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(12) United States Patent (10) Patent No.: US 7,662,770 B2 Kinch (45) Date of Patent: Feb. 16, 2010
(54) LOW MOLECULARWEIGHT PROTEIN WO WOO1? 12172 A1 2, 2001 TYROSINE PHOSPHATASE (LMW-PTP) ASA WO WO 01/12840 A2 2, 2001 DAGNOSTIC AND THERAPEUTIC TARGET WO WOO3,O94859 A2 11/2003 WO WOO3,O993 13 A1 12/2003 75 WO WO 2004/O14292 A2 2, 2004 (75) Inventor: Michael S. Kinch, Laytonville, MD WO WO 2004/O14292 A3 2, 2004 WO WO 2005/051307 A2 6, 2005 (73) Assignee: Purdue Research Foundation, West W W SE A. 3. LaFayette, IN (US) WO WO 2005/055948 A3 6, 2005 - WO WO 2005/056766 A2 6, 2005 (*) Notice: Subject to any disclaimer, the term of this patent is extended or adjusted under 35 OTHER PUBLICATIONS U.S.C. 154(b) by 350 days. Carles-Kinchet al., “Antibody targeting of the EphA2tyrosine kinase inhibits malignant cell behavior.” May 15, 2002 Cancer Research (21) Appl. No.: 10/515,358 62(10):2840-2847. Parket al., “Low-molecular-weight protein tyrosine phosphatase is a (22) PCT Filed: May 22, 2003 positive component of the fibroblast growth factor receptor signaling pathway.” May 15, 2002 Molecular and Cellular Biology (86). PCT No.: PCT/USO3A16269 22(10):3404-3414. Souza et al., “From immune response to cancer: a spot on the low S371 (c)(1), molecular weight protein tyrosine phosphatase.” Apr. 2009 Cellular (2), (4) Date: Aug. 11, 2005 and Molecular Life Sciences 66(6):1140-1153. Available online on Nov. 11, 2008. (87) PCT Pub. No.: WO03/0993 13 Supplementary European Search Report mailed Jun. 8, 2009, for EP Application No. 03734142.7 (3 pgs). PCT Pub. Date: Dec. 4, 2003 Cann, "Genetic clues to dispersal in human populations: retracting the past from the present.” Mar. 2, 2001 Science 291: 1742-1748. O O Garcia-Closas et al., “Collection of genomic DNA from adults in (65) Prior Publication Data epidemiological studies by buccal cytobrush and mouthwash.” Jun. US 2006/0093608A1 May 4, 2006 2001 Cancer Epidemiology, Biomarkers & Prevention 10:687-696. National Center for Biotechnology Information, National Library of Related U.S. Application Data Medicine, National Institutes of Health. GenBank Locus NM 007099, Accession No. NM 007099, Version NM 007099.1 (60) Provisional application No. 60/382.988, filed on May GI:6005987, "Homo sapiens acid phophatase 1, soluble (ACP1), 23, 2002. transport variant b, mRNA. online). Bethesda, MD retrieved on Mar. 26, 2008). Retrieved from the Internet:
OTHER PUBLICATIONS Gilardi-Hebenstreitet al., “An Eph-related Receptor Protein Tyrosine Kinase Gene Segmentally Expressed in the Developing Mouse Andres et al., “Expression of Two Novel eph-related Receptor Pro Hindbrain.” Oncogene, Dec. 1992;7(12):2499-2506. tein Tyrosine Kinases in Mammary Gland Development and Gustafson et al., “Solution Structure of the Low Molecular Weight Carcinogenesis.” Oncogene, May 1994:9: 1461-1467. Protein Tyrosine Phosphatase From Tritrichomonas foetus Reveals a Arakawa et al., “Genomic Organization of the Klebsiella Flexible Phosphate Binding Loop.” Protein Sci., Oct. 2005; pneumoniae cps Region Responsible for Serotype K2 Capsular 14(10):2515-2525. Polysaccharide Synthesis in the Virulent Strain Chedid.” J. Heinrikson, "Purification and Characterization of a Low Molecular Bacteriol. Apr. 1995; 177(7):1788-1796. Available online at Journal Weight Acid Phosphatase from Bovine Liver.” J. Biol. Chem. of Bacteriology retrieved Oct. 25, 2006). Retrieved from the 1969:244(2):299-307. Available online at JBC Online retrieved Oct. Internet: Sajjadiet al., “Identification of a New eph-Related Receptor Tyrosine retrieved on Oct. 25, 2006. Retrieved from the Kinase Gene From Mouse and Chicken That Is Developmentally Internet: Zelinski et al., “EphA2 Overexpression Alters Cellular Adhesions: Zhang, “Crystallographic Structure Determination of Bovine Heart Implications for Metastasis.” Abstract. FASEB Experimental Biol and Human Cytosolic Phosphotyrosyl Phosphatase.” Ph.D. Thesis ogy Conference, Orlando, FL, Mar. 31-Apr. 4, 2001; FASEB.J. Mar. Purdue University, West Lafayette, Indiana, 1995; 173 pgs. 7, 2001; 15(4): A234. Abstract No. 211.4; 4 pages. Zhang et al., “Crystal Structure of Bovine Low Molecular Weight Zelinksiet al., “Estrogen and MycNegatively Regulate Expression of Phosphotyrosyl Phosphatase Complexed with the Transition State the EphA2Tyrosine Kinase.” J. Cell. Biochem., 2002:85(4):714-720. Analog Vanadate.” Biochemistry; Jan. 7, 1997:36(1): 15-23. Zhanget al., “Purification and Characterization of a Low-Molecular Zhang et al., “Crystal Structure of Human Low Molecular Weight Weight Acid Phosphatase—A Phosphotyrosyl-Protein Phosphatase Phosphotyrosyl Phosphatase. Implications for Substrate Specificity.” from Bovine Heart.” Arch. Biochem. Biophy.s., Oct. 1990:282(1):39 J. Biol. Chem. Aug. 21, 1998:273(34):21714-21720. Available 49. online at JBC Online retrieved on Sep. 22, 2006). Retrieved from the Internet: Fig. 1 A-150228 "There is an ATG upstream BSml of the Xbal site. U.S. Patent Feb. 16, 2010 Sheet 2 of 22 US 7,662,770 B2 0 :(Wrd)VOAeN U.S. Patent Feb. 16, 2010 Sheet 3 of 22 US 7,662,770 B2 l h 5 -s D2 MDAMB 231 MDA-MB 435 SK-BR-3 MCF 7 NeOST Neo : s Purified U.S. Patent US 7,662,770 B2 U.S. Patent US 7,662,770 B2 elLej-WMWTJO403A -MAWT d-WINTI V U.S. Patent Feb. 16, 2010 Sheet 7 of 22 US 7,662,770 B2 s d - CO L al a. h. s rt 9 C D - cro 5 E m O) 9. S cC. L. r. C. CN v O. O H/Seluoo) 3 : 3 CN v ve (Ox) equnNeo t an s U.S. Patent Feb. 16, 2010 Sheet 8 of 22 US 7,662,770 B2 U.S. Patent Feb. 16, 2010 Sheet 9 of 22 US 7,662,770 B2 (f) s 2 E D o) 2 CN y O H/SeluooC) t f s U.S. Patent Feb. 16, 2010 Sheet 10 of 22 US 7,662,770 B2 d.id -MWT OI 6h4; U.S. Patent Feb. 16, 2010 Sheet 11 of 22 US 7,662,770 B2 d.Ld-MWT -MAWTI II'64' U.S. Patent Feb. 16, 2010 Sheet 12 of 22 s 5 wild | s U.S. Patent Feb. . 16, 2010 Sheet 13 of 22 US 7,662,770 B2 U.S. Patent Feb. 16, 2010 Sheet 14 of 22 US 7,662,770 B2 CN C R L t p WLSOS W62G A. p WLSD 92 N 90. U.S. Patent Feb. 16, 2010 Sheet 16 of 22 US 7,662,770 B2 coMWT ugeS U.S. Patent Feb. 16, 2010 Sheet 17 of 22 US 7,662,770 B2 ugeS U.S. Patent US 7,662,770 B2 U.S. Patent Feb. 16, 2010 Sheet 19 of 22 US 7,662,770 B2 U.S. Patent Feb. 16, 2010 Sheet 20 of 22 US 7,662,770 B2 o c an as d O) . . c shal CO .s CD -N4 O s O 3. L. () d O didWWI UOOeJSUe U.S. Patent Feb. 16, 2010 Sheet 21 of 22 US 7,662,770 B2 cy an as d O . . s (f) .will C CD E D as y () D cuopoeSue U.S. Patent Feb. 16, 2010 Sheet 22 of 22 US 7,662,770 B2 ZQUOIO IQUOIO€QUOIOZQUOIO JO409A IQUOIO O O O O C d CN O o VO s N (cuu) eunOA US 7,662,770 B2 1. 2 LOW MOLECULARWEIGHT PROTEIN treatment. Accordingly, the invention provides novel cancer TYROSINE PHOSPHATASE (LMW-PTP) ASA diagnostic, prognostic and treatment methods. DAGNOSTIC AND THERAPEUTIC TARGET In one aspect, the invention provides a method for the treatment of cancer in a mammal, preferably a human. In one This application claims the benefit of U.S. Provisional embodiment of the treatment method of the invention, the Application Ser. No. 60/382.988; filed May 23, 2002, which method is useful for treating cancer in a mammal wherein the is incorporated herein by reference in its entirety. cancer cell expresses a low molecular weight protein tyrosine This application also incorporates by reference the follow kinase (LMW-PTP). The treatment method involves admin ing U.S. patent applications in their entirety: Ser. No. 09/640, istering to the mammal a treatment agent effective to inhibit 952, filed Aug. 17, 2000; Ser. No. 09/640,935, filed Aug. 17, 10 the activity of LMW-PTP. 2000; and Ser. No. 09/952,560, filed Sep. 12, 2001. In another embodiment of the treatment method of the invention, the method is useful for treating cancer in a mam BACKGROUND OF THE INVENTION mal wherein the cancer cell expresses a low molecular weight protein tyrosine kinase (LMW-PTP) and an EphA2 receptor Cancer arises when a population of cells gains the ability to 15 molecule. The treatment method involves administering to inappropriately grow and Survive. These biological behaviors the mammal a first treatment agent effective to inhibit the often result from genetic and environmental abnormalities activity of LMW-PTP and a second treatment agent effective that work together to trigger specific signaling pathways that to favorably alter the biological activity of the EphA2 recep promote the inappropriate growth and Survival of malignant tor molecule. Preferably, the biological activity of EphA2 is cells. In particular, protein tyrosine phosphorylation is under favorably altered by increasing the phosphotyrosine content stood to initiate powerful signals that govern many different aspects of cell behavior. A popular paradigm suggests that a of the EphA2 receptor molecule. balance between tyrosine kinase and phosphatase activities The cancer treated using the method of the invention is serves to dictate the cellular levels of protein tyrosine phos preferably a metastatic carcinoma. Optionally in the treat phorylation and thereby governs cellular decisions regarding 25 ment method of the invention, the treatment agent is growth, Survival and invasiveness. This paradigm generally covalently linked to a cytotoxic agent. predicts that tyrosine kinases would be oncogenic whereas Overexpression of LMW-PTP is indicative of the presence tyrosine phosphatases negatively regulate malignant transfor of cancer cells in the mammal. Thus, in another aspect, the mation. Although this portioning is generally correct, emerg invention provides a method for diagnosis of cancer in a ing evidence reveals a more complex interplay between 30 mammal based upon overexpression of LMW-PTP. In one tyrosine kinases and phosphatases. For example, the PTP embodiment, the diagnostic method of the invention involves CAAX tyrosine phosphatase has been recently shown to lysing cells in a biological material obtained from the mam function as a powerful oncogene. Moreover, the enzymatic mal to yield a cell lysate; contacting the cell lysate with a activity of Src family kinases is liberated by phosphatase diagnostic agent that binds LMW-PTP to form a bound com mediated dephosphorylation of important tyrosine residues. 