an indication that the subject's or patient's prognosis is improving.
Where detected levels of PCDH8 expression and/or PCDH9 expression in an assayed diagnostic sample of the subject or patient do not increase significantly through successive assessments, it may be an indication that the subject's or patient's prognosis is not improving. Similarly, where detected levels of PCDH17 expression in an assayed diagnostic sample of the subject or patient do not decrease significantly through successive assessments, it may be an indication that the subject's or patient's prognosis is not improving. Where PCDH8 expression and/or PCDH9 expression is normal (e.g. detectable, high, etc.) in a diagnostic sample of the subject or patient, a physician may conclude that the subject's or patient's prognosis is favorable. Finally, where PCDH17 expression is normal (e.g. low, absent, etc.) in a diagnostic sample of the subject or patient, a physician may conclude that the subject's or patient's prognosis is favorable.
The present invention also provides a method for assessing the prognosis of a subject who has neoplasia, comprising assaying a diagnostic sample of the subject for methylation of the PCDH8 promoter and/or methylation of the PCDH9 promoter, wherein the subject's prognosis improves with a decrease in methylation of the PCDH8 promoter and/or the PCDH9 promoter in the diagnostic sample, and wherein the subject's prognosis worsens with an increase in methylation of the PCDH8 promoter and/or the PCDH9 promoter in the diagnostic sample. The neoplasia may be any of those described above, including breast cancer and prostate cancer.
The present invention further provides a method for determining whether a subject has neoplasia, by assaying a diagnostic sample of the subject for at least one mutation in a PCDH gene or protein (e.g. a PCDH8 gene mutation, a PCDH8 protein mutation, a PCDH17 gene mutation, a PCDH17 protein mutation, etc.). In one embodiment, the PCDH8 mutation and/or the PCDH17 mutation results in a loss of heterozygosity in chromosome 13. Exemplary PCDH8 mutations include, without limitation, a G436A (E146K) mutation (predicted to change aspartate46 to lysine), a G1028A (R343H) mutation (predicted to change arginine343 to histidine), a G748A (V250M) mutation (predicted to change valine250 to alanine), a C-IT mutation, a A1099G (T367A) mutation (predicted to change threonine367 to alanine) mutation, and a T2015A (L672Q) mutation. See also Table 4. Exemplary PCDH117 mutations include, without limitation, a deletion in exon 1, a C1364T (P222S) mutation, and a G2899T (E733D) mutation. See also Table 5.
The discovery that PCDHs exhibit differential expression in cells displaying neoplasias provides a means of identifying patients with neoplasias, and presents the potential for commercial application in the form of a test for the diagnosis of neoplasias. The development of such a test provides general screening procedures. Such procedures can assist in the early detection and diagnosis of cancers, and can provide a method for the follow-up of patients in whom there has been detection of PCDH8 expression and/or PCDH9 expression decreased below normal, and/or in whom there has been detection of PCDH17 expression elevated above normal.
Accordingly, the present invention further provides a kit for use as an assay of neoplasia, comprising: (a) at least one agent reactive with PCDH8, PCDH9, and/or PCDH17; and (b) reagents suitable for detecting expression of at least one protocadherin selected from the group consisting of PCDH8, PCDH9, and PCDH17. The agents may be any of those described above; in a preferred embodiment, the agent reactive with PCDH8, PCDH9, and/or PCDH17 is an anti-PCDH8, -PCDH9, and/or -PCDH17 antibody. The agents may be used in any of the above-described assays or methods for detecting or quantifying PCDH expression. Preferably, at least one agent of the present invention is labeled with a detectable marker or label.
Similarly, the present invention provides a kit for use as an assay to detect neoplasia, comprising: (a) at least one nucleic acid probe that hybridizes to nucleic acid encoding PCDH8, PCDH9, and/or PCDH17; and (b) reagents suitable for detecting expression of at least one nucleic acid encoding at least one protocadherin (e.g. PCDH8, PCDH9, PCDH17, etc.). Preferably, at least one nucleic acid of the present invention is labeled with a detectable marker or label.
Significant research efforts and resources have been directed toward the elucidation of anti-neoplastic measures, including chemotherapeutic agents, which are effective in treating patients suffering from neoplasia. Effective anti-neoplastic agents include those which inhibit or control the rapid proliferation of cells associated with neoplasms, those which effect regression or remission of neoplasms, and those which generally prolong the survival of patients suffering from neoplasia. Successful treatment of malignant neoplasia, or cancer, requires elimination of all malignant cells, whether they are found at the primary site, or have extended to local/regional areas, or have metastasized to other regions of the body.
Based upon data presented herein, it is believed that protocadherins play a role in the formation of neoplasms. Thus, it is expected that modulation of the levels of PCDHs in cells provides a means for treating and/or preventing cancer and other neoplasias. Accordingly, the present invention further provides a method for treating or preventing neoplasia in a subject, comprising modulating the level and/or function of at least one protocadherin (e.g. PCDH8, PCDH9, PCDH17, etc.) in the subject. Preferably, neoplasia is treated or prevented in a subject by modulating the level and/or function of PCDH8, PCDH9, and/or PCDH17 in the subject. For example, neoplasia may be treated or prevented in a subject by increasing the level and/or function of PCDH8 and/or PCDH9 in the subject, and/or by decreasing the level and/or function of PCDH 17 in the subject. The neoplasia may be any of those described above, but is preferably breast cancer or prostate cancer.
In general, the level and/or function of a PCDH in a subject may be modulated by administering to the subject a modulator of expression of the PCDH. As used herein, a “modulator of expression” may be any agent or combination of agents that has an antagonistic (inhibitory) or agonistic (facilitatory) effect on expression of a specified protein. Thus, a modulator of PCDH expression may be a PCDH agonist or a PCDH antagonist. The modulators of the present invention include any protein, polypeptide, peptide, nucleic acid (including DNA or RNA), antibody, Fab fragment, F(ab′)2 fragment, molecule, compound, antibiotic, or drug. Furthermore, the modulators of the present invention include any agent reactive with a PCDH of interest (e.g. PCDH8, PCDH9, etc.) that induces or upregulates expression of that PCDH, and any agent reactive with a PCDH of interest (e.g. PCDH17, etc.) that downregulates expression of that PCDH.
Modulators of PCDHs may be identified using a simple screening assay, including screening procedures well known in the art or disclosed herein. For example, to screen for candidate modulators of PCDHs, cells from a breast tumor line (e.g. MDA-MB-330, MDA-MB-436, MDA-MB-453, HCC-1395, DU4475, Hs578T, ZR75-30, UACC812, etc.) may be plated onto microtiter plates, then contacted with a library of drugs. Any resulting increase in (or upregulation of) PCDH expression, and/or any resulting decrease in (or downregulation of PCDH expression, then may be detected using nucleic acid hybridization and/or immunological techniques known in the art, including an ELISA. Modulators of PCDHs will include those agents/drugs that induce or upregulate expression of a PCDH, and those agents/drugs which decrease or downregulate expression of a PCDH. In this manner, candidate modulators also may be screened for their ability to inhibit proliferation of neoplasms, using PCDH expression as an indicator that cell division or growth of cells in a neoplasm is decreasing in rate, or has stopped.
It is within the confines of the present invention that the modulator of PCDH expression may be linked to another agent, or administered in combination with another agent (such as an anti-neoplastic drug or a ribozyme), in order to increase the effectiveness of the treatment of neoplasia and/or increase the efficacy of targeting. Examples of anti-neoplastic drug to which the modulator of PCDH expression may be linked include, without limitation, carboplatin, cyclophosphamide, doxorubicin, etoposide, and vincristine.
In accordance with the method of the present invention, neoplasia also may be treated or prevented in a subject by increasing the level and/or function of a PCDH (e.g. PCDH8, PCDH9, etc.) in the subject. Preferably, the level and/or function of a PCDH of interest (e.g. PCDH8, PCDH9, etc.) in the subject is increased by at least 10% in the method of the present invention. More preferably, the level and/or function of a PCDH of interest (e.g. PCDH8, PCDH9, etc.) is increased by at least 20%.
