Patent Application
20050276757
Biomarkers are molecular indicators of biological events, or phenomena, in organisms. Changes in the level of a biomarker can evidence an organism's biological response to a stimulus, such as a chemical compound. The biological responses may include events at a molecular, cellular or whole organism level.
Measured changes in biomarker levels are useful to gauge whether or not a particular effect has been achieved, or is likely to be achieved, in an organism. Thus, changes in biomarker levels can indicate whether or not a compound induces a particular biological response in an organism. Similarly, changes in biomarker levels can indicate that an organism has been exposed to a particular compound. In a clinical context, changes in biomarker levels can indicate whether an organism is experiencing, or will experience, a therapeutic effect or toxic event in response to a compound.
In the study and treatment of cancer, a continuing need exists for identification of new biomarkers. Cancer-related biomarkers can be employed to identify new effective cancer treatments, to monitor patient tolerance and/or therapeutic response to a cancer treatment, and to predict effective cancer treatments for individual patients.
The present inventors have discovered endogenous substrates for ZC1 and ZC3 kinases, both of which promote cell transformation and tumor growth. According to the invention, the preferred substrate of ZC1 is p120 catenin and the preferred substrate of ZC3 is Abi-1. Phosphorylation of the ZC1 and ZC3 substrates constitutes a new biomarker for cancer.
Thus, the present invention provides methods of determining whether a test compound modulates ZC1 kinase activity in a mammal. In one embodiment, the determination of whether a compound modulates ZC1 kinase activity in a mammal involves the following steps:
In another embodiment, the determination of whether a compound modulates ZC1 kinase activity in a mammal involves the following steps:
In another aspect, the invention provides a method of determining whether a mammal is responding to a compound that modulates ZC1 kinase activity. This involves comparing a base level of ZC1 substrate phosphorylation in a mammal against a level of ZC1 substrate phosphorylation after the mammal is exposed to a compound that modulates ZC1 kinase activity. A difference in the base and post-exposure levels indicates that the mammal is responding to the compound. Preferably, phosphorylation levels are derived from tumor samples in the mammal, and therefore can indicate whether therapy with a ZC1 kinase modulator is operating effectively.
In yet another aspect, the invention provides methods of predicting whether a mammal with cancer will respond therapeutically to administration of a modulator of ZC1 kinase. The method of predicting involves (a) measuring the level of ZC1 substrate phosphorylation in a mammal with cancer, and (b) comparing the level of ZC1 substrate phosphorylation in the mammal against a standard level of ZC1 substrate phosphorylation for the mammal. A difference in the phosphorylation levels indicates that the mammal will respond therapeutically to a modulator of ZC1 kinase. Preferably, the phosphorylation level is measured in a tumor sample.
The present invention also provides methods for determining whether a compound modulates ZC3 kinase activity in a mammal. In one embodiment, this determination can be made by performing the following steps:
In another embodiment, the determination of whether a compound modulates ZC3 kinase activity in a mammal involves the following steps:
In another aspect, the invention provides a method of determining whether a mammal is responding to a compound that modulates ZC3 kinase activity. This involves comparing a base level of ZC3 substrate phosphorylation in a mammal against a level of ZC3 substrate phosphorylation after the mammal is exposed to a compound that modulates ZC3 kinase activity. A difference in the base and post-exposure levels indicates that the mammal is responding to the compound. Preferably, phosphorylation levels are derived from tumor samples in the mammal, and therefore can indicate whether therapy with a ZC3 kinase modulator is operating effectively.
In yet another aspect, the invention includes methods of predicting whether a mammal with cancer will respond therapeutically to administration of a modulator of ZC3 kinase. The method of predicting involves (a) measuring the level of ZC3 substrate phosphorylation in a mammal with cancer, and (b) comparing the level of ZC3 substrate phosphorylation in the mammal against a standard level of ZC3 substrate phosphorylation for the mammal. A difference in the phosphorylation levels indicates that the mammal will respond therapeutically to a modulator of ZC3 kinase. Preferably, the phosphorylation level is measured in a tumor sample.
The present inventors have discovered endogenous substrates for ZC1 and ZC3 kinases, both of which promote cell transformation and tumor growth. According to the invention, the preferred substrate of ZC1 is p120 catenin and the preferred substrate of ZC3 is Abi-1. Phosphorylation of the ZC1 and ZC3 substrates constitutes a new biomarker for cancer.