35 plex; detecting the bound complex; and determining whether In the latter situation, phosphatases can actually up-regulate LMW-PTP is overexpressed in the biological sample relative protein tyrosine phosphorylation by increasing the enzymatic to a noncancerous biological material. Optionally, the method activity of kinases: also includes obtaining the biological material from the mam The EphA2 receptor tyrosine kinase is overexpressed in a mal. In another embodiment of the diagnostic method of the large number of human cancers. High levels of EphA2 apply 40 invention, expression levels of LMW-PTP are analyzed by to a large number of different cancers, including breast, pros assaying the biological material to determine whether LMW tate, colon and lung carcinomas as well as metastatic mela PTP is overexpressed in the biological material relative to a nomas. Moreover, the highest levels of EphA2 are consis noncancerous biological sample. This diagnostic method can tently found on the most aggressive cell models of human be performed in the mammal or outside of the mammal. Any cancer. EphA2 is not simply a marker of malignant disease as 45 biological material of the mammal can be analyzed, for ectopic overexpression of EphA2 is sufficient to confer tum example a tissue, organ or fluid of the mammal. origenic and metastatic upon non-transformed epithelial In yet another aspect, the invention provides a screening cells. method for evaluating the efficacy of a candidate cancer Cancer cells also display differences in EphA2 function as therapeutic agent that targets the EphA2 receptor molecule. compared with non-transformed epithelia. Despite being 50 In one embodiment, the screening method involves contact present at relatively low levels in non-transformed epithelial ing a cancer cell expressing EphA2 receptor molecule and cells, EphA2 in these cells is prominently tyrosine phospho LMW-PTP with a candidate therapeutic agent to yield a rylated. In contrast, the EphA2 in malignant cells is not treated cancer cell; determining the amount or activity of tyrosine phosphorylated even though it is grossly overex LMW-PTP in the treated cancer cell; and comparing the pressed in these cells. These differences in EphA2 phospho 55 amount or activity of LMW-PTP in the treated cancer cell tyrosine content are important because tyrosine phosphory with the amount or activity of LMW-PTP in an analogous lated EphA2 negatively regulates tumor cell growth and untreated cancer cell. A reduction in the amount or activity of invasiveness whereas unphosphorylated EphA2 promotes LMW-PTP in the treated cell is indicative of the efficacy of these same behaviors in malignant cells. The association of EphA2 targeting of the candidate therapeutic agent. EphA2 with malignancy is further detailed in international 60 patent applications WO 01/12172 and WO 01/12804. BRIEF DESCRIPTION OF THE FIGURES SUMMARY OF THE INVENTION FIG. 1 shows a schematic map of the eukaryotic expression vector pcDNA3, a 5.4 kb mammalian expression vector. The present invention identifies low molecular weight pro 65 Unique restriction sites are indicated. The HPTP gene was tein tyrosine phosphatase (LMW-PTP) as a novel diagnostic cloned into the HindIII/BamHI sites of this vector. Expres and therapeutic target in cancer diagnosis, prognosis and sion of the gene was driven by the CMV promoter. US 7,662,770 B2 3 4 FIG. 2 shows that EphA2 is regulated by an associated FIG. 7 shows that LMW-PTP enhances malignant charac phosphatase. (A) Monolayers of MCF-10A human mammary ter. (A) To evaluate anchorage-dependent cells growth, 1x10 epithelial cells were incubated in the presence or absence control or LMW-PTP transfected MCF-10A cells were (denoted as “C” for control) of 4 mM EGTA for 20 minutes seeded into monolayer culture and cell numbers were evalu before detergent extraction. The samples were resolved by ated microscopically at the intervals shown. (B) In parallel SDS-PAGE and probed with phosphotyrosine-specific anti studies, the control and LMW-PTP transfected cells were bodies (PY20 and 4G10; top). The membranes were stripped Suspended in soft agar. Shown is colony formation (per high and reprobed with EphA2 specific antibodies to confirm powered field) after five days of incubation at 37°C. These equal sample loading (below). (B) MCF-10A cells were results were representative of at least three separate experi treated with EGTA, as detailed above, in the presence of 10 ments. * Indicates p-0.01. absence of NaVO to inhibit phosphatase activity. (B)Eph A2 FIG. 8 shows that EphA2 retains enzymatic activity in was immunoprecipitated from MDA-MB-231 cells that had LMW-PTP transformed cells. Equal amounts of EphA2 were been incubated in the presence of the indicated concentra immunoprecipitated from control or LMW-PTP transformed tions of NaVO for 10 minutes at 37° C. MCF-10A cells and subjected to in vitro kinase assays. (A) FIG.3 shows that LMW-PTP protein levels are elevated in 15 Autophosphorylation with Y-P-labeled ATP was evaluated malignant cell lines. Detergent lysates (lanes 2-7) were har by autoradiography. To confirm equal sample loading, a por vested from non-transformed (MCF-10Aneo), oncogene tion of the immunoprecipitated materials was evaluated by transformed (MCF-10AneoST), and tumor derived (MCF-7, Western blot analyses with (B) EphA2 or (C) phosphoty SK-BR-3, MDA-MB-435, MDA-MB-231) mammary epi rosine antibodies. Whereas EphA2 is not tyrosine phospho thelial cells. The samples were resolved by SDS-PAGE and rylated in LMW-PTP transformed cells, it retains enzymatic subjected to Western Blot analysis using LMW-PTP specific activity. Note that equal amounts of EphA2 were utilized for antibodies (top). Purified LMW-PTP (lane 1) provided a posi these results to overcome differences in endogenous EphA2 tive control for western blot analyses. The membranes were expression (for example, See FIG. 5B). then stripped and reprobed with antibodies specific to Vincu FIG.9 shows that malignant transformation by LMW-PTP linto evaluate sample loading (bottom). Note that LMW-PTP 25 is related to EphA2 overexpression. MCF-10A cells were is overexpressed in tumor-derived cells despite the relative treated with EphA2 antisense (AS oligonucleotides, with over-loading of the non-transformed (MCF-10Aneo) inverted antisense (IAS) oligonucleotides or transfection samples. reagents alone providing negative controls. (A) Western blot FIG. 4 shows that EphA2 and LMW-PTP form a molecular analysis using EphA2 specific antibodies confirmed that the complex in vivo. (A) Complexes of EphA2 were immunopre 30 antisense treatment decreased EphA2 protein levels (top). cipitated from 5x10 MCF-10A or MDA-MB-231 cells, The membrane was then stripped and reprobed for B-catenin resolved by SDS-PAGE and subjected to Western blot analy to confirm equal sample loading (bottom). (B) Parallel ses with antibodies specific for LMW-PTP. (B) To confirm samples were suspended and incubated in Softagar for 5 days. complex formation, complexes of LMW-PTP were similarly Shown are the average number colonies per high-powered isolated by immunoprecipitation and probed with Eph A2 35 microscopic field (HPF). * Indicates p<0.01. specific antibodies. FIG. 10 shows that LMW-PTP overexpression alters two FIG. 5 shows that EphA2 can serve as a substrate for dimensional morphology in transfected MCF-10A cells. LMW-PTP in vitro. EphA2 was immunoprecipitated from FIG. 11 shows that LMW-PTP overexpressing transfected MCF-10A cells form foci at high cell density. 5x10 MCF-10A cells before incubation with the indicated 40 amounts of LMW-PTP protein for 0-30 minutes at 37° C. The FIG. 12 shows that LMW-PTP inactivation in transformed samples were then resolved by SDS-PAGE and subjected to cells results in decreased soft agar colonization. Western blot analysis with phosphotyrosine-specific antibod FIG. 13 shows that LMW-PTP inactivation in transformed ies. The membranes were stripped and reprobed with Eph A2 cells alters two-dimensional morphology and EphA2 distri specific antibodies to confirm equal sample loading. 45 bution. FIG. 6 shows that LMW-PTP dephosphorylates EphA2 in FIG. 14 shows the results of EGTA treatment of trans vivo. (A) MCF-10A cells were stably transfected with expres formed cells that have been transfected with D129A to inac sion vectors that encode for wild-type LMW-PTP. Detergent tivate LMW-PTP lysates were resolved by SDS-PAGE and subjected to West FIG. 15 shows a summary of immunofluorescence find ern blot analyses with LMW-PTP antibodies to confirm 50 ings. LMW-PTP overexpression, with purified LMW-PTP provid FIG. 16A shows co-localization of EphA2 and LMW-PTP ing a positive control. Parallel samples were then probed with in control and transfected MCF-10A cells. antibodies specific for C-catenin as a loading control. (B) FIG. 16B shows co-localization of EphA2 and LMW-PTP EphA2 was immunoprecipitated and Western blot were per intransformed cells that have been transfected with D129A to formed using EphA2 (top) and P-Tyr (bottom)-specific anti 55 inactivate LMW-PTP. bodies. (C) The overall levels of phosphotyrosine in control and LMW-PTP-transfected cells were compared using spe FIG. 17 shows that altered organization of actin cytoskel cific antibodies. Note that equal amounts of EphA2 were eton relates to LMW-PTP expression and function. utilized for these results to overcome differences in endog FIG. 18 shows that altered focal adhesion formation relates enous EphA2 expression (in contrast to part B). (D) The 60 to LMW-PTP expression and function. protein levels (top) and phosphotyrosine content of EphA2 in FIG. 19 shows cytokeratin expression altered by LMW MDA-MB-231 cells that had been transfected with a domi PTP expression. nant negative form of LMW-PTP (D129A) or a matched FIG. 20 shows vimentin expression altered by LMW-PTP vector control were evaluated by Western blot analyses. Note expression. the consistent findings that LMW-PTP activity relates to 65 FIG. 21 shows data relating to tumor development in mice decreased EphA2 phosphotyrosine content and increased injected with 5x10 EphA2 overexpressing MCF-10A cells EphA2 protein levels. and controls, 20 days after injection. US 7,662,770 B2 5 6 DETAILED DESCRIPTION OF THE PREFERRED Cell Acid Phosphatase: Purification and Properties of the A. EMBODIMENTS B, and CIsozymes. Biochem. Biophys. Acta. 1041,232-242: Waheed, A., Laidler, P. M., Wo, Y.-Y. P., and Van Etten, R. L. Tyrosine phosphorylation is controlled by cell membrane (1988), “Purification and Physiochemical Characterization of tyrosine kinases (i.e., enzymes that phosphorylate other pro a Human Placental Acid Phosphatase Possessing Phosphoty teins or peptides), and increased expression of tyrosine rosyl Protein Phosphatase Activity. Biochemistry 27, 42.65 kinases is known to occur in metastatic cancer cells. We have 4273; Boivin, P. and Galand, C. (1986), “The Human Red made the Surprising finding, however, that an enzyme that Cell Acid Phosphatase Is a Phosphotyrosine Protein Phos catalyzes the reverse reaction, dephosphorylation, is a pow phatase Which Dephosphorylates the Membrane Protein erful oncoprotein. This enzyme is low molecular weight pro 10 Band 3.’ Biochem. Biophys. Res. Commun. 134, 557-564), tein tyrosine phosphatase (LMW-PTP), and its oncogenic and BPTP (Zhang, Z-Y. and Van Etten, R. L. (1990), “Puri potential is in at least some instances linked to the receptor fication and Characterization of a Low-Molecular Weight tyrosine kinase EphA2, which is also implicated in oncogen Acid Phosphatase—A Phosphotyrosyl Protein Phosphatase esis and metastasis. from Bovine Heart.” Arch. Biochem. Biophys. 282, 3949; LMW-PTP is thus established as a new and unusual target 15 Chernoff, J. and Li, H.-C. (1985), A Major Phosphotyrosyl for treatment methods directed to cancer therapy. LMW-PTP Protein Phosphatase From Bovine Heart is Associated with a can be targeted either alone or in combination with treatments Low-Molecular-Weight Acid Phosphatase. Arch. Biochem. that target EphA2, other oncogenic tyrosine kinases, or other Biophys. 240, 135-145). These proteins, as well as other oncogenes or oncoproteins. LMW-PTP levels can also serve PTPases, share a common active site sequence motif. Cys as a marker in cancer detection, or as a surrogate marker to (Xaa)s-Arg. Some proteins that share a high degree of analyze the impact of treatments that target EphA2 or other sequence identity with the higher vertebrate enzymes include tyrosine kinases associated with the development or progres the low molecular weight PTPases from Escherichia coli sion of cancer. (Stevenson, G. Andrianopopoulos, K. Hobbs, M., and Reeves, P. R. (1996), “Organization of the Escherichia coli Low Molecular Weight Protein Tyrosine Phosphatase 25 K-12 Gene Cluster Responsible for Production of the Extra Protein tyrosine phosphatases (sometimes also referred to cellular Polysaccharide Colanic Acid.” J. Bact. 178, 4885 phosphotyrosine phosphatases), known as PTPases, catalyze 4893), Klebsiella (Arakawa, Y. Washarotayankun, R., Nagat the hydrolysis of phosphomonoesters, specifically, the suka, T., Ito, H., Kato, N., and Ohta, M. (1995), “Genomic dephosphorylation of protein phosphotyrosyl residues. There Organization of the Klebsiella pneumoniae CPS Region are three major classes of PTPases: dual-specificity PTPases, 30 Responsible for Serotype K2 Capsular Polysaccharide Syn high molecular weight PTPases and low molecular weight thesis in the Virulent Strain Chedid, J. Bacteriol. 177, 1788 PTPases (Zhang, M., Stauffacher, C., and Van Etten, R. L. 1796), Synechococcus (Wilbanks, S. M. and Glazer, A. N. (1995), “The Three Dimensional Structure, Chemical (1993), “Rod Structure of a Phycoerythrin II-containing Phy Mechanism and Function of the Low Molecular Weight Pro cobilisdome. I. Organization and Sequence of the Gene Clus tein Tyrosine Phosphatase. Adv. Prot. Phosphatases 9, 1-23). 35 ter Encoding the Major Phycobilirotein Rod Components in Several different acronyms are used interchangeably for low the Genome of Marine Synechococcus sp. WH8020.” J. Biol. molecular weight (LMW) PTPase and include LMW-PTP, Chem. 268, 1226-1234), and Tritrichomonas foetus (gb LMW PTP, LMW-PTPase and LMW PTPase. U66070). LMW-PTPs represent a family of PTPases that includes Some mammalian low molecular weight PTPases exist as members isolated from many different organisms. They typi 40 isozymes. Within specific species, the amino acid sequence cally have a relative molecular mass of about 18 kD. Members identity between the isozymes is greater than 95%. One such of the LMW-PTP family found in higher organisms include species is human, where the human red cell protein tyrosine bovine (Heinrikson, R. L. (1969), “Purification and Charac phosphatase (HPTP) is expressed. The two forms of this terization of a Low Molecular Weight Acid Phosphatase from protein, A (fast) and B (slow), differ in their electrophoretic Bovine Liver.” J. Biol. Chem. 244, 299-307), Erwinia Burg 45 mobility when resolved during starch gel electrophoresis. ert, P. and Geider, K. (1997), “Characterization of the ams I Except for the variable region, residues 40-73, the isozymes Gene Product as a Low Molecular Weight Acid Phosphatase have an identical amino acid sequence. Controlling Exopolysaccharide Synthesis of Erwinia Amylo The human isozymes (A and B) have a high level of amino vora,' FEBS Lett. 400, 252-256), budding yeast (Ltp1) (Osta acid sequence identity when compared to BPTP, 81% and nin, K., Pokalsky, C. Wang, S., and Van Etten, R. L. (1995), 50 94%, respectively. The crystal structure of BPTP, the proto “Cloning and Characterization of a Saccharomyces cerevi type of low molecular weight PTPases, has been solved siae Gene Encoding the Low Molecular Weight Protein-Ty (Zhang, M., Van Etten, R. L., and Stauffacher, C. V. (1994), rosine Phosphatase.” J. Biol. Chem. 270, 18491-18499), fis “Crystal Structure of Bovine Heart Phosphotyrosyl Phos sion yeast (Stp1) Mondesert, O. Moreno, S., and Russell, P. phatase at 2.2-A Resolution. Biochemistry 33, 11097 (1994), “Low Molecular Weight Protein Tyrosine Phos 55 11105). The structure consists of C-helices on both sides of a phatases are Highly Conserved Between Fission Yeast and four-stranded central parallel B-sheet. This structure incorpo Man.” J. Biol. Chem. 269,27996-27999), rat ACP1 and ACP2 rates a portion of a Rossman fold, the classic nucleotide isozymes (Manao, G., PaZZagli, L., Cirri, P., Caselli, A., binding fold consisting in part of two right-handed BOB Camici, G., Cappugi, G., Saeed, A., and Ramponi, G. (1992), motifs. The crystal structure of HPTP-A and yeast LTP1 have “Rat Liver Low M. Phosphotyrosine Protein Phosphatase 60 been solved (Wang, S., Stauffacher, C. and Van Etten, R. L. Isoenzymes: Purification and Amino Acid Sequences. J. (2000), "Structural and Mechanistic Basis for the Activation Prot. Chem. 11, 333-345), human (HPTP) (Wo, Y-Y. P. of a Low Molecular Weight Protein Tyrosine Phosphatase by Zhou, M.-M., Stevis, P., Davis, J. P. Zhang, Z.-Y., and Van Adenine.” Biochemistry 39, 1234-1242; Zhang, M. (1995), Etten, R. L. (1992), “Cloning. Expression, and Catalytic Ph.D. Thesis, Purdue University), and resemble BPTP. Low Mechanism of the Low Molecular Phosphotyrosyl Protein 65 molecular weight PTPases have eight conserved cysteines Phosphatase From Bovine Heart.” Biochemistry 31, 1712 (all in free thiol form), seven conserved arginines, and two 1721: Dissing, J. and Svensmark, O. (1990), “Human Red conserved histidines (Davis, J. P. Zhou, M. M., and Van US 7,662,770 B2 7 8 Etten, R. L. (1994), “Kinetic and Site-Directed Mutagenesis EphA2 has characteristic differences in normal and trans Studies of the Cystein Residues of Bovine Low Molecular formed breast epithelia (Zantek, N. D. (1999), Ph.D. Thesis, Weight Phosphotyrosyl Protein Phosphatase.J. Biol. Chem. Purdue University). In normal breast epithelia, EphA2 is 269,8734-8740). present in low protein levels, it is tyrosine phosphorylated, Tyrosine-phosphorylated proteins and peptides, as well as and, finally, it is localized in the sites of cell-cell adhesion. In simpler molecules such as phosphotyrosine and pNPP. are all transformed breast epithelia, high protein levels of EphA2 candidates for substrates of the low molecular weight exist, it is no longer tyrosine phosphorylated, and it is local PTPases. ized in the membrane ruffles. Natural and synthetic inhibitors of these enzymes also EphA2 has been found to have a functional role in cancer. exist. Among the strongest inhibitors of low molecular weight 10 When overexpressed, EphA2 is a powerful oncoprotein (Ze PTPases are the ions Vanadate, tungstate, and molybdate. linski. D. P. Zantek, N. D., Stewart, J., Irizarry, A. & Kinch, M. S. (2001) Cancer Res 61,2301-2306). Overexpression of EphA2 Receptor Tyrosine Kinase EphA2 in MCF-10A cells causes malignant transformation. EphA2, a 130 kD protein, is a member of the largest family Also, injection of these overexpressing cells into nude mice of receptor tyrosine kinases (Andres, A. C., Reid, H. H., 15 causes tumors. Interestingly, the EphA2 in cancer cells and in Zurcher, G., Blaschke, R. J., Albrecht, D., and Ziemiecki, A. EphA2-overexpressing cells is not tyrosine phosphorylated, (1994), “Expression of Two Noveleph-related Receptor Pro whereas EphA2 in nontransformed cells is tyrosine phospho tein Tyrosine Kinases in Mammary Gland Development and rylated. Carcinogenesis. Oncogene 9, 1461-1467; Lindberg et al., LMW-PTP shown hereinto regulate EphA2, has also been Mol. Cell. Biol. 10:6316-6324 (1990)). It is expressed prima shown to interact with another member of the Eph family, rily in cells of epithelial cell origin such as breast, lung, ovary, EphB1 (Stein, E., Lane, A. A. Cerretti, D. P. Schoecklmann, colon, etc. This protein, also known as ECK, Myk2, and Sek2. H. O., Schroff, A. D., Van Etten, R. L., and Daniel, T. O. was isolated from an erythropoietin-producing hepatocellu (1998), “Eph Receptors Discriminate Specific Ligand Oligo lar carcinoma cell line (Hirai, H., Maru, Y. Hagiwara, K., mers to Determine Alternative Signaling Complexes, Attach Nishida, J., and Takaku, F. (1987), “A Novel Putative 25 ment, and Assembly Responses.” Genes & Dev. 12, 667-678). Tyrosine Kinase Receptor Encoded by the Eph Gene.” Sci ence 238, 1717-1720). Due to multiple names and a growing Therapeutic Inhibition of LMW-PTP Activity family of different but related Eph proteins, a nomenclature Low-molecular weight protein tyrosine phosphatase committee met to officially name the proteins (Eph. Nomen (LMW-PTP) is overexpressed in a large number of tumor clature Committee (Flanaga, J. G., Gale, N.W., Hunter, T., 30 cells. The Examples below demonstrate that the phosphoty Pasquale, E. B., and Tessier-Lavgne, M.) (1997), “Unified rosine content of EphA2 is negatively regulated by LMW Nomenclature for Eph Family Receptors and Their Ligands, PTP establishing a role for this phosphatase in oncogenesis. the Ephrins. Cell 90, 403-404). The proteins were named They further demonstrate that overexpression of LMW-PTP either Eph A or EphB, depending on whether they bind induces a concomitant increase in EphA2 levels and is suffi ligands that are GPI-linked or transmembrane, respectively. 