By way of example, the level and/or function of a PCDH (e.g. PCDH8, PCDH9, etc.) in a subject may be increased by directly or indirectly increasing levels of the PCDH in vivo within the subject. For example, the level of a PCDH (e.g. PCDH8, PCDH9, etc.) in a subject may be increased by administering the PCDH protein to the subject, in an amount effective to treat neoplasia in the subject. Similarly, the level of a PCDH (e.g. PCDH8, PCDH9, etc.) in a subject may be increased by administering to the subject a nucleic acid sequence encoding the PCDH, in a manner permitting expression of the PCDH protein in the subject, and in an amount effective to treat the neoplasia.
Additionally, the level and/or function of a PCDH of interest (e.g. PCDH8, PCDH9, etc.) in a subject also may be increased by directly or indirectly causing, inducing, or stimulating the upregulation of expression of the PCDH within a subject. In the method of the present invention, a PCDH (e.g. PCDH8, PCDH9, etc.) in a subject may be unregulated, for example, by administering to the subject a modulator of PCDH expression, such as a small molecule or protein mimetic. Accordingly, in one embodiment of the present invention, the level and/or function of a PCDH of interest (e.g. PCDH8, PCDH9, etc.) is increased in a subject by administering to the subject a modulator of PCDH expression (e.g. a modulator of PCDH8 expression, a modulator of PCDH9 expression, etc.), in an amount effective to treat the neoplasia in the subject.
In accordance with the method of the present invention, the level and/or function of a PCDH of interest (e.g. PCDH8, PCDH9, etc.) in a subject also may be increased by targeting the PCDH directly or indirectly. The level and/or function of the PCDH may be increased indirectly by targeting an enzyme or other endogenous molecule that regulates or modulates the function of the PCDH in the subject. By way of example, the level and/or function of a PCDH of interest (e.g. PCDH8, PCDH9, etc.) in a subject may be increased by directly or indirectly activating, facilitating, or stimulating one or more functions of the PCDH in the subject (e.g. by the modulation or regulation of enzymes, other proteins, lipids, etc., that interact with the PCDH). The term “activating”, as used herein, means stimulating or inducing one or more functions of the PCDH in the subject, particularly those functions associated with the suppression or inhibition of neoplasias, as disclosed herein. In the method of the present invention, a PCDH (e.g. PCDH8, PCDH9, etc.) in a subject may be activated, for example, by administering to the subject a small molecule or protein mimetic that stimulates the PCDH (e.g. PCDH8, PCDH9, etc.) or that is reactive with the PCDH (e.g. PCDH8, PCDH9, etc.), in an amount effective to treat the neoplasia in the subject.
Activators of PCDH function may be identified using a functional screening assay, including any well known in the art and/or disclosed herein. For example, to screen for candidate activators of PCDH function, cells known to exhibit at least one PCDH function (e.g. function of PCDH8, PCDH9, etc.) may be plated onto microtiter plates, then contacted with a library of candidate agents/drugs. Any resulting increase in PCDH function (e.g. synaptic function, contact-inhibition of cell proliferation, suppression of tumor formation, control of gene expression, etc.) then may be detected using techniques known in the art. Activators of PCDH function will include those agents/drugs which increase at least one function of a PCDH. The function of a PCDH of interest may be measured using standard techniques known to one of skill in the art.
Once the candidate agent of the present invention has been screened, and has been determined to have a suitable activating effect on the PCDH of interest (e.g. PCDH8, PCDH9, etc.), it may be evaluated for its effect on tumor-cell proliferation. In particular, the candidate agent may be assessed for its ability to act as an inhibitor to cell division or to otherwise function as an appropriate tumor-suppressing agent. It is expected that the PCDH activator of the present invention will be useful to treat neoplasms, including those disclosed herein. Preferably, the PCDH activator of the present invention is a PCDH8 activator and/or a PCDH9 activator.
A PCDH protein (e.g. PCDH8, PCDH9, etc.), a PCDH nucleic acid, a modulator of PCDH expression, and an activator of PCDH function may be administered to a subject who has neoplasia, either alone or in combination with one or more anti-neoplastic drugs used to treat neoplasias. Examples of anti-neoplastic drugs with which the PCDH protein may be combined have already been described.
Furthermore, the level and/or function of a PCD1:I of interest (e.g. PCDH8, PCDH9, etc.) may be increased in a subject by altering chromatin silencing or PCDH methylation in the subject, or by otherwise reversing gene silencing of the PCDH. Chromatin is the natural substrate for the control of gene expression; it contains DNA, transcriptional machinery, and structural proteins (such as histories). It has been shown that transcriptional activity of a gene is largely controlled by packaging of the template within chromatin (Jones and Wolffe, Relationships between chromatin organization and DNA methylation in determining gene expression. Semin. Cancer Biol., 9(5):339-47, 1999). Histone acetylation and DNA methylation alter the nucleosomal infrastructure, thereby repressing or activating transcription. These covalent modifications have causal roles in promoter-specific events, and in the global control of chromosomal activity. Histone acetylation and DNA methylation also have a major impact in both oncogenic transformation and normal development. See, e.g. Jones and Wolffe, Relationships between chromatic organization and DNA methylation in determining gene expression. Semin. Cancer Biol., 9(5):339-47, 1999.
It is also known that two epigenetic processes—densely-methylated DNA in association with transcriptionally-repressive chromatin characterized by the presence of underacetylated histones—are dynamically linked (Cameron et al., Synergy of demethylation and histone deacetylase inhibition in the re-expression of genes silenced in cancer. Nat. Genet., 21:103-07, 1999). MeCP2, a methyl-CpG-binding protein, appears to reside in a complex with histone deacetylase activity. MeCP2 can mediate formation of transcriptionally-repressive chromatin on methylated promoter templates in vitro, and this process can be reversed by trichostatin A (TSA), a specific inhibitor of histone deacetylase. It has also been shown that, following minimal demethylation and slight gene reactivation in the presence of low-dose 5-aza-2′deoxycytidine (5Aza-dC), TSA treatment results in robust re-expression and transcriptional reactivation of hypermethylated genes MLH1, TIMP3 (TIMP3), CDKN2B (INK4B, p15), and CDKN2A (INK4, p16). Accordingly, in one embodiment of the present invention, activity of PCDH8 or PCDH9 is increased in the subject by administering 5-aza-deoxycytidine (e.g. 5-aza-2′deoxycytidine) and/or trichostatin A to the subject.
In accordance with the method of the present invention, neoplasia also may be treated or prevented in a subject by decreasing the level and/or function of a PCDH (e.g. PCDH17, etc.) in the subject. Preferably, the level and/or function of a PCDH of interest (e.g. PCDH17, etc.) in the subject is decreased by at least 10% in the method of the present invention. More preferably, the level and/or function of a PCDH of interest (e.g. PCDH17, etc.) is decreased by at least 20%.
By way of example, the level and/or function of a PCDH (e.g. PCDH17, etc.) in a subject may be decreased by directly or indirectly decreasing levels of the PCDH in vivo within the subject. A decrease in the level of the PCDH in the subject may be achieved, for example, by directly or indirectly causing, inducing, or stimulating the downregulation of expression of the PCDH within a subject. Accordingly, in one embodiment of the present invention, the level and/or function of a PCDH of interest (e.g. PCDH17, etc) is decreased in a subject by administering to the subject a modulator of PCDH expression (e.g. a modulator of PCDH17 expression, etc.), such as a small molecule or protein mimetic, in an amount effective to treat the neoplasia in the subject.
In accordance with the method of the present invention, the level and/or function of a PCDH of interest (e.g. PCDH17, etc.) in a subject also may be decreased by targeting the PCDH directly or indirectly. The level and/or function of the PCDH may be decreased indirectly by targeting an enzyme or other endogenous molecule that regulates or modulates the function of the PCDH in the subject.
By way of example, the level and/or function of a PCDH of interest (e.g. PCDH17, etc.) in a subject may be decreased by directly or indirectly blocking, deactivating, inhibiting, or preventing one or more functions of the PCDH in the subject (e.g. by the modulation or regulation of proteins that interact with the PCDH), or by diminishing the amount of the PCDH in the subject. The term “inhibiting”, as used herein, means disabling, disrupting, or inactivating one or more functions of the PCDH in the subject, particularly those functions associated with the formation and growth of neoplasias, as disclosed herein.