In accord with the inventors' discovery, the present invention includes methods for determining whether a test compound modulates ZC1 or ZC3 kinase activity in a mammal. It also includes methods of determining whether a mammal is biologically responding to a compound that modulates ZC1 or ZC3 kinase activity. Further, the invention includes methods of accurately predicting whether a mammal with cancer will respond therapeutically to administration of a modulator of either ZC1 or ZC3 kinase.
In one embodiment of the present invention, the determination of whether a compound modulates ZC1 kinase activity in a mammal involves the following steps:
In another embodiment, the determination of whether a compound modulates ZC1 kinase activity in a mammal involves the following steps:
In another aspect, the invention also provides a method of determining whether a mammal is responding to a compound that modulates ZC1 kinase activity. This involves comparing a base level of ZC1 substrate phosphorylation in a mammal against a level of ZC1 substrate phosphorylation after the mammal is exposed to a compound that modulates ZC1 kinase activity. A difference in the base and post-exposure levels indicates that the mammal is responding to the compound. Preferably, phosphorylation levels are derived from tumor samples in the mammal, and therefore can indicate whether therapy with a ZC1 kinase modulator is operating effectively.
In yet another aspect, the invention includes methods of predicting whether a mammal with cancer will respond therapeutically to administration of a modulator of ZC1 kinase. The method of predicting involves (a) measuring the level of ZC1 substrate phosphorylation in a mammal with cancer, and (b) comparing the level of ZC1 substrate phosphorylation in the mammal against a standard level of ZC1 substrate phosphorylation for the mammal. A difference in the phosphorylation levels indicates that the mammal will respond therapeutically to a modulator of ZC1 kinase. Preferably, the phosphorylation level is measured in a tumor sample.
The present invention also provides methods for determining whether a compound modulates ZC3 kinase activity in a mammal. In one embodiment, this determination can be made by performing the following steps:
In another embodiment, the determination of whether a compound modulates ZC3 kinase activity in a mammal involves the following steps:
In another aspect, the invention provides a method of determining whether a mammal is responding to a compound that modulates ZC3 kinase activity. This involves comparing a base level of ZC3 substrate phosphorylation in a mammal against a level of ZC3 substrate phosphorylation after the mammal is exposed to a compound that modulates ZC3 kinase activity. A difference in the base and post-exposure levels indicates that the mammal is responding to the compound. Preferably, phosphorylation levels are derived from tumor samples in the mammal, and therefore can indicate whether therapy with a ZC3 kinase modulator is operating effectively.
In yet another aspect, the invention includes methods of predicting whether a mammal with cancer will respond therapeutically to administration of a modulator of ZC3 kinase. The method of predicting involves (a) measuring the level of ZC3 substrate phosphorylation in a mammal with cancer, and (b) comparing the level of ZC3 substrate phosphorylation in the mammal against a standard level of ZC3 substrate phosphorylation for the mammal. A difference in the phosphorylation levels indicates that the mammal will respond therapeutically to a modulator of ZC3 kinase. Preferably, the phosphorylation level is measured in a tumor sample.
ZC1 kinase, or HGK (hematopoietic progenitor kinase-like/germinal center kinase-like kinase), is a member of the human STE20/mitogen-activated protein kinase family of serine/threonine kinases. It is the ortholog of mouse NIK (Nck-interacting kinase). The structure of ZC1 and its relationship to other kinases are thoroughly described in U.S. patent application Ser. No. 09/291,417 (published Mar. 13, 2003) and Wright, J. H. et al., Mol. Cell. Biol., 23(6): 2068-82 (2003), both of which are incorporated herein by reference. Several splice variants of ZC1 exist.
ZC1 is understood to play a role in human cancers. More particularly, ZC1 functions in cellular adhesion, invasion and transformation. It is highly expressed in most tumor cell lines relative to normal tissue. Moreover, ZC1 plays an active role in transformation. This has been demonstrated in experiments showing that expression of inactive, dominant-negative mutants of ZC1 in both fibroblast and epithelial cell lines inhibits H-Ras-V12-induced focus formation. Additionally, expression of inactive mutant ZC1 inhibits anchorage-independent growth of cells, modulates integrin receptor expression and inhibits hepatocyte growth factor-stimulated epithelial cell invasion.
ZC3 also is a member of the human STE20/mitogen-activated protein kinase family of serine/threonine kinases. Its structure and relationship to other kinases are thoroughly described in U.S. patent application Ser. No. 09/291,417 (published Mar. 13, 2003), which is incorporated herein by reference. As with ZC1, several splice variants of ZC3 exist. ZC3 shares a high degree of homology with ZC1, and also is understood to play a role in human cancers. It is highly expressed in most breast cancers and leukemias, relative to normal tissues.