35 cient to confer malignant transformation upon non-trans EphA proteins bindephrin-Aligands, whereas EphB proteins formed epithelial cells. Oncogenesis that is associated with bind ephrin-B ligands. The number represents the order in increased activation or expression of LMW-PTP (whether or which they were discovered. not the cancer cells express EphA2) can be treated or pre Different methods have been used to isolate EphA2. First, vented by inhibiting the activity of LMW-PTP in accordance hybridization techniques were used to isolate EphA2 from 40 with the invention. DNA libraries (Lindberget al., Mol. Cell. Biol. 10:6316-6324 These findings establish LMW-PTP as a target for prophy (1990); Hirai, H., Maru, Y., Hagiwara, K., Nishida, J., and lactic and therapeutic methods. By inhibiting the activity of Takaku, F. (1987), “A Novel Putative Tyrosine Kinase Recep LMW-PTP, dephosphorylation of EphA2 can be slowed or tor Encoded by the Eph Gene.” Science 238, 1717-1720). prevented, thereby favorably altering the activity of EphA2 Secondly, the polymerase chain reaction (PCR) was 45 and preventing or reversing cancer progression. employed using primers for the kinase domain (Andres, A.C., Treatments that result in an inhibition in the activity of Reid, H. H., Zurcher, G., Blaschke, R. J., Albrecht, D., and LMW-PTP are therefore expected to be accompanied by a Ziemiecki, A. (1994), “Expression of Two Noveleph-related favorable change in the disease state of a cancer patient. Receptor Protein Tyrosine Kinases in Mammary Gland Favorable changes in the disease state of a cancer patient Development and Carcinogenesis.” Oncogene 9, 1461-1467; 50 include, for example, a reduction in the tumor burden, a Gilardi-Hebenstreit, P., Nieto, M.A., Frain, M., Mattei, M. slowing of tumor growth, prevention or deferral of disease G., Chestier, A., Wilkinson, D. G., and Charnay, P. (1992), stage progression and prevention or deferral of metastasis. “An Eph-related Receptor Protein Tyrosine Kinase Gene Seg Favorable changes in the disease state of a patient can be mentally Expressed in the Developing Mouse Hindbrain.” detected using any convenient method including radiography, Oncogene 7, 1499-2506). Next, cDNA expression libraries 55 Sonography, biochemical assay, and the like. were probed with antibodies specific for phosphotyrosine The invention thus provides a method for treating cancer in (Zhou, R., Copeland, T. D. Kromer, L. F., and Schulz, N. T. a mammal, preferably a human. The method is also well (1994), “Isolation and Characterization of Bsk, a Growth Suited for veterinary applications such as treatment of cancer Receptor-like Tyrosine Kinase Associated with the Limbic in a pet such as a cat or a dog. The method is effective to treat System. J. Neuro. Res. 37, 129-143). Lastly, monoclonal 60 a cancer characterized by cells that overexpress LMW-PTP, antibodies were screened against proteins that are tyrosine particularly metastatic carcinoma cells of the breast, prostate, phosphorylated in oncogenic transforming cells (Zantek, N. colon, lung, bladder, ovary, pancreas and skin (melanoma) D. (1999), Ph.D. Thesis, Purdue University). that additionally possess overexpressed or functionally EphA2 binds ligands known as ephrinA, with the physi altered EphA2 tyrosine kinase receptor (see, e.g., Kinchet al., ological ligand identified as Ephrin A1. Ligand binding 65 Clin. Cancer Res., 2003, 9(2):613-618: Kinch et al., Clin. induces tyrosine phosphorylation of the Eph protein. EphA2, Exp. Metastasis, 2003, 2001):59-68; Walker-Daniels et al., in particular, is able to bind five different ephrin ligands. Am J. Pathol., 2003, 162(4): 1037-1042: Zelinski, D. P. US 7,662,770 B2 10 Zantek, N. D., Stewart, J., Irizarry, A. & Kinch, M. S. (2001) binding of LMW-PTP to its substrate, particularly those that Cancer Res 61, 2301-2306; Zantek, N. D. (1999), Ph.D. interfere with EphA2-LMW-PTP interactions. Small mol Thesis, Purdue University). A treatmentagent that inhibits the ecules that resemble pyridoxyl phosphate are particularly biological activity of LMW-PTP is introduced into the mam preferred. Such as those that Substitute phosphonic acid or mal, either systemically or at the site of a cancer tumor, in an 5 Sulfonic acid for the phosphate group in pyridoxal phosphate. amount effective to inhibit the biological activity of EphA2. The active site of LMW-PTP can be targeted, particularly Optionally, the treatment agent can be linked to a drug, pref Tyr131, Tyr132 and Asp129. For example, as shown in the erably a cytotoxic drug, thereby possessing the dual activities examples below, the substrate-trapping mutant LMW-PTP of inhibiting LMW-PTP and serving as a carrier molecule for protein D129A (Asp to Ala at position 129) effectively com the cytotoxic drug. Where the resulting molecular complex 10 petes against wild-type LMW-PTP to restore normal epithe includes a cleavable therapeutic agent, treatment can include lial morphology to transformed cells. Because the BPTP delivery of yet another agent to effect cleavage. X-ray crystal structure has been solved, rational drug design “Inhibition' in LMW-PTP activity can be assessed in com can be used to identify or design highly specific inhibitors of parison to LMW-PTP activity prior to treatment. Typically LMW-PTP which are expected to be especially useful thera this is assessed in a laboratory setting using cell lines such as 15 peutically. those described in the Examples below. Administration to a A preferred embodiment of the treatment method includes patient of a treatment agent that causes inhibition of LMW administration to cancer patient of a first treatment agent that PTP activity in a laboratory setting in model systems rou targets LMW-PTP and a second treatment agent that targets tinely used for human cancer research is fully expected to EphA2. The treatment agents may be administered in any cause inhibition of LMW-PTP activity in the patents cells in order or may be administered simultaneously (co-administra vivo. It should be understood that the method of the invention tion). Multiple treatment agents that target LMW-PTP or is not limited by the way in which, or the extent to which, EphA2 may be administered. LMW-PTP activity is inhibited in the target cells. The treatment agent that targets EphA2 can be, for Methods for inhibiting LMW-PTP activity include, for example, an antibody, a Small molecule, a peptide, a ligand or example, those that act directly on the gene encoding one or 25 ligand mimic, or an antisense nucleic acid. In one aspect of more of the LMW-PTP enzymes (such as HPTP-A and the method, the second treatment agent “activates’ EphA2 by HPTP-B), those that act on the mRNA transcript produced by binding to an extracellular epitope on the receptor molecule. the gene, those that interfere with the translation of the mRNA Ligand-mediated activation is characterized by increased transcript into the protein, and those that directly impair the EphA2 phosphotyrosine content and is accompanied by a activity of the translated protein. 30 favorable alteration in EphA2 activity. A “favorable alteration Transcription of a gene can be impeded by delivering to the in EphA2 activity” means a change in the activity, number cellan antisense DNA or RNA molecule or a double stranded (i.e., protein levels) and/or function of EphA2 receptors in RNA molecule. Examples include siRNA and iRNA. Another cancer cells So as to arrest or reverse cell growth or prolifera way the activity of an enzyme can be inhibited is by interfer tion, or to initiate or cause killing of the cancer cell. Arrest or ing with the mRNA transcription product of the gene. For 35 reversal of cell growth or proliferation can be evidenced by example, a ribozyme (or a DNA vector operably encoding a various phenotypic changes in the cancer cells such as ribozyme) can be delivered to the cell to cleave the target increased differentiation, decreased affinity for ECM pro mRNA. Antisense nucleic acids and double stranded RNAs teins, increased cell-cell adhesions, slower growth rate, may also be used to interfere with translation. reduced numbers of EphA2 and/or increased localization of The invention is not limited to the method used to deliver 40 EphA2, decreased cell migration or invasion, and can be the treatment agent. When the treatment agent is a polypep caused either directly or indirectly. Optionally, the second tide, such as D129A LMW-PTP, it can be conveniently deliv treatmentagent causes EphA2 crosslinking, and/or and accel ered in a gene therapy embodiment by introducing into the eration in the degradation of EphA2. In another aspect of the cell a DNA molecule operably encoding the treatment agent invention, the second treatment agent reduces expression of such that it is transcribed and translated once delivered to the 45 EphA2 in a target cancer or precancerous cell at the DNA/ cell. The DNA can be naked DNA or it can be provided as the RNA level, for example via the binding of an antisense oli part of a vector. The vector can be viral or nonviral (e.g., gonucleotide. plasmid or cosmid), integrating or nonintegrating. Examples of viral vectors include retroviral vectors and adenoviral vec Detection of Cancer or Precancerous Conditions tOrS. 50 LMW-PTP can also serve as a marker for cancer or pre Peptides, ligands, ligand mimics, peptidomimetic com cancerous conditions in a mammal, preferably a human. The pounds and other Small molecules are examples of those that invention therefore also includes a method for diagnosing a can be used to directly compromise the activity of the trans cancerous or precancerous condition, or staging a cancer, by lated protein. Optionally, these agents can be introduced detecting and, optionally, quantifying the amount or activity using a delivery vehicle Such as a liposome. Alternatively, a 55 of LMW-PTP in a biological sample. The diagnostic method proteinaceous intracellular agent that inhibits the activity of of the invention can be used to obtain or confirm an initial LMW-PTP can be delivered as a nucleic acid, for example as diagnosis of cancer, or to provide information on cancer RNA, DNA, or analogs or combinations thereof, using con localization, cancer metastasis, or cancer prognosis. The ventional methods, wherein the therapeutic polypeptide is method is applicable to both human and veterinary medical encoded by the nucleic acid and operably linked to regulatory 60 USS. elements such that it is expressed in the target mammalian In one embodiment of the diagnostic method, a biological cell. sample such as a tissue, organ or fluid that has been obtained Preferred treatment agents for use in inhibiting LMW-PTP from the mammal is analyzed. The method optionally activity include Small molecules, peptides, antisense oligo includes the step of removing the biological material from the nucleotides, and Substrate mimics (e.g., non-hydrolyzable or 65 mammal. Cells present in the biological material are lysed, Substrate trapping inhibitors). Treatment agents can include and the lysate is contacted with a polyclonal or monoclonal antagonists that resemble substrate or that interfere with the LMW-PTP antibody. The resulting antibody/LMW-PTP US 7,662,770 B2 11 12 bound complex is either itself detectable or capable of asso concerning the presence or absence of cancer and the State of ciating with another compound to form a detectable complex. the disease. For example, the use of anti-EphA2 or phospho Bound antibody can be detected directly in an ELISA or tyrosine-specific antibodies provides additional data for similar assay; alternatively, the diagnostic agent can comprise determining detecting or evaluating malignancies. a detectable label, and the detectable label can be detected using methods known in the art. LMW-PTP Activity as an Indicator of the Efficacy of Cancer In embodiments of the diagnostic method wherein LMW Treatments PTP is detected via the binding of a detectably labeled diag LMW-PTP can serve as a surrogate marker to evaluate the nostic agent Such as an antibody, preferred labels include efficacy of cancer therapeutic agents, particularly those that chromogenic dyes, fluorescent labels and radioactive labels. 