In the method of the present invention, a PCDH (e.g. PCDH17, etc.) in a subject may be inhibited, for example, by administering to the subject a small molecule or protein mimetic that inhibits the PCDH (e.g. PCDH17, etc.) and/or that is reactive with the PCDH (e.g. PCDH17, etc.), in an amount effective to treat the neoplasia in the subject. By way of example, a PCDH of interest (e.g. PCDH17, etc.) may be inhibited in a subject who has neoplasia by administering a PCDH inhibitor to the subject. As used herein, “a PCDH inhibitor” shall include a protein, polypeptide, peptide, nucleic acid (including DNA, RNA, and an antisense oligonucleotide), antibody (monoclonal and polyclonal, as described above), Fab fragment (as described above), F(ab′)2 fragment (as described above), molecule, compound, antibiotic, drug, or any combination thereof. PCDH inhibitors (e.g. inhibitors of PCDH17) provide novel and valuable tools for treating and preventing neoplasia. For example, since PCDH17 is generally not expressed in non-diseased subjects, inhibition of PCDH17 should have a therapeutic effect without resulting in the harmful or deleterious side-effects that frequently accompany therapy using other anti-neoplastic drugs or radiation. Exemplary PCDH inhibitors include, without limitation, oligonucleotides antisense to the PCDH of interest (e.g. PCDH17, etc.), RNAi directed against the PCDH of interest (e.g. PCDH17, ctc.), agents reactive with the PCDH of interest (e.g. PCDH17, etc.), a dominant-negative form of the PCDH of interest (e.g. PCDH17, etc.), and a constitutively-active form of the PCDH of interest (e.g. PCDH17, etc.).
It is well understood in the art that a gene may be silenced at a number of stages, including, without limitation, pre-transcription silencing, transcription silencing, translation silencing, post-transcription silencing, and post-translation silencing. Accordingly, in one embodiment of the present invention, the level and/or function of a PCDH of interest (e.g. PCDH17, etc.) is decreased in the subject by gene-silencing the PCDH using antisense technology or interference RNA (RNAi).
RNA interference (RNAi) is an RNA-mediated, sequence-specific gene-silencing mechanism. RNAi, a double-stranded (ds) interference RNA, was discovered by Guo and Kemphues in 1995, when they reported that both the sense and antisense strands of test oligonucleotides disrupted the expression of par-1 in Caenorhabditis elegans, following injection into a cell (Guo et al., Par-1, A gene required for establishing polarity in C. elegans embryos, encodes a putative Ser/Thr kinase that is asymmetrically distributed. Cell, 81:61 1-20, 1995). In 1998, Fire et al. clearly proved the existence and efficacy of RNAi by injecting into the gut of C. elegans a dsRNA that had been prepared in vitro (Fire et al., Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans, Nature, 391:806-11, 1998). The injection of dsRNA into C. elegans resulted in loss of expression of the homologous target gene, not only throughout the worm, but also in its progeny. It is now well accepted that the phenomenon of RNAi is ubiquitous among bacteria, fungi, plants, and animals.
As used herein, “RNAi” refers to a double-stranded RNA (dsRNA) duplex of any length, with or without single-strand overhangs, wherein at least one strand, putatively the antisense strand, is homologous to the target mRNA to be degraded. As further used herein, a “double-stranded RNA” molecule includes any RNA molecule, fragment, or segment containing two strands forming an RNA duplex, notwithstanding the presence of single-stranded overhangs of unpaired nucleotides. Additionally, as used herein, a double-stranded RNA molecule includes single-stranded RNA molecules forming functional stem-loop structures, such that they thereby form the structural equivalent of an RNA duplex with single-strand overhangs. The double-stranded RNA molecule of the present invention may be very large, comprising thousands of nucleotides; preferably, however, it is small, in the range of 21-25 nucleotides. In a preferred embodiment, the RNAi of the present invention comprises a double-stranded RNA duplex of at least 19 nucleotides.
The RNAi of the present invention may be included within a plasmid, although it is to be understood that other types of nucleic acids, such as viral vectors, may also be used for the purposes of the present invention. The term “plasmid”, as used herein, refers generally to circular double-stranded DNA that is not bound to a chromosome. The DNA may be a chromosomal or episomal-derived plasmid. The plasmid of the present invention may optionally contain a terminator of transcription; a promoter, and/or a discrete series of restriction-endonuclease recognition sites, located between the promoter and the terminator. In a preferred embodiment, a polynucleotide insert of interest (e.g. one encoding the RNAi) should be operatively linked to an appropriate promoter, such as its native promoter or a host-derived promoter, such as a tissue-specific promoter or an inducible promoter. Other suitable promoters will be known to the skilled artisan.
In one embodiment of the present invention, RNAi is produced in vivo by an expression vector containing a gene-silencing cassette coding for RNAi or any other a post-transcription gene-silencing composition. See, e.g. U.S. Pat. No. 6,278,039, C. elegans deletion mutants; U.S. Patent Application No. 2002/0006664, Arrayed transfection method and uses related thereto; WO 99/326197 Genetic inhibition by double-stranded RNA; WO 01/29058, RNA interference pathway genes as tools for targeted genetic interference; WO 01/68836, Methods and compositions for RNA interference; and WO 01/96584, Materials and methods for the control of nematodes. In another embodiment of the present invention, RNAi is produced in vitro, synthetically or recombinantly, and transferred into the subject using standard molecular-biology techniques. Methods of making and transferring RNAi are well known in the art: see, e.g. Ashrafi et al., Genome-wide RNAi analysis of Caenorhabditis elegans fat regulatory genes. Nature, 421:268-72, 2003; Cottrell et al., Silence of the strands: RNA interference in eukaryotic pathogens. Trends Microbiol., 11:37-43, 2003; Nikolaev et al., Parc: A cytoplasmic anchor for p53. Cell, 112:29-40, 2003, Wilda et al., Killing of leukemic cells with a BCR/ABL fusion gene RNA interference (RNAi), Oncogene, 21:5716-24, 2002; Escobar et al., RNAi-mediated oncogene silencing confers resistance to crown gall tumorigenesis. Proc. Natl. Acad. Sci. USA, 98:13437-42, 2001; and Billy et al., Specific interference with gene expression induced by long, double-stranded RNA in mouse embryonal teratocarcinoma cell lines. Proc. Natl. Acad. Sci. USA, 98:14428-33, 2001.
A PCDH of interest (e.g. PCDH17, etc.) also may be silenced with antisense technology. An antisense oligonucleotide (e.g. DNA, RNA, etc.) is a molecule with a sequence complementary to a specific RNA transcript, or mRNA; upon binding, the antisense oligonucleotide prevents further processing of the transcript or translation of the mRNA. In accordance with this embodiment of the present invention, the PCDH inhibitor is an oligonucleotide antisense to the PCDH of interest (e.g. PCDH17, etc.).
Oligonucleotides antisense to a PCDH of interest (e.g. PCDH17, etc.) may be designed based on the nucleotide sequence of the PCDH. For example, a partial sequence of the PCDH nucleotide sequence (generally, 18-20 base pairs), or a variation sequence thereof, may be selected for the design of an antisense oligonucleotide. This portion of the PCDH nucleotide sequence may be within the 5′ domain. A nucleotide sequence complementary to the selected partial sequence of the PCDH gene, or the selected variation sequence, then may be chemically synthesized using one of a variety of techniques known to those skilled in the art, including, without limitation, automated synthesis of oligonucleotides having sequences which correspond to a partial sequence of the PCDH nucleotide sequence, or a variation sequence thereof, using commercially-available oligonucleotide synthesizers, such as the Applied Biosystems Model 392 DNA/RNA synthesizer.