The present inventors have discovered that ZC1 kinase localizes to cell-cell junctions, where it associates with p120 catenin. Moreover, they have discovered that ZC1 kinase phosphorylates p120 catenin, both in vivo and in vitro. Indeed, p120 catenin constitutes a very efficient substrate for ZC1 kinase.
Thus, according to the present invention, p120 catenin constitutes a useful and preferred ZC1 kinase substrate. p120 catenin belongs to the Armadillo/β-catenin gene superfamily, originally described as a substrate for src and other receptor tyrosine kinases, and its expression is often altered or lost in tumors of the breast, prostate, colon, stomach, pancreas and bladder. Loss and change in localization of p120 is also associated with poor prognosis and advanced disease, suggesting a role in cancer progression.
In vivo, p120 catenin it is often localized to cellular adherins junctions, but also has been found in the cell nucleus, particularly in metastatic tumor cells that have lost cadherin expression. Tyrosine and serine phosphorylation of p120 catenin occurs after stimulation of cells by epidermal growth factor (EGF), colony-stimulating growth factor (CSF-1) and platelet-derived growth factor (PDGF), and correlates with transformation in cells transfected with v-Src. p120 catenin associates with the cytoplasmic domain of E-cadherin, which targets p120 catenin to cell-cell junctions, where it functions in development, morphogenesis and tumorigenesis. Additionally, p120 catenin regulates Rho GTPases, inhibiting RhoA and activating Rac and cdc42. Evidence indicates that p120 can both positively and negatively regulate adhesion, depending on expression levels, localization, association with E-cadherin/Rho GTPases, and isoform (alternatively spliced) expressed.
The present inventors also have discovered that ZC3 kinase associates with Abi-1 and Abi-2 proteins, via the SH3 domains of those proteins, in cells. Moreover, the inventors have discovered that ZC3 kinase strongly phosphorylates Abi proteins.
Thus, according to the present invention, Abi-1 and Abi-2 constitute useful ZC3 kinase substrates. Abi-1 and Abi-2 were first identified by their ability to interact with the c-Abl tyrosine, and were initially described as a suppressors of v-Abl transforming activity. Abi-1, also known as E3b1, was shown to be involved in cytoskeletal reorganization by transducing signals between Ras and Rac. The role of Abi proteins in modulating the actin cytoskeleton is further demonstrated by the fact that they localize to sites of actin polymerization at the tips of lamellipodia and filopodia.
Abi-1 and Abi-2 contain SH3 domains and polyproline motifs (PXXP) that allow them to interact with various proteins. They can interact with SH3-containing proteins through their PXXP motifs and with PXXP-containing proteins through their own SH3 domain. Indeed, the present inventors have found that the PXXP motifs in ZC3 interact with Abi-1 and Abi-2.
As used herein, the term “modulates” refers to the ability of a compound to alter kinase activity. Thus, modulation includes activation and inhibition of kinase activity. “Activation” refers to increasing the cellular activity of a kinase, whereas “inhibition” refers to decreasing the cellular activity of a kinase. Preferably, a modulator inhibits kinase activity. Modulation may result directly from interaction of a compound with a kinase molecule, or indirectly from interaction of a compound with other elements that impact kinase activity. For example, a modulator may alter the function of a kinase by increasing or decreasing the probability that a complex forms between the kinase and a natural binding partner.
The “base level of phosphorylation” refers to the level of substrate phosphorylation in a mammal before exposure to a compound that modulates the relevant kinase activity. For example, in a method for determining whether a compound modulates ZC1 kinase activity in a mammal, the base level of phosphorylation might be a measured level of p120 catenin phosphorylation prior to administration of a test compound. Similarly, in a method for determining whether a mammal is responding to a compound that modulates ZC3 kinase activity, a base level of phosphorylation might be a measured level of Abi-2 phosphorylation from prior to administration of the modulating compound. Methods for determining a base level of phosphorylation are described herein.
In comparison, a “standard level of phosphorylation” refers to the level of substrate phosphorylation in a mammal of the same species that has not been exposed to a compound that modulates the relevant kinase activity. The standard level may be determined from substrate phosphorylation in an individual mammal, but is more meaningful if determined from substrate phosphorylation in a population of mammals. By way of example, in a method for determining whether a compound modulates ZC1 kinase activity in a mammal, a standard level of phosphorylation might be the level of p120 catenin phosphorylation in another mammal of the same species or an average level of p120 catenin phosphorylation in a group of mammals of the same species. Similarly, in a method of predicting whether a mammal will respond therapeutically to a ZC3 kinase modulator, a standard level of phosphorylation might be the level of Abi-1 phosphorylation in another mammal of the same species or an average level of Abi-1 phosphorylation in a group of mammals of the same species.