10 target EphA2. The amount or activity of LMW-PTP in a Among the most commonly used chromagens are 3-amino cancer cell that overexpresses LMW-PTP (the control) is 9-ethylcarbazole (AEC) and 3,3'-diaminobenzidine tetrahy compared to the amount or activity of LMW-PTP in an analo drocholoride (DAB). These can be detected using light gous cancer cell that has been treated with a candidate thera microscopy. peutic agent. Reduction in the amount or activity of LMW The most commonly used fluorescent labeling compounds 15 PTP in the treated cell is indicative of an efficacious cancer are fluorescein isothiocyanate, rhodamine, phycoerythrin, treatment. phycocyanin, allophycocyanin, o-phthaldehyde and fluores The present invention is illustrated by the following camine. Chemiluminescent and bioluminescent compounds examples. It is to be understood that the particular examples, Such as luminol, isoluminol, theromatic acridinium ester, imi materials, amounts, and procedures are to be interpreted dazole, acridinium salt, oxalate ester, luciferin, luciferase, broadly in accordance with the scope and spirit of the inven and aequorin also may be used. When the fluorescent-labeled tion as set forth herein. antibody is exposed to light of the proper wavelength, its presence can be detected due to its fluorescence. EXAMPLES Radioactive isotopes which are particularly useful for labeling the antibodies of the present invention include H, 25 Example I I, I, S. P. and C. The radioactive isotope can be detected by Such means as the use of a gamma counter, a Protein-Protein Interactions Involving Low Scintillation counter, or by autoradiography. Molecular Weight Protein Tyrosine Phosphatase and Antibody-antigen complexes can be detected using west Eph A2 ern blotting, dot blotting, precipitation, agglutination, 30 enzyme immunoassay (EIA) or enzyme-linked immunosor Materials and Methods bent assay (ELISA), immunohistochemistry, in situ hybrid Protein Production. Ampicillin, N-Z-amine A (casein ization, flow cytometry on a variety of tissues or bodily fluids, hydrolysate), IPTG, and SP-Sephadex C-50 all were obtained and a variety of sandwich assays. These techniques are well from Sigma. The SP-Sephadex G-50 was purchased from known in the art. See, for example, U.S. Pat. No. 5,876,949, 35 hereby incorporated by reference. In an enzyme immunoas Pharmacia The YM3 membranes were from Amicon. All say (EIA) or enzyme-linked immunosorbent assay (ELISA), other materials were purchased either from Sigma or BioRad. the enzyme, when Subsequently exposed to its Substrate, Cell lines. The cell models used for this study were breast reacts with the Substrate and generates a chemical moiety epithelia. A commonly used cell line in this research labora which can be detected, for example, by spectrophotometric, 40 tory is MCF-10A. This cell line is a part of the family of fluorometric, or visual means. Enzymes which can be used to MCF-10 cells, an established immortal human mammary detectably label antibodies include, but are not limited to epithelial cell line. MCF-10 cells were isolated from the malate dehydrogenase, staphylococcal nuclease, delta-5-ste mammary tissue of an adult woman who had fibrocystic roid isomerase, yeast alcohol dehydrogenase, alpha-glycero disease. MCF-10A cells grow as attached cells. The MCF phosphate dehydrogenase, triose phosphate isomerase, 45 10A (Neo) cell line is the parent cell line, MCF-10A, with a horseradish peroxidase, alkaline phosphatase, asparaginase, neomycin resistance gene. The MDA-MB-231 cell line is a glucose oxidase, beta-galactosidase, ribonuclease, urease, highly invasive and metastatic mammary cell line. These cells catalase, glucose-6-phosphate dehydrogenase, glucoamy were isolated from an adult woman with breast cancer. lase, and acetylcholinesterase. Other methods of labeling and Care for these cells consist of handling them every two detecting antibodies are known in the art and are within the 50 days, either by refreshing the media or splitting them. To split Scope of this invention. the cells, the media was first removed by aspiration. The cells In another embodiment of the diagnostic method, a bio were washed in 2-3 ml PBS, then trypsin solution (2-3 ml logical material is assayed for LMW-PTP phosphatase activ diluted 1:50 in PBS) was added and the plates were placed in ity. Depending on the assay used, this method can be per the incubator at 37° C. for 10-30 minutes. Next, 2-3 ml of formed on biological material that is present in the mammal 55 media was added to each plate. The cells in the PBS/trypsin or that has been removed from the mammal. For example, Solution and media were spun to a pellet in a tabletop centri biological material obtained from the mammal can be Sub fuge for 5 minutes. The PBS/trypsin solution and media were jected to a biochemical assay for LMW-PTP phosphatase aspirated, and the cells were resuspended in media The cells activity. Detection can also be accomplished by employing a were then plated in the tissue culture dishes. detectable reagent that binds to DNA or RNA coding for the 60 The growth medium for the MCF-10A (Neo) cells consists LMW-PTP protein. of DMEM/F12, 5.6% horse serum, 20 ng/ml epidermal LMW-PTP can be used as a marker for cancer, precancer growth factor, all from Upstate Biotechnology, Inc., 100 ous or metastatic disease in a wide variety of tissue samples, ug/ml streptomycin, 100 units/ml penicillin, 10 g/ml insulin including biopsied tumor tissue and a variety of body fluid from Sigma, 0.25 g/ml fungizone, and 2 nML-Glutamine. samples, such as blood, plasma, spinal fluid, saliva, and urine. 65 The growth medium for the MDA-MB-231 cells consists of Other antibodies may be used in combination with anti RPMI, 2 nML-Glutamine, 100 lug/ml streptomycin, and 100 bodies that bind to LMW-PTP to provide further information units/ml penicillin. US 7,662,770 B2 13 14 Antibodies. An antibody that recognizes the intracellular The cell pellet was thawed (if applicable) then resuspended domain of EphA2 is D7 (Upstate Biochemicals, New York). in 100 mM CHCOONa buffer, pH 5.0, containing 1 mM This monoclonal antibody (MAb) was produced from a bulk EDTA and 1 mM DTT. The DTT was added just prior to use. culture as stated in Zantek, Ph.D. Thesis, Purdue University, The cells were disrupted by passing them twice through a 1999. For immunoprecipitations with this antibody, 30 ul pre-chilled French pressure cell set at a pressure gauge of were used. For immunoblotting, a dilution of 1:1 in TBSTB 1000 psi. The lysates were spun to a pellet in the refrigerated (30 ml 5M NaCl, 50 ml 1 M Tris, pH 7.6, 1 ml Tween-20, 1 g centrifuge at 16,000 rpm for 15 minutes. The supernatant was BSA, and 920 ml ddHO) was used. For immunofluorescence poured into a new centrifuge tube, then loaded onto an SP microscopy, the antibody was used without dilution. Sephadex C-50 cation-exchange column (1.5x30 cm) that The monoclonal antibodies directed against HPTP (10.1 10 was pre-equilibrated with 10 mMCHCOONabuffer, pH4.8, and 7.1) were developed as stated in Alfred Schroff, Ph.D. containing 30 mMNaH2PO 1 mM EDTA and 60 mMNaCl. Thesis, Purdue University, 1997. For immunoprecipitations, The C-50 column was washed with 10 bed-volumes of 10 with 10.1 (C.-HPTP-B) 10ul were used. For immunoblotting, mMCHCOONa buffer until the Aso was roughly zero. The the antibody was diluted in TBSTB at 1:100. For immunof protein was then eluted with a high salt solution, 300 mM luorescence microscopy, the antibody was diluted at 1:10 in 15 NaH2PO and 1 mM EDTA at pH 5.1. The flow-rate was set PBS. The same conditions were used for the other MAb at 30-40 mL/hr. Each fraction collected contained approxi directed against HPTP, 7.1 (O-HPTP-A/B). For the poly mately 6 ml. The fractions with the highest Aso were clonal antibodies against HPTP, 10ul of antibody were used resolved on a 15% SDS-polyacrylamide gel to access protein for immunoprecipitation. For immunoblotting, the antibody purity. The purest fractions were combined, then concen was diluted in TBSTB at 1:2000. For immunofluorescence trated to roughly 5 ml using an Amicon ultrafiltration appa microscopy, the antibody was diluted in PBS at 1:100. ratus. The concentrate was loaded on a Sephadex G-50 size To detect phosphotyrosine, the antibody known as 4G10 exclusion column that was pre-equilibrated with 10 mM was used. This antibody was produced from a bulk culture. CHCOONa buffer at pH4.8, containing 30 mMNaHPO, 1 For immunoblotting, a dilution of 1:1 in TBSTB was used. mM EDTA and 60 mMNaCl. The flow-rate was set at 15-25 The secondary antibodies used for immunofluorescence 25 ml/hr and fractions of approximately 6 mL were collected. microscopy were DAR-Flat a 1:40 dilution and/or DAM-Rh The fractions with the highest Aso were tested on a 15% at a 1:100 dilution in PBS. For immunoblotting experiments, SDS-polyacrylamide gel to assess protein purity. The purest either Goat Anti-Mouse (for MAb) or Goat Anti-Rabbit (for fractions were combined and stored at 4°C. in G-50 buffer. PAb) was used at a 1:10,000 dilution in TBSTB. Immunofluorescence microscopy. Up to five glass cover 30 slips were placed in a 3.5 cm dish. The cell line(s) appropriate Affinity matrices. Protein-A Sepharose was purchased for the particular study was plated into those dishes 24 hours from Sigma. The Affi-gel 10 was purchased from BioRad. prior to use. The cells usually reached a confluence of 60-70% Other materials. Other materials were purchased from by this time. The cells were fixed in a 3.7% formaldehyde Fisher, Pierce, Malinckrodt, New England Biolabs, solution for 2 minutes, then permeabilized in 1% Triton for 5 QIAGEN, and Roche Diagnostics. 