By way of example, antisense molecules may be generated, synthetically or recombinantly, with a nucleic-acid vector expressing an antisense gene-silencing cassette. Such antisense molecules may be single-stranded RNAs or DNAs, with lengths as short as 15-20 bases or as long as a sequence complementary to the entire mRNA. RNA molecules are sensitive to nucleases, and have half-lives of 15-30 min in serum. To afford protection against nuclease digestion, an antisense deoxyoligonucleotide may be synthesized as a phosphorothioate, in which one of the nonbridging oxygens surrounding the phosphate group of the deoxynucleotide is replaced with a sulfur atom (Stein et al., Oligodeoxynucleotides as inhibitors of gene expression: a review. Cancer Res., 48:2659-68, 1998). Antisense molecules designed to bind to the entire mRNA may be made by inserting cDNA into an expression plasmid in the opposite or antisense orientation. Antisense molecules may also function by preventing translation initiation factors from binding near the 5′ cap site of the mRNA, or by interfering with interaction of the mRNA and ribosomes. See, e.g. U.S. Pat. No. 6,448,080, Antisense modulation of WRN expression; U.S. Patent Application No. 2003/0018993, Methods of gene silencing using inverted repeat sequences; U.S. Patent Application No., 2003/0017549, Methods and compositions for expressing polynucleotides specifically in smooth muscle cells in vivo; Tavian et al., Stable expression of antisense urokinase mRNA inhibits the proliferation and invasion of human hepatocellular carcinoma cells. Cancer Gene Ther., 10:112-20, 2003; Maxwell and Rivera, Proline oxidase induces apoptosis in tumor cells and its expression is absent or reduced in renal carcinoma. J: Biol. Chem., 278(11):9784-89, 2003; Ghosh et al., Role of superoxide dismutase in survival of Leishmania within the macrophage. Biochem. J., 369:447-52, 2003; and Zhang et al., An anti-sense construct of full-length ATM cDNA imposes a radiosensitive phenotype on normal cells. Oncogene, 17:811-18, 1998.
Once the desired antisense oligonucleotide has been prepared, its ability to inhibit the PCDH of interest (e.g. PCDH17, etc.) then may be assayed. For example, the oligonucleotide antisense to the PCDH may be contacted with tumor cells derived from a breast tumor cell line, and the levels of PCDH expression (e.g. PCDH17 expression, etc.) in the cells may be determined using standard techniques, such as Western-blot analysis. Alternatively, the antisense oligonucleotide may be delivered to tumor cells derived from a breast tumor cell line using a liposome vehicle, and the levels of PCDH expression (e.g. PCDH17 expression, etc.) in the cells may then be determined using standard techniques, such as Western-blot analysis. Where the level of PCDH expression (e.g. PCDH17 expression, etc.) in tumor cells is reduced in the presence of the designed antisense oligonucleotide, it may be concluded that the oligonucleotide could be a useful PCDH inhibitor.
It is within the confines of the present invention that an oligonucleotide antisense to a PCDH of interest (e.g. PCDH17, etc.), or RNAi directed against the PCDH of interest, may be linked to another agent, such as an anti-neoplastic drug or a ribozyme, in order to increase the effectiveness of the treatment, increase the efficacy of targeting, and/or increase the efficacy of degradation of PCDH RNA. Examples of anti-neoplastic drugs to which the antisense or R:NAi oligonucleotide may be linked include, without limitation, carboplatin, cyclophosphamide, doxorubicin, etoposide, and vincristine. Moreover, oligonucleotide antisense to the PCDH of interest (e.g. PCDH17, etc.) may be prepared using modified bases (e.g. a phosphorothioate) to make the oligonucleotide more stable and better able to withstand degradation.
In another embodiment of the present invention, a PCDH of interest (e.g. PCDH17, etc.) in a subject is inhibited by administering to the subject a small molecule or protein mimetic that is reactive with the PCDH. In accordance with this embodiment, the PCDH inhibitor would be an agent reactive with the PCDH of interest (e.g. PCDH17, etc.). Examples of such reactive agents have already been provided. In one preferred embodiment, the agent is an antibody that binds to the PCDH of interest (e.g. PCDH17, etc.). Methods for making anti-PCDH antibodies have been described above.
Additional PCDH inhibitors may be identified using screening procedures well known in the art. For example, to screen for candidate inhibitors of PCDH function, cells known to exhibit at least one PCDH function (e.g. function of PCDH17, etc.) may be plated onto microtiter plates, then contacted with a library of candidate agents/drugs. Any resulting decrease in PCDH function (e.g. synaptic function, contact-inhibition of cell proliferation, suppression of tumor formation, control of gene expression, etc.) then may be detected using techniques known in the art. Inhibitors of PCDH function will include those agents/drugs which decrease at least one function of a PCDH. The function of a PCDH of interest may be measured using standard techniques known to one of skill in the art.
Once the candidate agent of the present invention has been screened, and has been determined to have a suitable inhibitory effect on the PCDH of interest (e.g. PCDH17, etc.)—i.e., it is reactive with the PCDH, it binds the PCDH, or it otherwise inactivates the function of the PCDH)—it may be evaluated for its effect on tumor-cell proliferation. In particular, the candidate agent may be assessed for its ability to act as an inhibitor to cell division or to otherwise function as an appropriate tumor-suppressing agent. It is expected that the PCDH inhibitor of the present invention will be useful to treat neoplasms, including those disclosed herein. Preferably, the PCDH inhibitor of the present invention is a PCDH17 inhibitor.
It is within the confines of the present invention that the agent reactive with a PCDH of interest (e.g. PCDH17, etc.), or any other PCDH inhibitor, may be co-administered to a subject along with an additional therapeutic agent. By way of example, anti-neoplastic drugs with which the reactive agent or other PCDH inhibitor may be co-administered include, without limitation, carboplatin, cyclophosphamide, doxorubicin, etoposide, and vincristine. The reactive agent or other PCDH inhibitor also may be administered to a subject in combination with a fusion protein, such as a VEGF-Trap.
Vascular endothelial growth factor (VEGF) is a critical promoter of blood-vessel growth during embryonic development and tumorigenesis. The VEGF-Trap is a high-affinity, soluble decoy receptor for VEGF. To date, studies of VEGF antagonists have primarily focused on halting progression in models of minimal-residual cancer. Consistent with this focus, recent clinical trials suggest that blockade of VEGF may impede cancer progression, presumably by preventing neoangiogenesis. However, VEGF is also a key mediator of endothelial-vascular mural-cell interactions, a role that may contribute to the integrity of mature vessels in advanced tumors. The VEGF-Trap can achieve high-affinity blockade of VEGF, and abolish mature, preexisting vasculature, in established xenografts, such eradication of vasculature is followed by marked tumor regression. Thus, the contribution of relatively low levels of VEGF to vessel integrity may be critical to maintenance of even very small tumor masses (Huang et al., Regression of established tumors and metastases by potent vascular endothelial growth factor blockade. Proc. Natl. Acad. Sci. USA, 100(13):7785-90, 2003). See also Saishin et al., VEGF-TRAP(R1R2) suppresses choroidal neovascularization and VEGF-induced breakdown of the blood-retinal barrier. Cell Physiol., 195(2):241-48, 2003. Exemplary VEGF-Traps include, without limitation, VEGF-TRAP(R1R2)—a composite decoy receptor that combines ligand binding elements, taken from the extracellular domains of VEGF receptors 1 and 2, fused to the Fc portion of IgG1—and vascular endothelial growth factor Trap(A40).
The present invention contemplates the use of proteins and protein analogues generated by synthesis of polypeptides in vitro (e.g. by chemical means or by in vitro translation of mRNA). For example, PCDHs (e.g. PCDH8, PCDH9, etc.), PCDH inhibitors (e.g. inhibitors of PCDH17, etc.), and other peptides for use in the methods of the present invention may be synthesized by methods commonly known to one skilled in the art (Modern Techniques of Peptide and Amino Acid Analysis (New York: John Wiley & Sons, 1981; Bodansky, M., Principles of Peptide Synthesis (New York: Springer-Verlag New York, Inc., 1984). Examples of methods that may be employed in the synthesis of the amino acid sequences, and analogues of these sequences, include, but are not limited to, solid-phase peptide synthesis, solution-method peptide synthesis, and synthesis using any of the commercially-available peptide synthesizers. Amino acid sequences for use in the present invention may contain coupling agents and protecting groups, which are used in the synthesis of protein sequences, and which are well known to one of skill in the art.
In the method of the present invention, a PCDH protein, a nucleic acid sequence encoding a PCDH, a modulator of PCDH expression, an activator of PCDH function, an agent reactive with a PCDH, a PCDH inhibitor, or any other therapeutic agent for use in treating neoplasia as disclosed herein (referred to herein as any “anti-neoplastic therapeutic agent”), is administered to a subject who has neoplasia in an amount effective to treat the neoplasia in the subject. As used herein, the phrase “effective to treat the neoplasia” means effective to ameliorate or minimize the clinical impairment or symptoms resulting from the neoplasia. For example, the clinical impairment or symptoms of the neoplasia may be ameliorated or minimized by diminishing any pain or discomfort suffered by the subject; by extending the survival of the subject beyond that which would otherwise be expected in the absence of such treatment; by inhibiting or preventing the development or spread of the neoplasia, or by limiting, suspending, terminating, or otherwise controlling the maturation and proliferation of cells in the neoplasm. The amount of the anti-neoplastic therapeutic agent of choice that is effective to treat neoplasia in a subject will vary depending on the particular factors of each case, including the type of neoplasia, the stage of neoplasia, the subject's weight, the severity of the subject's condition, and the method of administration. These amounts can be readily determined by the skilled artisan.