Methods for measuring substrate phosphorylation are well known in the art. As the accompanying examples illustrate, one method involves isolating substrate proteins by immunoprecipitation, followed by measurement of reactivity with a labeled phospho-threonine specific antibody. A more streamlined, and preferred, method for measuring substrate phosphorylation employs labeled antibodies that specifically bind phosphorylated residues on a substrate molecule. Examples would include monoclonal antibodies or antibody fragments that specifically bind to residues on p120 catenin or Abi-1 that have been phosphorylated by ZC1 and ZC3, respectively. Such antibodies also are useful for performing in situ hybridization. Phosphorylation levels can be visually evaluated or can be quantified using well known technologies, such as ELISA, SDS PAGE, Western Blot and immunoprecipitation
According to the present invention, a mammal responds “therapeutically” when factors contributing to an abnormal condition are ameliorated to some degree. A therapeutic response can refer to one or more of the following: (a) an increase or decrease in cell proliferation, growth, and/or differentiation; (b) an increase or decrease in cell death; (c) a decrease in degeneration; (d) relief of symptoms associated with an abnormal condition; and (e) enhancing the function of an affected population of cells. Preferably, the abnormal condition is cancer, and the therapeutic response is a decrease in malignant cell proliferation and/or growth.
Methods of the present invention may be performed on any mammal, including a human, rat, mouse, dog, rabbit, pig, sheep, cow, horse, cat, primate or monkey. Preferably, the mammal is a rat, mouse or primate. Most preferably, the mammal is a human.
The inventive methods may be performed in vitro. In some embodiments, phosphorylation is measured in at least one biological tissue, such as buccal mucosa tissue, skin, hair follicles, tumor tissue or bone marrow. In other embodiments, phosphorylation is measured in at least one biological fluid, such as whole blood, peripheral blood mononuclear cells, plasma, urine or saliva.
In some embodiments, the inventive methods are performed on mammals that have cancer. The cancers may be, but are not limited to, prostate cancer, colorectal cancer, thyroid cancer, an advanced solid malignancy, pancreatic cancer, breast cancer, parotid cancer, synovial cell cancer or sarcoma, gastrointestinal stromal tumor, laryngeal cancer, testicular cancer, leiomyosarcoma, rectal cancer, gall bladder cancer, hepatocellular cancer, melanoma, ovary cancer, lung cancer, colon cancer, renal cell carcinoma, sarcoma, retropero sarcoma, pelvis sarcoma, uterine cancer, pelvic angiosarcoma, pleural mesothelioma, neuroendocrine cancer, bronchial adenocarcinoma, head and neck cancer or thymic cancer.
“Exposing,” “administer,” and “administration” refer to the delivery of a compound to a mammal. Suitable routes of administration include oral, rectal, transmucosal or intestinal administration or intramuscular, subcutaneous, intramedullary, intrathecal, direct intraventricular, intravenous, intravitreal, intraperitoneal, intranasal, or intraocular injections. The preferred routes of administration are oral and parenteral.
Administration of a compound may occur in any of numerous forms known in the art. Proper formulation is dependent upon the route of administration chosen. The compounds may be administered alone or as part of a pharmaceutical composition. A “pharmaceutical composition” refers to a mixture of at least one compound with physiologically/pharmaceutically acceptable carrier(s) and/or excipient(s). The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.
“Physiologically/pharmaceutically acceptable carrier” refers to a carrier or diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound.
“Pharmaceutically acceptable excipient” refers to an inert substance added to a pharmaceutical composition to further facilitate administration of a compound. Examples include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, polyethylene glycols and the like.
Determination of a proper dose is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein. For any compound used in the methods of the invention, the dose can be estimated initially from cell culture assays. Then, the dosage can be formulated for use in animal models so as to achieve a circulating concentration range that includes the IC50 as determined in cell culture. Such information can be used to more accurately determine useful doses in humans.
Methods of the invention may be used to individually adjust dosage amounts and intervals, to provide plasma levels of the active species that are sufficient to maintain the kinase modulating effects. These plasma levels are referred to as minimal effective concentrations (MECs). The MEC will vary for each compound but can be estimated from in vitro data, e.g., the concentration necessary to achieve 50-90% inhibition of a kinase may be ascertained using the assays described herein. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration.