35 minutes, and washed in Universal Buffer (UB) for 5 minutes. LMW-PTP expression and purification. The growth The cells were then incubated at room temperature with the medium (M9ZB) was prepared as follows: in a 4 L flask, 20 g primary antibody for 30 minutes. Next, the cells were washed N-Z-amine A (casein hydrolysate), 10 g NaCl, 2 g NHCl, 6 in UB (12 ml 5 MNaCl, 20 ml 1 M Tris, pH 7.6, 4 ml 10% g KHPO, and 12 g Na HPOHO were dissolved in 2 L of Azide) for 5 minutes. The cells were then incubated with a ddHO. The pH of the medium was then adjusted to 7.4 with 40 secondary antibody for 30 minutes. After a brief wash for 5 NaOH pellets. To a 500-ml flask, 200 ml of M9ZB solution seconds in ddHO, the coverslips were placed face down on were poured. The two containers of media were then auto approximately 5 ul of FluorSave (Calbiochem) on a glass claved for 20 minutes. After cooling to room temperature, slide. The cells were allowed to dry at room temperature for filter sterilized solutions of 20 mL 40% glucose and 2 mL 1M approximately 15 minutes; then they were placed under a hair MgSO were added per 2 L of medium. Just prior to inocu 45 dryer set on “low” for an additional 15 minutes or until dry. lation 200 ul of 50 mg/mL Amp was added to the flask The cells were viewed under an oil immersion lens (60x) of a containing 200 ml (M97B) medium. A 200-ml culture of the fluorescence microscope. BL21 strain of E. coli containing the recombinant plasmid Immunoprecipitation. For immunoprecipitations (IPs) with the gene of interest was grown overnight on a gyratory with monoclonal antibodies, Rabbit Anti-Mouse Protein-A shaker set at 250-300 rpm at 37° C. 50 Sepharose (RAMPAS) was used. For those with polyclonal The next day, 1.8 ml of 50 mg/mL Amp was added to the antibodies, Protein-A Sepharose (PAS) was used. The beads remaining 1.8 L of fresh medium. The overnight culture was were prepared by, first, adding Protein-A Sepharose to the then diluted 1:10 in the fresh (M9ZB) medium, and the cells 100 ul-mark of a 1.5 mL microfuge tube. Next, 1 ml of UB were allowed to grow an additional 3 hours. When the optical was added to swell the beads. For RAMPAS, 50 ul/ml Rabbit density at 600 nm (OD) reached between 0.6 and 1.0, 2 ml 55 Anti-Mouse (RAM) IgG was also added to the tube of beads of 4 mMIPTG were added to induce protein expression. The and UB. The mixture(s) were allowed to rotate on a rotary culture was incubated at 37° C. for an additional 3 hours for stirrer overnight at 4°C. The next day, the beads were washed WT-PTPase or 6 hours for mutant PTPases. The cells were three times in 1 ml of UB. The beads were then brought to a harvested by refrigerated centrifugation for 15 minutes at 50% slurry in UB. 5000 rpm. The supernatant was poured back into the 4-L 60 The plate of cells was placed on ice. The cells were washed flask, then autoclaved for 20 minutes before discarding. The once with 2-3 ml of PBS. Afterwards, the cells lysed in a 1% cell pellet was resuspended and washed in 10 mL 0.85% Triton lysis buffer (5 ml 1 M Tris, pH 7.6, 3 ml 5 M NaCl, 1 NaCl, spun to a pellet again at 500 rpm, then resuspended in ml 10% NaN, 1 ml 200mMEDTA, 10 ml 10% TritonX-100, 2 mL 0.85% NaCl. The mixture was placed in a small centri 80 ml dd HO) or RIPA lysis buffer (5 ml 1M Tris, pH 7.6, 3 fuge tube then spun at 5000 rpm for 10 minutes. The super 65 ml 5 MNaCl, 1 ml 10%, NaNs, 1 ml 200 mM EDTA, 10 ml natant was poured off, and the pellet was either stored at -20° 10% TritonX-100, 5 ml 10% deoxycholate, 500 ul 20% SDS, C. overnight or lysed immediately. 74.5 ml ddHO) lysis buffer containing 1 mM NaVO, 10 US 7,662,770 B2 15 16 ug/ml leupeptin, and 10 ug/ml aprotinin for 5 minutes on ice. on the membrane were blocked with a solution of Teleostean The lysates were collected, and each set of lysates was nor gelatin (50 ml of TBSTB and enough gelatin to give a “tea' malized for equal protein content using Coomassie Protein color). The membrane was incubated in the blocking solution Assay Reagent. A plate-reader was used to measure the absor at room temperature for 30 minutes. Next, the membrane was bance of 590 nm. After equalizing the lysates with the appro incubated with primary antibody for 30 minutes. The mem priate lysis buffer, the samples were prepared. brane was washed three times for 10 minutes each in TBSTB, For each sample, 30 ul PAS (or RAMPAS) was added to which was followed by a 30-minute incubation with second each sample tube. Next, the appropriate primary antibody was ary antibody. Afterwards, the membrane was washed three added. Finally, 150-200 ul portions of the lysates were added. times for 8 minutes each in TBSTB, then twice for 6 minutes The samples were allowed to rotate at 4°C. for either 1.5 10 each in TBS (30 ml 5 MNaCl, 50 ml 1 M Tris, pH 7.6, 920 ml hours or overnight. The samples were then washed three ddHO). Next, the chemiluminescent reagents were added to times in 1 ml of the same lysis buffer that had been used to lyse the membrane (1:1). Finally, the film was exposed to the the cells. After the final wash, 15 ul Laemmli buffer was membrane, which was wrapped in Saran Wrap, and devel added to the pelleted beads, and the samples were boiled for oped. 10 minutes. Afterwards, the samples were loaded and 15 Small-scale DNA purification. The plasmid plT-11d con resolved on a 15% SDS-polyacrylamide gel set at 220 V for taining the gene for HPTP, was purified from the BL21 strain 1.75 hours. After protein resolution, the proteins were trans of E. coli using the commercially available QIAprep Mini ferred to nitrocellulose overnight. prep from QIAGEN. E. coli containing HPTP-A and HPTP-B Substrate trapping. Purified, catalytically-inactive LMW were both, but separately, streaked onto LB/Amp plates. Both PTP recombinant mutants, D129A-BPTP and C12A-BPTP, plates were placed in a 37°C. incubator overnight. The next were used to create potential substrate trap(s). The affinity day, 3 ml of LB medium and 6 ul Amp were placed into two support was prepared by, first, washing 1-1.5 ml of Affi-gel 10 sterile snap-top tubes. The tubes were then labeled HPTP-A in several volumes of cold ddH.O. Next, the moist gel was or HPTP-B. One colony from each plate was used to inoculate added to a 15-ml conical tube, along with a 5 mg/ml of pure the respectively labeled tube with a colony containing the PTPase mutant. The tube was rotated at 4°C. for 4 hours to 25 HPTP-A gene or the HPTP-B gene. The tubes were placed on allow the protein to couple to the beads. Afterwards, 100 ul a shaker set at 250 rpm overnight (12-16 hours). The next day, ethanolamine per 1 ml Affi-gel was added to block reactive the cultures were spun to a pellet, and the Supernatant was gel sites that had not been bound by protein, then the tube was removed by aspiration. rotated for an additional hour. The slurry was poured into a To purify the DNA from bacterial pellets using the small plastic column. The beads were allowed to settle in the 30 QIAprep Miniprep protocol, the bacterial pellets were resus column, they were thenwashed with 20 ml ofddH.O.The pH pended in 250 ul of a buffered RNase A solution (Buffer P1). of the wash was measured. If greater than or equal to seven, Next, the cell Suspension was placed in a microfuge tube and the pH was adjusted with 10 mM HCl. Next, the Aso was lysed in 2501 ul of an alkaline lysis buffer (Buffer P2) con measured. If not near Zero, the washes were continued until sisting of NaOH and SDS. The tubes were inverted gently five the Aso read near Zero. The column was stored at 4°C. until 35 used. times. Lysis was carried out for 5 minutes. The mixture was Prior to application of the lysates, the column was washed then neutralized by adding 350 ul neutralizing buffer (Buffer in 10 ml ofddHO three times, then equilibrated in the appro N3). priate lysis buffer. The cells were lysed in the appropriate After spinning the tubes at 13,000 rpm for 10 minutes, the lysis buffer for 5 minutes on ice. The lysates were collected 40 Supernatant was transferred to the QIAprep spin column. The and added to the column to incubate for various times at 4°C. spin column was placed in a 2-ml collection tube. Together, The beads were then washed in the appropriate lysis buffer they were placed in a centrifuge and spun at 13,000 rpm for 1 three times. Laemmli buffer was added to the beads, which minute. The flow-through was discarded. Next, the spin col were boiled for 10 minutes. The samples were resolved on a umn was washed with 750 ul of Buffer PE, and spun at 13,000 15% SDS-polyacrylamide gel, and finally transferred to 45 rpm for 1 minute. After discarding the flow-through, the spin nitrocellulose overnight. column was spun once more at 13,000 rpm for 1 minute. The Dephosphorylation. MCF-10A (Neo) cells were grown to spin column was placed in a clean microfuge tube, and the 80% confluence. The cells were lysed in 1% Triton lysis DNA was eluted with 601 ul of Buffer EB and stored at -20° buffer for 5 minutes on ice. The lysates were collected and C. combined. EphA2 IPs were prepared: 30 ul D7, 30 ul RAM 50 Amplification of the coding regions. Polymerase chain PAS, and 200 ul lysates. The IPs were mixed for 1.5 hours at reaction was used to amplify the coding regions of the genes. 