In accordance with the method of the present invention, the anti-neoplastic therapeutic agents disclosed herein (e.g. modulators of PCDH expression, PCDH proteins, nucleic acid sequences encoding PCDHs, agents reactive with PCDHs, PCDH inhibitors, etc.) may be administered to a human or animal subject by known procedures, including, without limitation, oral administration, parenteral administration (e.g. epifascial, intracapsular, intracutaneous, intradermal, intramuscular, intraorbital, intraperitoneal, intraspinal, intrasternal, intravascular, intravenous, parenchymatous, or subcutaneous administration), transdermal administration, and administration by osmotic pump. One preferred method of administration is parenteral administration, by intravenous or subcutaneous injection.
For oral administration, the formulation of the anti-neoplastic therapeutic agent of choice may be presented as capsules, tablets, powders, granules, or as a suspension. The formulation may have conventional additives, such as lactose, mannitol, cornstarch, or potato starch. The formulation also may be presented with binders, such as crystalline cellulose, cellulose derivatives, acacia, cornstarch, or gelatins. Additionally, the formulation may be presented with disintegrators, such as cornstarch, potato starch, or sodium carboxymethylcellulose. The formulation also may be presented with dibasic calcium phosphate anhydrous or sodium starch glycolate. Finally, the formulation may be presented with lubricants, such as talc or magnesium stearate.
For parenteral administration, the anti-neoplastic therapeutic agent of choice may be combined with a sterile aqueous solution, which is preferably isotonic with the blood of the subject. Such a formulation may be prepared by dissolving a solid active ingredient in water containing physiologically-compatible substances, such as sodium chloride, glycine, and the like, and having a buffered pH compatible with physiological conditions, so as to produce an aqueous solution, then rendering said solution sterile. The formulation may be presented in unit or multi-dose containers, such as sealed ampules or vials. The formulation also may be delivered by any mode of injection, including any of those described above.
For transdermal administration, the anti-neoplastic therapeutic agent of choice may be combined with skin penetration enhancers, such as propylene glycol, polyethylene glycol, isopropanol, ethanol, oleic acid, N-methylpyrrolidone, and the like, which increase the permeability of the skin to the anti-neoplastic therapeutic agent, and permit the anti-neoplastic therapeutic agent to penetrate through the skin and into the bloodstream. The composition of enhancer and anti-neoplastic therapeutic agent also may be further combined with a polymeric substance, such as ethylcellulose, hydroxypropyl cellulose, ethylene/vinylacetate, polyvinyl pyrrolidone, and the like, to provide the composition in gel form, which may be dissolved in solvent, such as methylene chloride, evaporated to the desired viscosity, and then applied to backing material to provide a patch. The anti-neoplastic therapeutic agent may be administered transdermally, at or near the site on the subject where the neoplasm is localized. Alternatively, the anti-neoplastic therapeutic agent may be administered transdermally at a site other than the affected area, in order to achieve systemic administration.
The anti-neoplastic therapeutic agent of the present invention also may be released or delivered from an osmotic mini-pump or other time-release device. The release rate from an elementary osmotic mini-pump may be modulated with a microporous, fast-response gel disposed in the release orifice. An osmotic mini-pump would be useful for controlling release, or targeting delivery, of the anti-neoplastic therapeutic agent of choice.
Where the anti-neoplastic therapeutic agent of choice is a protein, the protein may be administered to a subject by introducing to the subject the protein itself, or by introducing to the subject a nucleic acid encoding the protein, in a manner permitting expression of the protein. Accordingly, in one embodiment of the present invention, the level and/or function of at least one PCDH (e.g. PCDH8, PCDH9, PCDH17, etc.) in a subject may be modulated, and neoplasia in the subject may be treated, by administering to the subject an amount of the anti-neoplastic therapeutic protein effective to treat neoplasia in the subject. This amount may be readily determined by the skilled artisan. In a further embodiment of the present invention, the level and/or function of at least one PCDH (e.g. PCDH8, PCDH9, PCDH17, etc.) in a subject may be modulated, and neoplasia in the subject may be treated, by administering to the subject a nucleic acid sequence encoding the anti-neoplastic therapeutic protein, in a manner permitting expression of the protein in the subject. The nucleic acid sequence encoding the anti-neoplastic therapeutic protein is administered to the subject in an amount effective to treat neoplasia in the subject. This amount may be readily determined by the skilled artisan.
Anti-neoplastic therapeutic proteins for use in the present invention may be administered or introduced to a subject by known techniques used for the introduction of proteins and other drugs, including, for example, injection and transfusion. Where a neoplasm is localized to a particular portion of the body of the subject, it may be desirable to introduce the therapeutic protein directly to that area by injection or by some other means (e.g. by introducing the protein into the blood or another body fluid).
Anti-neoplastic therapeutic nucleic acids for use in the present invention may be introduced to a subject using conventional procedures known in the art, including, without limitation, electroporation, DEAL dextran transfection, calcium phosphate transfection, lipofection, monocationic liposome fusion, polycationic liposome fusion, protoplast fusion, creation of an in vivo electrical field, DNA-coated microprojectile bombardment, injection with recombinant replication-defective viruses, homologous recombination, in vivo gene therapy, ex vivo gene therapy, viral vectors, naked DNA transfer, or any combination thereof. Recombinant viral vectors suitable for gene therapy include, but are not limited to, vectors derived from the genomes of such viruses as retrovirus, HSV, adenovirus, adeno-associated virus, Semiliki Forest virus, cytomegalovirus, and vaccinia virus. Additionally, it is within the confines of the present invention that a nucleic acid encoding an anti-neoplastic therapeutic protein may be introduced into suitable cells in vitro, using conventional procedures, to achieve expression of the therapeutic protein in the cells. Cells expressing the therapeutic protein then may be introduced into a subject to treat neoplasia in vivo. In such an ex vivo gene therapy approach, the cells are preferably removed from the subject, subjected to DNA techniques to incorporate nucleic acid encoding the therapeutic protein, and then reintroduced into the subject.
It is also within the confines of the present invention that a formulation containing an anti-neoplastic therapeutic agent for use in treating neoplasia (e.g. a modulator of PCDH expression, a PCDH protein, a nucleic acid sequence encoding a PCDH, an agent reactive with a PCDH, a PCDH inhibitor, etc.) may be further associated with a pharmaceutically-acceptable carrier, thereby comprising a pharmaceutical composition. Accordingly, the present invention further provides a pharmaceutical composition, comprising an anti-neoplastic therapeutic agent for use in treating neoplasia, as disclosed herein (e.g. a modulator of PCDH expression, a PCDH protein, a nucleic acid sequence encoding a PCDH, an agent reactive with a PCDH, a PCDH inhibitor, etc.), and a pharmaceutically-acceptable carrier. The pharmaceutically-acceptable carrier must be “acceptable” in the sense of being compatible with the other ingredients of the composition, and not deleterious to the recipient thereof. Examples of acceptable pharmaceutical carriers include carboxymethyl cellulose, crystalline cellulose, glycerin, gum arable, lactose, magnesium stearate, methyl cellulose, powders, saline, sodium alginate, sucrose, starch, talc, and water, among others. Formulations of the pharmaceutical composition may be conveniently presented in unit dosage.
The pharmaceutical formulations of the present invention may be prepared by methods well-known in the pharmaceutical arts. For example, the anti-neoplastic therapeutic agent of choice may be brought into association with a carrier or diluent, as a suspension or solution. Optionally, one or more accessory ingredients (e.g. buffers, flavoring agents, surface active agents, and the like) also may be added. The choice of carrier will depend upon the route of administration. The pharmaceutical composition would be useful for administering the anti-neoplastic therapeutic agent of the present invention to a subject to treat neoplasia. The anti-neoplastic therapeutic agent would be provided in an amount that is effective to treat neoplasia in a subject to whom the pharmaceutical composition is administered. That amount may be readily determined by the skilled artisan, as described above.