Methods of the invention are performed so that the step where a mammal is exposed to a compound includes administration of at least one dose of the compound, or at least two doses, or at least 5 doses or at least 10 doses, up to at least 55 or 56 doses. In certain embodiments, these doses are administered during a period of 4 hours, 6 hours, or 24 hours to about 100 days. In further embodiments, the doses are administered over a period of 24 hours, 2 days, or 28 days. In other embodiments, two doses are administered per every 24 hours, and in other embodiments, the doses are administered about every 12 hours. It will be understood by those of skill in the art that the administration of a compound, according to the exposure steps of the methods, can be varied to suit individual needs of the mammal being treated, the compound being administered, the method of administration and the disease being treated. For example, in a typical dosing regimen, the patient receives one dose per day of the compound, for a number of days, such as about 28 or about 56 days. In other dosing regimens, the compound is administered about once per day, twice per week, or once per week.
The measurement of substrate phosphorylation, following the exposure step in the methods, can be performed on a sample from the mammal taken about 4 or 6 hours following the first dose (exposure) of the mammal to the compound. In other embodiments, this measurement is performed on a sample taken 12 hours, 1 day, 2 days, up to about 100 days, after the first dose (exposure) of the mammal to the compound. In other embodiments, the phosphorylation measurements are taken from samples from the mammals 4 or 6 hours after the first dose of compound or 24 hours after the first dose of compound, or 15 or 28 days after the first dose of compound. Typically, dosing of compound will be periodic between the first and last dose of compound that precedes the sample taken for measurement of phosphorylation. For example, the compound is administered once a day, every day for 28 days. Typically, the mammal sample taken (for measurement of phosphorylation) will be taken shortly following the most recent dose of compound, for example within 24 of the most recent dose of compound.
The difference in the level of phosphorylation measured in the inventive methods (e.g., “base” level vs. post-exposure or “standard” vs. tumor) may be an increase or decrease of at least about 10%, 15%, 20%, 25%, 30%, 35%, 50%, 75% or 100%. Preferably, it is an increase or decrease of at least about 25%. In some embodiments, the difference may be an increase or decrease of at least 2-, 3- 5-, 10-, 15-, or 20- fold. In still other embodiments, the difference in the level of phosphorylation is an increase or decrease of at least 1.1-, 1.2-, 1.3-, 1.4-, 1.5-, 1.6-, 1.7-, 1.8-, 1.9-, 2.0-, 2.5-, 3.0-, 3.5-, 4.0-, 5.0-, 7.5-, 10.0-, 15.0- or 20.0- fold.
In addition to the above-described methods, the invention includes a kit comprising: (a) a phospho-threonine-specific antibody or antibody fragment, and (b) instructions for performing one of the methods described herein.
Reference to the following illustrative examples will help to provide a more complete understanding of the invention.
This example demonstrates that ZC1 kinase co-localizes with p120 catenin at cell-cell junctions.
ZC1 antibodies were raised and used to determine where ZC1 is localized in cells. In H1299 cells, as well as in rat intestinal epithelial cells (RIE-1), anti-ZC1 antibodies showed a punctate cytoplasmic staining, with a significant amount of the staining also at points of cell-cell contact (
To corroborate the conclusion that a portion of the cellular ZC1 is localized to adheren junctions, we demonstrated that ZC1 and p120 associate in cell lysates. Confluent plates of each of four cell lines, H1299, MCF-7, RIE-1, and SKOV-3 were lysed in detergent, and the cellular supernatants were subjected to immunoprecipitation with anti-ZC1 antisera. When immunoprecipitates were analyzed in Western blots probing for endogenous p120, two bands corresponding to two isoforms of p120 were observed to co-purify with ZC1 in the immunoprecipitate (
This example demonstrates that ZC1 kinase phosphorylates p120 catenin in vitro.
Co-transfection with a plasmid encoding p120, with and without a plasmid encoding active full-length ZC1, into 293T cells with lipofectamine (Invitrogen) was performed. After 48 hours, transfected cells were harvested in lysis buffer (50 mM HEPES (pH 7.0), 150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 10% glycerol, 1% Triton, 10 mM pyrophosphate, 1 mM Na3VO5, 1 mM DTT, 0.1 mg/ml of 4-(2-aminoethyl)benzenesulfonyl fluoride, 2-10 mg/ml each of aproptonin, pepstatin, leupeptin and E-64), incubated on a rocker for 30 minutes and clarified by centrifugation. Supernatants were precleared with protein A sepharose and immunoprecipitated with anti-p120 antibody (and protein A sepharose) for 2 hours at 4° C. After incubation, immune complexes were washed four times in lysis buffer and then 2 times in kinase assay buffer. Washed complexes were incubated with kinase assay buffer containing 100 micromolar 32P-ATP with or without 4 micrograms purified GST-ZC1 kinase domain for 30 minutes at 30° C. Reactions were stopped with Laemmli SDS sample buffer and analyzed by SDS-PAGE, stained with GelCoat coomassie stain (Bio-Rad) and analyzed by autoradiography.