4°C. They were washed two times in 500 ul Triton lysis The primer designed for the forward strand contains a Hind buffer, then twice in 500 ul ofddH.O. Each pellet was resus III restriction site: AAT TTA AAG CTT CCA TGG CGG pended in 10 mM 50 ul CHCOONa buffer, and the tubes AAC AGG CTA CCA AG (SEQ ID NO:1). The primer were placed in a 37°C. waterbath for 5 minutes to adjust the 55 designed for the reverse strand contains an EcoRI restriction temperature to physiological conditions. Next, 500 ul of Site: CGTTCTTGG AGA AGG CCC ACT GAG AATTCT PTPase solution at the chosen concentration, was added to the TCGT (SEQID NO:2). An additional primer designed for the tubes to react with EphA2 for the chosen times. At the end of reverse strand contains a BamHI restriction site: GCGCGC the reaction, the beads were pelleted and the Supernatant was GGA TCC TCA GTG GGCCTT CTCC (SEQID NO:3). removed by aspiration. Laemmli buffer was added to each 60 Briefly, 50l reaction mixtures consisting of 40 ul ddHO, sample and they were boiled for 10 minutes. Finally, the 5ul 10x buffer, 1 ul forward primer, 1 ul reverse primer, 1 ul proteins were separated on a 10% SDS-polyacrylamide gel, dNTPs and 1 Julpfu polymerase were prepared. The reaction and transferred to nitrocellulose overnight. mixtures were placed in a thermal cycler set for the following Immunoblotting. The nitrocellulose membrane was cycle: 94° C. for 2 minutes, 94° C. for 1 minute, 55° C. for 1 stained with Ponceau S to identify and mark the location of 65 minute, 65° C. for 1 minute, 65° C. for 10 minutes, then hold the molecular weight markers. The membrane was rinsed at 4°C. Steps two through four were repeated 30 times prior several times in ddHO to remove the dye. Non-specific sites to proceeding to the next step, 65° C. for 10 minutes. US 7,662,770 B2 17 18 At the end of the cycle, the PCR products were analyzed on LB/Amp plates. The plates were placed in the 37°C. incuba a 1% agarose gel (600mg agarose, 1.2 ml 50xTAE, 58.8 ml tor lid side up for 10 minutes, then lid side down overnight ddHO). The PCR products were then purified using the (16-18 hours). commercially available QIAquick PCR purification kit from Screen of colonies. The QIAprep Miniprep protocol was QIAGEN. Briefly, five volumes of Buffer PB were added to 5 used to purify the DNA from each of the six bacterial cultures. one volume of the PCR product reaction mixture and mixed Tubes containing the purified DNA were labeled appropri briefly. The mixture was added to the QIAquick spin column ately: colony A1, colony A2, colony B1, colony B2, etc. To and spun for 1 minute at 13,000 rpm. After discarding the screen the colonies, purified DNA from each was digested flow-through, 750 ul of PE Buffer was added to the column with HindIII and BamHI; Nde I and EcoRI; and AccI. The 10 Hind III/BamH I digestion reactions were prepared: 5 ul and spun for 1 minute more at 13,000 rpm. The column was vector/insert, 1 ul NEB-Buffer 2, 1 ul 10xBSA, 1.8 ul Hind placed in a clean microfuge tube, and 30 ul of Buffer EB was III/BamHI stock, 6.2 pulddHO. The Hind III/BamHI stock added to the column. The column incubated at room tempera was prepared as follows: 7.2 ul BamHI added to 9.6 ul Hind ture with the buffer for 1 minute before being spun at 13,000 III. Next, the Nde I/EcoRI digestion reactions were prepared: rpm for 1 minute to elute the DNA. 15 5ul vector/insert, 0.5 ul EcoRI, 0.3 ul Nde I, 1.5 ul NEB Removal of the extensions. The PCR product reaction mix Buffer 4, 7.7 ul ddH.O. Finally, the AccI digestion reactions tures were prepared for digestion: 5 ul PCR product, 1 ul were prepared: 5 ul vector/insert, 0.5 ul Acc I, 1.5 ul NEB NEB-Buffer 2, 1 Jul 10xBSA, and 0.9 ul Hind III/BamH I Buffer 3, and 8 ul ddHO. All digests were done overnight at stock. The HindIII/BamHI stock consisted of 2.4 ul HindIII 37°C. The digests were resolved on a 1% agarose gel and a and 1.2 ul Bam HI. The reaction mixture for digestion for the photograph was taken of the gel. pcDNA3 vector (FIG. 1) from Invitrogen was prepared: 1 ul Medium-scale DNA purification. A six-hour 5 ml culture pcDNA3, 1.5 ul NEB-Buffer 2, 1.5ul 10xBSA, 0.5ul Hind grew at 37°C. on the shaker set at 250 rpm. The 5-ml culture III, and 0.5 ul BamH I. The plasmid pcDNA3 is a 5.4 kb was diluted with 50 ml of LB. The tube was placed on the mammalian expression vector. The HPTP gene was cloned shaker overnight. On the next day, 40 ml of the overnight into the HindIII/BamHI sites of this vector, and expression of 25 culture was transferred to a 5-ml Screw-cap centrifuge tube the gene was driven by the CMV promoter. The PCR products and pelleted by centrifugation for 5 minutes at 5000 rpm. The and the mammalian expression vector, pcDNA3, were commercially available QUANTUM Midi Prep from BioRad digested at 37°C. for 2.5 hours. The digests were analyzed on was used to purify DNA on a medium scale. Briefly, the a 1% agarose gel. After resolution, a photograph was taken of supernatant was poured off and 5 ml of Cell Resuspension the gel. Digestion of the PCR products and the pcDNA3 30 solution were added to the cell pellet. The tube was vortexed vector were expected to generate fragments that were 491 bp to resuspend the cells. Next, 5 ml of Cell Lysis solution were and 5,428 bp, respectively. added to the tube, then inverted six to eighttimes. The mixture was neutralized by adding 5 ml Neutralization solution, fol Gel pieces containing the digested products were removed lowed by inverting the tube six to eight times, neutralized the from the gel and placed in a microfuge tube. To remove the 35 solution. The mixture was spun for 10 minutes at 8000 rpm. digested products from the gel, a commercially available The supernatant was transferred to a new tube along with 1 ml QIAquick Gel Extraction kit from QIAGEN was used. of Quantum-Prep matrix. The mixture was gently swirled for Briefly, 210 ul of Buffer QG were added to the tubes. The 15 to 30 seconds, then spun for 2 minutes at 8000 rpm. The tubes were placed in a 50° C. waterbath for approximately 10 supernatant was poured off, then 10 ml of wash buffer were minutes, with mixing every 2-3 minutes. Next, 70 ul of iso 40 added to the matrix and mixed by shaking. The tube was spun propanol were added to the tubes and mixed. The samples for 2 minutes at 8000 rpm. After pouring the washbuffer from were then placed in a column attached to a collection tube and the pellet, 600 ul Wash Buffer were added to the tube to spun at 13,000 rpm for 1 minute. After discarding the flow resuspend the pellet. The spin column was attached to a through, 500 ul of Buffer QG were added to the column, and microfuge tube and a hole was punctured in the lid of the the column was spun for 1 minute at 13,000 rpm. The flow 45 microfuge tube. After spinning the tube for 30 seconds at through was discarded, and the column was washed with 750 12,000 rpm, the flow-through was discarded. Next, 500 ul of ul of Buffer PE then spun at 13,000 rpm for 1 minute. The Wash Buffer was added to the tube, and the tube was spun for flow-through was discarded, and the column was spun once 30 seconds at 12,000 rpm. The flow-through was discarded, more at 13,000 rpm for 1 minute to elute the DNA. The DNA was stored at -20°C. then the column was spun for 2 minutes more at 12,000 rpm 50 to remove residual Wash Buffer. The column was transferred Ligation and transformation. The amplified HPTP-A and to a clean microfuge tube. The DNA was eluted with 600 ul of HPTP-B genes were both, but separately, ligated with the TE (pH 8). pcDNA3 vector. The ligation reaction mixture was prepared: Next, the DNA was ethanol-precipitated by adding /10 the 10 ul insert, 5ul vector, 2 Jul 10x ligation buffer, 2 Jul 10xATP, volume of 5MNaCl following by two times that total volume and 1 Julligase. The ligation mixtures were placed in a thermal 55 (NaCl plus DNA) of 100% ethanol. The microfuge tube was cycler set at 16° C. for 18 hours followed by holding the gently inverted a few times and incubated at -20°C. for 20 temperature at 4°C. minutes. The DNA was spun to a pellet for 10 minutes at The DH5C. strain of competent E. coli was transformed 13,000 rpm. Under sterile conditions, the ethanol/NaCl was with the ligation mixtures. Two microfuge tubes each with aspirated from the pellet. The pellet was left to air dry in the 200 ul of competent E. coli (DH5C.) were thawed on ice. The 60 hood. Afterwards, the DNA was resuspended in 100 ul sterile ligation mixture was added to each tube of cells, the tubes TE (pH 8). To determine the concentration of the DNA were vortexed briefly, and then incubated on ice for 20 min sample, the absorbance at 260 nm (Ago) was measured. The utes. The tubes were placed in a 42°C. waterbath for 1.5 DNA was stored at -20° C. minutes, then placed on ice for 2 minutes. The contents of the Transfection. To overexpress HPTP in the MCF-10A tubes were placed separately into tubes containing 1 ml of 65 (Neo) cell line, the commercially available FuGENER) trans LB. The mixtures were placed on the shaker set at 250 rpm for fection kit from Roche Diagnostics was used. The cells were 45 minutes. Next, 200 ul of each culture were spread onto two plated 18 hours prior to use in 6-well plates such that their US 7,662,770 B2 19 20 confluence would be approximately 50% on the day of trans might be a competition between phosphorylation and dephos fection. In a microfuge tube, 97 ul serum-free dilution media phorylation of EphA2, and that in MDA-MB-231 cells the was added to 3 ul of the FuGENE reagent. The diluted balance is tilted toward dephosphorylation. However, in the FuGENE was incubated at room temperature for 5 minutes. MCF-10A (Neo) cells, the balance is not tilted substantially Next, 1 lug of DNA was added to a second microfuge tube. in one direction or the other. As a result, EphA2 retains some Dropwise, the diluted FuGENE reagent was added to the of its tyrosine phosphorylation in the MCF-10A (Neo) cell DNA. The tube was gently tapped to mix the contents of the line. tube. The tube was then incubated for 15 minutes at room Subcellular localization. A panel of polyclonal and mono temperature. The media on the cells was replaced with 2 ml of clonal antibodies, all directed against the LMW-PTP, was fresh media. Dropwise, the FuGENE/media solution was 10 used to stain MCF-10A (Neo) and MDA-MB-231 cells. To added to the plated cells, then the plate was swirled to dis determine the Subcellular localization of the LMW-PTP in the tribute the contents around the plate. The cells were incubated MCF-10A (Neo) and MDA-MB-231, cells were grown on at 37° C. for 36 to 48 hours. coverslips overnight. After fixation and permeabilization, the On the day of analysis, the cells were lysed in 1% Triton cells were stained with a primary antibody to detect the lysis buffer, HPTP and D7 immunoprecipitations were done. 15 LMW-PTP. A fluorescent tag attached to the secondary anti The samples were eventually resolved on a 15% SDS-poly body facilitated observation of the subcellular location of the acrylamide gel, then transferred to nitrocellulose overnight. LMW-PTP on the fluorescence microscope. The LMW-PTP The next day, immunoblotting was done with antibodies was found to be diffuse and widely distributed in the MCF directed against EphA2, HPTP, and phosphotyrosine. 