Additionally, the present invention provides methods for identifying agents for use in treating and/or preventing neoplasia. In one embodiment of the invention, the method is performed in vitro. By way of example, the method may comprise the steps of: (a) obtaining a collection of cells having aberrant PCDH expression (e.g. having at least one of PCDH8 expression decreased below normal, PCDH9 expression decreased below normal, and PCDH17 expression elevated above normal); (b) contacting a candidate agent with the cells; and (c) determining the effect, if any, of the candidate agent on the aberrant PCDH expression (e.g. on PCDH8 expression, PCDH9 expression, and/or PCDH17 expression) in the cells. The method may further comprise the steps of: (d) contacting the candidate agent with neoplastic cells having aberrant PCDH expression (e.g. having at least one of PCDH8 expression decreased below normal, PCDH9 expression decreased below normal, and PCDH17 expression elevated above normal); and (e) determining if the agent has an effect on proliferation of the neoplastic cells. In particular, the candidate agent may be assessed for its ability to act as an inhibitor to cell division or to otherwise function as an appropriate tumor-suppressing agent.
The present invention also provides an in vivo method of identifying agents for use in treating and/or preventing neoplasia. By way of example, the method may comprise the steps of: (a) obtaining an animal comprising cells having aberrant PCDH expression (e.g. having at least one of PCDH8 expression decreased below normal, PCDH9 expression decreased below normal, and PCDH17 expression elevated above normal; (b) administering a candidate agent to the animal; and (c) determining the effect, if any, of the candidate agent on the aberrant PCDH expression (e.g. on PCDH8 expression, PCDH9 expression, and/or PCDH17 expression) in the animal. The method may further comprise the steps of: (d) contacting the candidate agent with an animal comprising neoplastic cells, wherein the neoplastic cells have aberrant PCDH expression (e.g. have at least one of PCDH8 expression decreased below normal, PCDH9 expression decreased below normal, and PCDH17 expression elevated above normal); and (e) determining if the agent has an effect on proliferation of the neoplastic cells. In particular, the candidate agent may be assessed for its ability to act as an inhibitor to cell division or to otherwise function as an appropriate tumor-suppressing agent.
The present invention is further directed to agents identified by the above-described identification methods. These agents are expected to be useful in treating or preventing neoplasia in a subject. Accordingly, the present invention further provides a method for treating or preventing neoplasia in a subject, comprising administering to the subject an agent of the present invention, in an amount effective to treat or prevent the neoplasia in the subject. This amount may be readily determined by one skilled in the art. Also provided is a use of an agent of the present invention in a method of treating or preventing neoplasia.
The present invention also provides a pharmaceutical composition comprising an agent identified by one of the above-described identification methods, and a pharmaceutically-acceptable carrier. Examples of suitable pharmaceutically-acceptable carriers, and methods of preparing pharmaceutical formulations and compositions, are described above. The pharmaceutical composition of the present invention would be useful for administering to a subject an agent of the present invention, in order to treat or prevent neoplasia in the subject. In such cases, the pharmaceutical composition is administered to a subject in an amount effective to treat or prevent the neoplasia.
The present invention is described in the following Examples, which are set forth to aid in the understanding of the invention, and should not be construed to limit in any way the scope of the invention as defined in the claims which follow thereafter.
UACC-812, UACC-893, MDA-MB-453, MDA-MB-175vii, MDA-MB-468, MDA-MB-361, MDA-MB-231, MDA-MB-436, MDA-MB-415, MDA-MB-330, MDA-MB-157, MDA-MB-134vi, MDA-MB-435s, ZR75-30, ZR75-1, BT-549, BT-483, T-47D, BT-474, DU4475, CAMA-1, MCF7, SK-BR-3, Hs578t, HCC38, HCC1143, HCC1187, HCC1395, HCC1428, HCC1806, HCC1937, HCC2157, HCC1500, HCC1599, HCC2218, HCC1419, HCC70, HCC202, HCC1954, HCC1569, HCC1008, LNCaP, DU145, MCF10A and PC-3 were purchased from the American Type Culture Collection (ATCC). SUM44P92, SUM52PE, SUM102PT, SUM149PT, SIJM159PT, SUM185PE, SUM225PE, SUM190PT, and SUM1315MO2 were acquired from the University of Michigan's Comprehensive Cancer Center. M2E6E7, M3E6E7, 70N, 70E6, 76N, and 76E6 were obtained from V. Band. HCC1395 and HCC1395 were obtained from Adi Gazdar. All cell lines, except MCF10A, were maintained per distributors' protocols. MCF10A were grown as described (Debnath, J., Muthuswamy, S. K. & Brugge, J. S. Morphogenesis and oncogenesis of MCF-10A mammary epithelial acini grown in three-dimensional basement membrane cultures. Methods 30, 256-68 (2003)). Breast tumor samples were from the Columbia University Tumor Bank. Female murine breast and brain tissue were from virgin and parous female mice.
Tissue was digested in SDS/proteinase K. DNA was extracted with phenol:chloroform:isoamyl alcohol (Invitrogen), and precipitated by NaCl/ethanol. The DNA pellet was resuspended in LoTE.
Genomic subtraction was performed using a modified method of representational-difference analysis (Lisitsyn et al., Cloning the differences between two complex genomes. Science, 259:946-51, 1993). Unique sequence was identified in 18 of 150 clones. Six fragments were derived from the EBV genome. Two of the fragments mapped to chromosome 13q21 and were absent in the tumor line.
Cells were lysed in solution D, and RNA was separated by CsCl. RNA was resuspended in sodium acetate, and reprecipitated with ethanol. The pellet was dissolved in DEPC-treated water. RNA was primed with random hexamers, and reverse-transcribed with Superscript II reverse transcriptase (Invitrogen).
cDNA was synthesized from RNA primed with random hexamers (Amersham). Primers used for RT-PCR are below: DIAPH3: ATCTCCCTGATCAAGACTCAAT (SEQ ID NO:74, ACTGTGAGAAAGTGGAAAGTA (SEQ ID NO:75). PCDH8: TGGCGGTGTGGAAAGGACA (SEQ ID NO:75), CGGAGTGACCTGTATATGTG (SEQ ID NO:76). For reactivation studies, cells were treated with 1 μM 5-aza-deoxycytidine for 72 hours.
Markers D13S1305, D13S155, and D13S1228 were amplified from genomic DNA. A reduction in peak intensity of 50% or greater was scored as a loss. PCDHs 8 and 17 were amplified from genomic DNA using Platinum Taq (Invitrogen). Sequences were analyzed using Mutation Surveyor (SoftGenetics). The primers that were used are set forth in Tables 1 and 2.
5-10 micrograms of genomic DNA were digested with RsaI alone or with RsaI in combination with HpaII, MspI, or CfoI. The digest was transferred to nylon membranes (Nytran), cross-linked, and baked. Membranes were hybridized with 32P-labeled probes in UltraHyb (Ambion), washed, and exposed to film. Probes were generated by PCR amplification from genomic DNA, and labeled randomly using Klenow polymerase (USB) and 32P-dCTP (Amersham).
Tissue was fixed in 4% paraformaldehyde overnight at 4° C., and dehydrated in 30% sucrose, 15-micron cryosections were cut and dried. Sections were fixed in 4% paraformaldehyde, digested in proteinase K, re-fixed, and acetylated. Sections were hybridized with DIG-labeled cRNA probes (for example from mouse clone ID 3813893), and then incubated with anti-DIG-AP antibody (Roche). Alkaline phosphatase activity was visualized with vector NBT/BCIP. Probes were transcribed with T7, SP6, or T3 RNA polymerases from linearized EST clones, and labeled with DIG-NTPs.
Cells were plated to 20% confluence, and treated with fresh 1 micromolar 5-aza-deoxycytidine for 72 h. RNA and cDNA were prepared from the cells and subjected to PCR.
Summarized below are results obtained by the inventors in connection with the experiments of Examples 1-8 above.