Results are shown in
This example demonstrates that ZC1 kinase phosphorylates p120 catenin in vivo.
Co-transfection with a plasmid encoding p120, with and without a plasmid encoding different forms of ZC1, all tagged with myc (WT=wild type; KR=kinase-inactive; 1230E=T1230E phosphorylation site mutant; 209E=T209E phosphorylation site mutant; 191E=T191E inactive phosphorylation site mutant; 187E=T187E phosphorylation site mutant; d2 and DC both contain deletion mutants of ZC1 that retain the kinase domain), into 293T cells with lipofectamine (Invitrogen) was performed. After 48 hours, transfected cells were harvested in lysis buffer (50 mM HEPES (pH 7.0), 150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 10% glycerol, 1% Triton, 10 mM pyrophosphate, 1 mM Na3VO5, 1 mM DTT, 0.1 mg/ml of 4-(2-aminoethyl)benzenesulfonyl fluoride, 2-10 mg/ml each of aproptonin, pepstatin, leupeptin and E-64), incubated on a rocker for 30 minutes and clarified by centrifugation. Supernantants were precleared with protein A sepharose, and immunoprecipitated with anti-p120 antibody (and protein A sepharose) for 2 hours at 4° C. After incubation, immune complexes were washed four times in lysis buffer and subjected to SDS-PAGE in parallel gels. Western blots of those gels were probed with anti pThr (Cell Signaling), anti-myc for ZC1 ((E10 in house hybridoma sup), and anti-p120 (Becton-Dickenson). Results are shown in
To show that p120 was also a substrate for ZC1 in the cell, the phosphorylation state of p120 in the cell was examined with a phospho-threonine antibody. A plasmid encoding p120 protein was co-transfected into 293T cells, alone and in combination with 8 different forms of ZC1, including both activate and inactive forms of the enzyme. When immunoprecipitated from cell extracts, p120 reactivity with the phospho-threonine antibody increased in those extracts prepared from cell also transfected with active forms of ZC1 (
This example demonstrates that ZC3 kinase selectively interacts with Abi-1 and Abi-2 proteins. More particularly, the linker region of ZC3 (aa 672-1032 of ZC3 allele), which contains several PXXP motifs, interacts with SH3 domains on Abi-1 and Abi-2.
A pGEX expression construct containing the linker domain of ZC3 was constructed. The linker domain (amino acids 672-1032 of ZC3 allele) was arbitrarily defined as the region that lies between the NCK-interaction domain and the CNH domain. The resulting GST-ZC3-linker protein was immobilized on glutathione-Sepharose resin and used to pan several phage display libraries (Novagen T7Select10-3- normal brain, breast tumor, colon tumor, liver tumor and lung tumor cDNA). After three rounds of enrichment, 32-40 individual plaques from each library were sequenced. Results are summarized in
This example demonstrates that ZC3 kinase selectively interacts with Abi-1 and Abi-2 proteins intracellularly.
COS7 cells were transfected with the following plasmids: ZC3: pcDNA-ZC3, ZC3B: pcDNA-ZC3B, Abi1: pCMV-Flag-Abi1, Abi2B: pCGN HA-Abi2B. After 48 hours, cells were lysed in HNTG buffer (50 mM HEPES pH7.5, 10% glycerol, 150 mM NaCl, 1% Triton X-100, 1.5 mM MgCl2, 1 mM EGTA) containing protease inhibitors. After measuring protein concentration using the BCA assay (Pierce), 200 ug of cell lysates were subjected to immunoprecipitation with either an antibody against ZC3 (292B), an anti-Flag antibody (Sigma) to bring down Abi1, or an anti-HA antibody (12CA5) to bring down Abi2B. Protein A and Protein G sepharose beads (Amersham Biosciences) were used to bring down the immune-complexes, which were washed four times in HNTG buffer before being processed for SDS-PAGE. Several gels were run and probed with the different antibodies. Total lysates were also subjected to western blotting to confirm that the various constructs transfected into the cells were correctly expressed (two lower blots on each panel). The anti-Abi antibody, #5421, used for western blotting was described in Dai, Quackenbush et al. 1998.
This example shows that ZC3 kinase phosphorylates Abi-1.
Abi-1 was immunoprecipitated from 293T cells using an anti-Flag antibody. Immunoprecipitations performed on lysates from 293T cells transfected with pcDNA3, and without any lysate were used as negative controls. The immunoprecipitated protein bound to Protein A Sepharose beads was subjected to a kinase assay (20 mM Tris HCl pH 7.4, 200 mM NaCl, 10 mM MgCl2, 1 mM DTT, 100 uM ATP) plus 5 uCi of [y32P]ATP per 50 ul reaction. The kinase domain of ZC3 fused to GST (190 ng of protein) was added to the samples. The kinase reaction was allowed to proceed for 20 min at 30° C., with agitation, and was stopped by adding SDS sample buffer and incubating 3 min at 95° C. GST-ZC3KD alone and plus Myelin Basic Protein (MBP, 5 ug) samples were also included as controls. The samples were processed for SDS-PAGE (4-20% gradient gel, Invitrogen), the gel was stained (Gel Code, Pierce), dried and autoradiographed.
This example shows that over-expression of HGK (ZC1) promotes anchorage-independent growth, that HGK siRNA reduces anchorage-independent growth, that HGK promotes tumor growth in vivo, and that HGK promotes cell rounding and loss of cell-cell contacts.
Cell lines and transfection methods
H1299, HeLa, and A549 human cell lines and other tumor cell lines were originally obtained from the ATCC (Manassas, Va.). All cell lines were grown in DMEM medium (Invitrogen, Carlsbad, Calif.), supplemented with 10% fetal bovine serum (FBS, Invitrogen) at 37° C. with 5% CO2. Construction of pcDNA3 plasmids encoding the wild type and K54R mutant can be found in Wright et al, 003. To express HGK wild type (WT) and inactive mutant (KR), H1299 cells were transfected with 5 ug pcDNA3/HGK construct using Lipofectamine 2000 (Invitrogen). Stable pools and clonal populations of transfected cells were selected using 500 ug/ml G418 (Invitrogen) over the course of one to two weeks post-transfection. Transfectants over-expressing either wild-type HGK (WT), kinase inactive HGK (KR) or vector alone (V) were cultured in an identical manner as the parental cells from which they were derived.
RNA interference
Oligos were prepared 2′-deprotected, annealed, purified and lyophilized. They were generated with dTdT/dTdT overhangs on the sense and antisense strands. Two siRNA duplexes against the HGK ORF were prepared (sense strand shown):
The duplexes were resuspended in sterile water at a stock concentration of 40 μM. A scrambled siRNA sequence also was prepared:
The scrambled target sequence is not present in mammalian cells as determined by BLAST search at NCBI: www.ncbi.nim.nih.gov/BLAST/. A LaminAC siRNA sequence also was prepared:
Cell lines (4.5×104 cells) were seeded in 12-well dishes and allowed to adhere for six hours. Cell lines were transfected with either varying concentrations of HGK siRNA reagents, scrambled siRNA or siRNA reagents against other genes using Oligofectamine (Invitrogen) in growth medium without antibiotics as follows: 5 μl of oligofectamine and 20 μl of Opti-MEM were mixed and incubated for 10 minutes at room temperature, then mixed with the siRNA reagents (19 μl volume or less) which had been diluted with 100 μl of Opti-MEM Growth medium. The oligofectamine/siRNA suspension was incubated for 25 minutes at room temperature. The medium in each well was replaced by 0.8 ml of fresh medium containing FBS. The lipofectamine-siRNA complexes were added drop wise. Seventy-two hours post-transfection a second round of transfection with siRNA was performed. Cells were allowed to recover for an additional 24 hours. siRNA transfected cells (1.0×103, 5×103 cells) were plated in 0.25% Bactoagar (Gibco-BRL)-ISCOVE's-10% FBS on top of a 0.75% Bactoagar-ISCOVE's-10% FBS base layer. For each cell type, treatments with or without siRNAs were performed in two 60 mm dishes. After seven days, plates were fed with 0.75% Bactoagar/ISCOVE's-10% FBS. Soft agar plates were stained with Neutral Red (Invitrogen) after 14 days and scanned and quantified with Prolmage Software.