Representational difference analysis and genomic subtraction revealed a homozygous deletion of a portion of chromosome 13q in the breast tumor cell line HCC1395 (
Since protocadherins 17 and 20 were missing in HCC1395, the inventors screened protocadherins 17, 8, 9, and 20 for homozygous deletions in 88 human breast tumor lines and xenografts. From this screen, an additional homozygous deletion was identified in PCDH9 in the breast tumor line, MDA-MB-436 (
Homozygous deletions, loss of heterozygosity (LOH), and linkage data in hereditary breast cancer are summarized in
The deletions overlap with regions of LOH found in breast and prostate cancer. Furthermore, they fall within the linked interval found in hereditary breast cancers that are free of BRCA1 and BRCA2 mutations. (For breast cancer breast cancer LOH, see Eiriksdottir et al., Mapping loss of heterozygosity at chromosome 13q: loss at 13q12-q13 is associated with breast tumour progression and poor prognosis. Eur. J. Cancer, 34(13):2076-81, 1998; for breast cancer linkage, see Kainu et al., Somatic deletions in hereditary breast cancers implicate 13q21 as a putative novel breast cancer susceptibility locus. Proc. Natl. Acad. Sci. USA, 97(17):9603-08, 2000; and for prostate cancer LOH, see Hyytinen et al., Three distinct regions of allelic loss at 13q14, 13q21-22, and 13q33 in prostate cancer. Genes Chromosomes Cancer, 25(2):108-14, 1999). A protocadherin cluster partly lies within the deletions. In HCC1395, PCDHs 8 and 17 are deleted; in MDA-MB-436, PCDH9 is deleted.
Invasive ductal carcinoma of the breast evolves from epithelial cells of breast ducts. These ducts are composed of luminal, basal, and myoopithelial components. The breast acinus or duct is formed by 2 layers of cells. A layer of polarized, cuboidal, luminal cells lines the lumen; a second layer of cells, consisting of basal and myoepithelial cells, is subjacent to the luminal cells. Most breast tumors arise from the luminal epithelium.
Examination of the expression of protocadherins in cultured breast epithelial cells demonstrated that protocadherin 9 was expressed in both luminal and basal/ myoopithelial cell types, while protocadherin 8 was restricted to the luminal cells (
In situ probes for the 4 protocadherins were prepared. The probes were then hybridized to normal mouse brain sections. In situ hybridization revealed that all four probes were strongly expressed in the hippocampus (
The same in situ probes were then annealed to frozen sections of wild-type mouse mammary tissue. In situ hybridization revealed different expression patterns for PCDHs 8, 9, 17, and 20 in mouse breast tissue. Specifically, PCDH8 was expressed in luminal cells; PCDH9 was expressed in luminal cells, and possibly in basal/myoepithelial cells; and PCDH20 was expressed in luminal cells, and possibly in basal/myoepithelial cells. PCDH17 expression was not detected in breast epithelial cells.
In contrast to the expression of protocadherin 8 seen in normal breast tissue, lack of PCDH8 expression was frequently observed in breast tumor xenografts and primary breast tumors (
As shown in Table 3, protocadherin 9 expression was reduced in about 35% of breast tumor cell lines and xenografts, and in 2 out of three prostate tumor cell lines. Reduced expression was observed in only 10% of primary tumors; this may reflect contamination from the high expression seen in normal ductal cells. Loss of protocadherin 8 expression occurred in 18% of cell lines, and in 39% of tumor xenografts and primary tumors. Loss of protocadherin 20 expression was less common, occurring in 8%, 19%, and 5% of tumor lines, xenografts, and primary biopsies, respectively. Since protocadherins 8, 9, and 20 are normally expressed in breast tissue, these data suggest that their genes were silenced during tumor evolution. Interestingly, this was also the case in 2 prostate cancer cell lines. The inventors observed that 90-100% of tumor samples expressed the PCDH17 gene; this gene is silent in normal breast epithelium.
*number of samples expressing a gene/total number of samples (% of tumors)
The data shown in Table 3 suggest that a switch in the pattern of protocadherin expression occurs in breast cancer development, where PCDH17 is turned on, and PCDHs 8 and 9 are silenced. Little change occurs for protocadherin 20.
Patterns of altered PCDH expression in tumors are also shown in
MDA-MB-330 was the only breast tumor line or xenograft with a normal pattern of 13q protocadherin expression (
As demonstrated by Southern-blot analysis, tumor cell lines that failed to express protocadherins—such as ZR75-30 and MDA-MB-435s—show strong methylation of the PCDH8 promoter (
Primary tumors were then screened for evidence of methylation (
To demonstrate that promoter methylation could be responsible for gene silencing of protocadherin 8, the inventors treated MDA-MB-435s cells with the DNA methyltransferase inhibitor, 5-aza-deoxycytidine. PCDH8 expression was restored in MDA-MB-435 cells treated with 5-aza-deoxycytidine (
The inventors then examined the possibility that protocadherin 8 could be mutated in breast cancer. Seventy-five cases of breast cancer were screened for loss of heterozygosity. Of these, 29 exhibited loss of one allele in the vicinity of protocadherin 8 (39%). The exons of protocadherin 8 were amplified and sequenced in 28 of these tumors, and in 23 cell lines that expressed protocadherin 8 by RT-PCR. Somatic mutations were found in two cases. Both occurred in the extracellular domain, and fell within the cadherin repeats. Breast tumor 68T exhibited loss of heterozygosity (LOH) at markers D13S 1305, D13S155, and D13S1228 (
Germline mutations were also found in two cases (
The mutation data for PCDH8 are summarized in Table 4.
*total number of primary tumors = 28; total number of cell lines = 23
Because protocadherin 17 was abnormally activated in nearly all breast tumor samples tested, the inventors performed amplification and sequencing of protocadherin 17 in 22 tumors and in 21 cell lines. A missense mutation was identified at amino acid 222 (P222S). This mutation was in cadherin domain 2 (
*total number of primary tumors = 22, total number of cell lines = 21
The possible results of the deregulation of protocadherins are shown in
The inventors' model suggests that activation of protocadherin 17, and inhibition of protocadherins 8 and 9, contribute to tumor formation. Since nearly all samples have abnormal patterns of expression, it is believed that protocadherins 8 and 9 regulate the normal morphology of ductal cells through cell-cell communication. When their expression is lost, and protocadherin 17 is activated, the tissue-organizing signals are lost, and cells no longer respond to each other appropriately. This can lead to inappropriate migration, proliferation, and apoptosis.
mRNA and protein expression of PCDH8 was detected in two breast luminal epithelial cells lines (M2 and M3), a spontaneously immortalized breast epithelial line, MCF10A (10A), and the breast tumor line MCF7 but not in HCC1395 the line with the homozygous deletion (
To look for changes in PCDH8 expression in breast cancer, a panel of 85 cancer cell lines and tumor biopsies were screened for PCDH8 message. As shown in
To determine whether PCDH8 is a tumor suppressor gene, 116 breast tumors as well as 21 breast tumor cell lines were screened for mutations. In a subset of cases, LOH was screened for, and was present in 39% of cases. Three cancer-specific somatic mutations were found that were all associated with loss of the wild type allele.
The PCDH8 gene is predicted to encode an open reading frame with a signal peptide sequence, 6 extracellular cadherin repeats (EC), a transmembrane domain, and a cytoplasmic tail (
The genetic changes in PCDH8 found in the breast cancer samples are consistent with a tumor suppressor. All of the missense changes clustered in the cadherin repeats, showing that they may disrupt adhesive function. Alignment of the E146K mutation to the analogous glutamic acid residue in C-cadherin predicts that it coordinates calcium ions, a function that is required for proper adhesive function (Shapiro, L. et al. Structural basis of cell-cell adhesion by cadherins. Nature 374, 327-37 (1995); Nagar, B., Overduin, M., Ikura, M. & Rini, J. M. Structural basis of calcium-induced E-cadherin rigidification and dimerization. Nature 380, 360-4 (1996); Boggon, T. J. et al. C-cadherin ectodomain structure and implications for cell adhesion mechanisms. Science 296, 1308-13 (2002)).
To determine the mechanism of PCDH8 silencing seen in some breast cancer cases, PCDH8 CpG island methylation was assessed by Southern blot. The PCDH8 CpG island was not methylated in normal breast (
TMA = tissue microarray,
DCIS = ductal carcinoma in situ
4/5 (80%) of cell lines with undetectable levels, of message exhibit methylation of the PCDH8 promoter.