Cell growth assays
Monolayer growth. Proliferation assays were performed as follows: 1×105 cells were plated in each well of a 6-well tissue culture plate. After 24 hours, growth medium was removed and replaced with DMEM containing 1% or 10% fetal calf serum (Invitrogen). Cells were removed from plates with trypsin and counted using a hemocytometer on days 1, 3 and 5 following.
Soft agar assay. This assay was performed to determine the ability of cells to grow in an anchorage-independent manner. Stock soft agar solution was prepared by autoclaving 1.6% bacto-agar (Difco, Becton Dickenson, Franklin Lakes, N.J.) in H2O. Stock agar was diluted in Iscove's Modified Dulbecco's Medium (IMEM, Invitrogen) to various concentrations. 6 cm2 plastic tissue culture dishes were coated with 2.5 ml 0.8% agar/FBS (5% or 10%), allowing for polymerization before a middle layer of 1×104 cells in a mixture of 0.4% agar (plus 5% or 10% FBS, 2.5 ml) was added. After polymerization of the middle layer, a final layer of 2.5 ml of 0.8% agar/FBS was layered on top of the cells. Assays were carried out for 4 weeks, with weekly “feedings” of 0.8% agar/FBS/IMEM. Colonies were observed and photographed using a Zeiss Axiovert 100 inverted microscope. Plates were fixed in 10% acetic acid+10% MeOH and stained with 0.1 % crystal violet in 20% MeOH. Colony number was tabulated by scanning the plates and counting manually.
In vivo cell inoculation
Cells were routinely prepared for inoculation by centrifugation and washing twice, followed by resuspension at 5 million cells/100 μl 1× PBS (cells were determined to be at least 90% viable). Cells were then injected subcutaneously into the right dorsal flank of 6-8 week old intact male NCr nu/nu mice (Charles River Laboratories, Wilmington, Mass.). Beginning approximately 2 weeks post-injection, xenografted tumors were measured weekly and monitored for growth and/or necrosis. Animals were sacrificed by CO2 asphyxiation as tumors reached 2 cm3 or before, if they showed any sign of discomfort, paralysis or emaciation. Tumor xenografts were excised and immediately snap frozen in liquid nitrogen, ground into a fine powder and stored for use at −80° C. When needed, powder was lysed in HGK lysis buffer and assayed by western blotting.
Immunofluorescence
Cells were plated on glass cover slips, grown to approximately 70% confluence, fixed with 4% para-formaldehyde (EMS, Ft. Washington, Pa.), permeabilized with 0.2% Triton-X100/PBS and nonspecific sites blocked by incubation with 3% FBS. Incubation with Texas red-conjugated phalloidin (dilution of 1:100) for one hour was followed by counterstaining with 10 ng/ml DAPI (Pierce). Cover slips were washed, mounted onto microscope slides and examined using the Nikon Microphoto FXA microscope.
H1299 is a lung carcinoma cell line that shows slow growth in soft agar. A plasmid encoding wild type HGK, kinase-inactive HGK, or an empty vector were transfected into H1299 cells. G418-resistant clones were isolated expressing active or inactive HGK or vector alone (
HGK is a large kinase with multiple domains in addition to the catalytic domain. To determine if the increase in anchorage-independent cell growth was linked to the kinase activity, we did a second study comparing clones over-expressing the inactive and inactive forms of HGK, along with vector controls.
These same clones were analyzed in parallel for growth in monolayer culture in both high and low serum (
To demonstrate that HGK was important for anchorage-independent growth using another approach, we screened cell lines for the ability to significantly lower the endogenous HGK levels in tumor cells using RNA interference (siRNA). One cell type where we succeeded in lowering HGK levels was in HeLa cervical carcinoma cells. As compared with the control oligos, scrambled HGK oligos, or untreated cells, we were able to observe a reduction in the level of HGK protein (
HGK kinase can promote tumor growth in vivo
To demonstrate that over-expression of active and inactive forms of HGK can also affect H1299 tumor cell growth in vivo, we analyzed the same set of clones tested for in vitro growth in
HGK promotes cell rounding and loss of cell-cell contacts
Immunofluorescence with anti-HGK antisera from two vector clones and four wild type HGK-expressing H1299 clones are shown in
The morphology of 293T transformed embryonic kidney cells, H1299 lung carcinoma, and A375 melanoma cells were examined after infection with adenoviruses expressing wild type HGK, K54R HGK, or vector alone (no insert). In 293T, dramatic morphological changes are apparent (
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| Number | Date | Country | |
|---|---|---|---|
| 60570083 | May 2004 | US |