M = methylated,
U = unmethylated,
− = no detection of message,
+ = detection of message.
Methylation in each case correlated with reduction of PCDH8 expression (
LOH+ = presence of LOH,
LOH− = absence of LOH,
Unmeth = unmethylated,
Meth = methylated.
To study the relationship between promoter methylation and PCDH8 silencing, MDA-MB-435s cells were treated with a DNA methyltransferase inhibitor, 5-aza-deoxycytidine. Treatment restored expression of PCDH8, showing that chromatin modification of the CpG island is involved in gene silencing in tumors (
Thirty-five of the breast tumors that had been evaluated for LOH and PCDH8 mutation were screened for loss of protein expression. Levels of PCDH8 protein were reduced relative to adjacent normal ducts and lobules in 8/35 (23%) tumors (
Having established that PCDH8 is a candidate tumor suppressor, studies were carried out to address whether introducing somatic mutations of PCDH8 into normal cells could alter cell growth (
To explore the function of PCDH8 in mammary cell growth and differentiation MCF10A cells were infected with retroviruses expressing epitope-tagged wild-type PCDH8, the somatic mutants E146K (PCDH8K) and R343H (PCDH8H), or the empty vector pBABEpuro. The expression of exogenous PCDH8 was readily detectable and appeared to be higher than the endogenous level expressed in MCF10A (
Considering that PAPC regulates embryonic cell movements and PCDH8 is expressed in membranes, studies were carried out to determine whether PCDH8 could alter adhesion and migration. While overexpression of PCDH8 improved plating efficiency, expression of PCDH8K or PCDH8H did not (
Studies were then carried out to determine whether PCDH8 could alter cell migration. In transwell migration assays, both wild type PCDH8 and mutant PCDH8H repressed migration (
Studies were next carried out to determine whether mutant PCDH8 could affect proliferation and differentiation. Since wild-type MCF10A cells require exogenous EGF to proliferate, studies were carried out to determine whether PCDH8 tumor mutants could supplant the requirement for EGF. While vector control, wild-type PCDH8, and PCDH8H cells were growth limited in the absence of EGF, PCDH8K cells were capable of modest proliferation (FIG. 14F). Similarly, in another immortalized breast line dependent on EGF signaling, M2, cells expressing PCDH8K had enhanced proliferation relative to empty vector control cells in the absence of exogenous EGF (
PCDH8-mediated proliferation was context dependent. In both MCF10A and ZR75-30, expression of PCDH8 increased proliferation in EGF-containing and serum-rich media compared to mutant PCDH8 or vector control (
On the other hand, under both enriched and limited growth factor conditions, PCDH8K conferred increased susceptibility to transformation. A subset of 10A-PCDH8K cells was able to form foci when grown on plastic, whereas control, wt PCDH8, and PCDH8H cells were not (
Given the phenotype of PCDH8K in Matrigel, studies were carried out to determine whether wild type PCDH8 could perturb acinus development and morphogenesis in transformed cells. MCF10A cells expressing RasV12 were used as a model because RasV12 promotes loss of E-cadherin expression (
Overexpression of wild type PCDH8 was able to inhibit the migration, consistent with its adhesive and anti-motility properties (
A human PCDH8 clone was purchased from Origene, clone ID FB1851_H03, pCMV6-XL4-PCDH8. This clone contained a missense change. The wild type sequence was created using QuikChange XL Site-Directed Mutagenesis Kit (Stratagene), primers CAGGACACCTACGAGCTGGACGTGCG (SEQ ID NO:67) and CGCACGTCCAGCTCGTAGGTGTCCTG (SEQ ID NO:68). pBABE-PCDH8-myc was generated by PCR amplification and cloned into pBABEpuro between EcoRI and SalI sites. PCDH8-E146K mutant was generated using primers
Phoenix-ampho cells for retrovirus production were used. A T75 flask of cells was transfected with 21 ug plasmid using Lipofectamine 2000 (Invitrogen). Virus was harvested 48-72 hours post-transfection, stabilized with FBS, and passed through a 0.45 um filter. Cells were infected with viruses in the presence of 8 μg/ml polybrene and selected with 1 μg/ml puromycin.
MCF10A cells expressing MYC epitope-tagged proteins were plated onto sterile cover slips in a 6-well dish. Sixteen hours after plating, cells were fixed in 2% paraformaldehyde in PBS pH 7.4 for 30 minutes at room temperature. Cells were washed for 20 minutes in PBS, permeabilized for 1 hour in buffer A (5% goat serum, 0.1% Triton X-100 in PBS), and incubated with 1:1000 dilution of mouse monoclonal anti-MYC (9E10) antibody (Santa Cruz) in buffer A. Cells were washed in PBS, and incubated with 1:600 dilution of Alexafluor 568 anti-mouse antibody (Molecular Probes) and counterstained with DAPI (0.15 μg/ml in water).
Whole cell lysates were used in all Western blots. Paraffin sections were stained with 1:5000 dilution of the anti-PCDH8 antibody. Slides were developed with ABC-DAB (Vector, Biogenics). Antibodies: Anti-PCDH8 was raised against amino acids 1052-1070 (YQSPPGRYLSPKKGANENV (SEQ ID NO:73)) in rabbits and affinity purified (NCBI accession number AAC70009). Other antibodies were commercially available: anti-tubulin (Tu27)(Covance), anti-vinculin (hVIN-1)(Sigma), anti-Myc (9E10)(Santa Cruz), anti-E-cadherin (BD Biosciences), anti-v-H-ras (Ab-1)(Calbiochem).
40 μl growth-factor reduced Matrigel (BD Biosciences) was plated on 8-chamber slides (Costar). MCF10A cell lines were grown in each chamber as described (Debnath, J., Muthuswamy, S. K. & Brugge, J. S. Morphogenesis and oncogenesis of MCF-10A mammary epithelial acini grown in three-dimensional basement membrane cultures. Methods 30, 256-68 (2003)).
MCF10A, MDA-MB-468 and ZR75-30:3,000 cells/well were plated on 48 well plates in growth media. For MCF10A, media was changed to assay media (DMEM:F12, 0.5% horse serum, 0.5 ug/ml hydrocortisone, 100 ng/ml cholera toxin, 10 μg/ml insulin, 1× penicillin/streptomycin) 12-16 hrs after plating. At day of harvest (12-16 hrs after plating for adhesion assays), cells were washed in PBS, stained with crystal violet, and solubilized with 10% acetic acid. Absorbance at 590 nm was read with a MicroQuant universal microplate spectrophotometer plate reader (BIO-TEK). For M2 assays: 100,000 cells were plated into 6 well plates. 12-16 hrs after plating, cells were grown in DFCI-1 media, without supplemental EGF. Band, V. & Sager, R. Distinctive traits of normal and tumor-derived human mammary epithelial cells expressed in a medium that supports long-term growth of both cell types (Proc Natl Acad Sci USA 86, 1249-53 (1989)). At day of harvest, cells were trypsinized and counted. All assays were performed in triplicate. Error bars of all adhesion assays and M2 proliferation assays represent standard deviations. Error bars of MCF10A and ZR75-30 proliferation assays represent standard errors of the mean.
Transwell migration assay: 50,000 cells were plated in the upper chambers of 8 micron transwell cell culture plates (Costar). Lower chambers contained either assay media (without chemoattractant) or growth media (with chemoattractant). After 16-24 hrs of incubation, cells on the upper surface of the filters were removed with swabs. Migrating cells were stained with crystal violet. For each filter, 20 high power fields were counted. Wound healing assay: Equal numbers of cells were plated on a 6-well plate. A single wound was introduced using a P20 pipette tip and media was replaced.
While the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be appreciated by one skilled in the art, from a reading of the disclosure, that various changes in form and detail can be made without departing from the true scope of the invention in the appended claims.
This application is a continuation-in-part of International Application No. PCT/US2005/021602, filed Jun. 17, 2005, and claims priority to U.S. Provisional Application Ser. No. 60/581,215, filed Jun. 17, 2004; both applications are herein incorporated by reference in their entireties.
This invention was made with government support under NIH Grant No. R01 CA082783. As such, the United States government has certain rights in this invention.
| Number | Date | Country | |
|---|---|---|---|
| 60581215 | Jun 2004 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US05/21602 | Jun 2005 | US |
| Child | 11637277 | Dec 2006 | US |