10A (Neo) cells. When MDA-MB-231 cells were stained, the LMW-PTP was found localized in the membrane ruffles. This Results was an exciting finding because EphA2 was known to local Expression and Purification of the LMW-PTP ize in the membrane ruffles in the MDA-MB-231 cell line, as LMW-PTPs can be purified using a two-step purification well (Zantek, N. D. (1999), Ph.D. Thesis, Purdue University). scheme involving cation-exchange chromatography (typi In Vitro Protein-Protein Interaction Between the LMW-PTP cally using a SP-Sephadex C-50 column) followed by size 25 and Eph A2 exclusion chromatography (typically using a Sephadex G50 Co-immunoprecipitation, Attempts were made to co-im column). A minor difference between the recombinant pro munoprecipitate the two proteins with separate antibodies tein (isolated after expression in E. coli) and the native bovine directed against either protein. Co-immunoprecipitation of or human protein is that the recombinant protein is not acety the LMW-PTP was readily detectable when immunoprecipi lated on the N-terminal alanine residue as in the native tissue 30 tating with D7, an EphA2-specific antibody, followed by protein. In this Example, WT-BPTP, D129A-BPTP, and immunoblotting analyses with either the 7.1 or 10.0 LMW C12A-BPTP were expressed and purified using the pET-11 PTP antibody. The co-immunoprecipitation was more evi expression system. A stock Supply of the purified proteins, dent when blotting with the 10.1 antibody. As would be pre HPTP-A and HPTP-B, was already on hand. dicted from the relative protein level analysis of the LMW Expression and purification of WT-BPTP from E. coli 35 PTP more LMW-PTP was co-immunoprecipitated from the occurred without difficulty and generated good quantities of MDA-MB-231 cell line than from the MCF-10A (Neo) cell protein (40-50 mg per liter of expression medium). Expres line. A somewhat less dramatic result was obtained when sion of recombinant, mutant PTPases resulted in less protein immunoprecipitating with either the 7.1 or 10.1 antibody, (approximately 10-15 mg per liter of expression medium). In followed by immunoblotting with the D7 antibody. Bands the case of the D129A bovine mutant, the induction period 40 appeared in the lanes of the 7.1 and 10.1 IPs that were roughly was increased to six hours, and the wash buffer was changed co-linear with those of the D7 IP control. This suggests that to 1 mM EDTA to increase the binding of the mutant proteins these bands might represent EphA2. to the C50-columns. Once purified, the protein was stable for It was somewhat Surprising that co-immunoprecipitation months at -20°C. in phosphate buffer. 45 of the proteins occurred in both of our cell lines. It was Comparison of the LMW-PTP in MCF-10A (Neo) and MDA predicted that an interaction would be detected, but this was MB-231 Cell Lines expected to be more likely in the MCF-10A (Neo) cell line Protein levels. EphA2 is tyrosine phosphorylated in the because EphA2 is tyrosine phosphorylated there. However, non-transformed MCF-10A (Neo) cell line, but not in the because the interaction of a phosphatase with its Substrate is malignant MDA-MB-231 cell line. 50 either so transient or so weak, it was also thought that the Endogenous protein levels of the LMW-PTP in the MCF interaction might not be easily detected. In our case, an inter 10A (Neo) and the MDA-MB-231 cell lines were first com action was detected in both cell lines. pared by immunoblotting analyses. The results revealed In Vitro Dephosphorylation lower protein levels of the LMW-PTP in the MCF-10A (Neo) Attempts at Substrate trapping to detect direct interaction cell line relative to the levels of the protein in the MDA-MB 55 between EphA2 and LMW-PTP failed, so an alternative in 231 cell line. This suggests that the higher protein levels of the vitro test was conducted. We examined the ability of pure LMW-PTP observed in the MDA-MB-231 cell line might LMW-PTP to dephosphorylate EphA2 isolated by immuno correlate with EphA2 being substantially more dephospho precipitation. We found that the LMW-PTP dephosphory rylated in that cell line compared to MCF-10A (Neo) cell line. lated EphA2 in an enzyme concentration-dependent and a Although EphA2 is tyrosine phosphorylated in MCF-10A 60 time-dependent manner. (Neo) cells, even higher levels of tyrosine phosphorylation of As would be expected, the extent of dephosphorylation of the cells can be achieved if EphA2 is treated with a soluble EphA2 by the LMW-PTP was found to be greater when larger form of its ligand or artificially activated at the cell surface amounts of phosphatase are used than in cases when Smaller (Zantek, N. D. (1999), Ph.D. Thesis, Purdue University). amounts are used. The enzyme concentration-dependent With this in mind, it may also be suggested that the LMW 65 dephosphorylation of EphA2 by the LMW-PTP is consistent PTP dephosphorylates EphA2 in MCF-10A (Neo) cells, but with the hypothesis that high levels of LMW-PTP suppress not to the same degree as in the MDA-MB-231 cells. There tyrosine phosphorylation of EphA2 in MDA-MB-231 cells. It US 7,662,770 B2 21 22 is thought that the higher LMW-PTP levels cause substantial cells are treated with the pervanadate ion (Zantek, N. D. dephosphorylation of EphA2 in those cells. The enzyme con (1999), Ph.D. Thesis, Purdue University). This gives a strong centration-dependent dephosphorylation of EphA2 follows indication that a PTPase is causing the loss of tyrosine phos basic kinetic behavior. The rate of the reaction increases with phorylation of EphA2. Also, treatment of EphA2 with a increasing enzyme concentration. As a result, there is greater 5 soluble form of the ephrin A1 ligand and cross-linking of turnover per unit time. When the progress of the reaction is EphA2 at the surface of the cell leads to transient tyrosine studied over longer periods of time, it is found that greater phosphorylation of EphA2. The loss of tyrosine phosphory enzyme concentrations continue to dephosphorylate Eph A2 lation of EphA2 that occurs over time with these treatments in comparison with Smaller enzyme concentrations. The lev could be due to a PTPase interacting with EphA2. eling off of dephosphorylation that is observed may be due to 10 Example II instability of the protein under very dilute conditions. In Vivo Protein-Protein Interaction Between the LMW-PTP Regulation of EphA2 by Low Molecular Weight and Eph A2 Protein Tyrosine Phosphatase Vector construction. To explore the effects of overexpress ing the LMW-PTP in the MCF-10A (Neo) cell line, a 15 pcDNA3 eukaryotic expression vector containing the coding Materials and Methods region of the LMW-PTP was constructed. Microgram Cell Lines and Antibodies. Human breast (MCF-10A, amounts of the pET-11d plasmid were isolated without diffi MCF 10A ST, MCF-7, MDA-MB-231, MDA-MB435, SK culty using a commercially available DNA purification kit. BR-3) epithelial cells were cultured as described in Example Primers were designed and used to amplify the coding region I and previously (Paine, T. M., Soule, H. D., Pauley, R. J. & of the A- and B-isoforms of the LMW-PTP. Dawson, P.J. (1992) IntJ Cancer 50, 463-473: Jacob, A. N., The PCR products were purified using a commercially Kalapurakal, J., Davidson, W. R. Kandpal, G., Dunson, N., available PCR product purification kit from QIAGEN. The Prashar, Y. & Kandpal, R. P. (1999) Cancer Detection & amplified coding regions of the LMW-PTP isoenzymes were Prevention 23, 325-332: Shevrin, D. H., Gorny, K. I. & digested with BamHI and HindIII to remove the extensions. 25 Kukreja, S. C. (1989) Prostate 15, 187-194). Monoclonal The “sticky-ends' that were produced allowed for directional antibodies specific for phospho-tyrosine (PY20) and B-cate cloning of the inserts in the mammalian expression vector, nin were purchased from Transduction Laboratories (Lexing pcDNA3, which was also digested with HindIII and BamHI. ton, Ky.). Monoclonal antibodies specific for phosphoty Digestion of the isoenzymes generated 491 bp fragment. rosine (4G10) and EphA2 (clone D7) were purchased from Digestion of the pcDNA3 vector generated an open vector 30 Upstate Biotechnology, Inc. (Lake Placid, N.Y.). Monoclonal with 18 fewer base pairs than the circular vector. antibodies against Vinculin were purchased from NeoMark After cell transformation, the constructed vectors were ers (Fremont, Calif.). isolated from the cells and screened with a panel of restriction Cell Lysates. Cell lysates were harvested and normalized enzymes to determine if the coding regions of the human A for equal loading as described previously (Kinch, M. S., and B-isoenzymes of LMW-PTP were present. The coding 35 Clark, G. J., Der, C. J. & Burridge, K. (1995) J Cell Biol regions were present in their respective vectors as indicated 130,461-471). To confirm equal loading, blots were stripped by the cuts produced by the restriction enzymes. as described previously and reprobed with antibodies specific Overexpression of the LMW-PTP in MCF-10A (Neo) to f-catenin or Vinculin (Kinch, M. S., Clark, G.J., Der, C.J. cells. Overexpression of the LMW-PTP in the MCF-10A & Burridge, K. (1995).J Cell Biol 130, 461-471). (Neo) cell line was attempted in order to explore the effects of increased protein levels of the phosphatase on EphA2's Immunoprecipitation and Western Blot Analyses: Immu tyrosine phosphorylation status. Large quantities of the con noprecipitation of EphA2 or LMW-PTP were performed structed vectors were isolated in a highly pure form using a using rabbit anti-mouse (Chemicon, Temecula, Calif.) con commercially available DNA purification kit. Isolation of the jugated Protein A Sepharose (Sigma, St. Louis, Mo.) as vectors using this procedure occurred without great difficulty. described previously (Zantek, N. D., Azimi, M., Fedor The commercially available transfection kit, FuGENE, was 45 Chaiken, M., Wang, B., Brackenbury, R. & Kinch, M. S. used to transfect the MCF-10A (Neo) cell line with “empty” (1999) Cell Growth & Differentiation 10, 629-638). To con pcDNA3, HPTP-A?pcDNA3 and HPTP-B/pcDNA3, respec firm equal loading, blots were stripped as described previ tively. The “empty” vector served as a control in the experi ously (Kinch, M. S., Clark, G.J., Der, C. J. & Burridge, K. ments such that any changes in EphA2s tyrosine phospho (1995).J Cell Biol 130, 461471) and reprobed with EphA2 or rylation status should be attributable to increased levels of the 50 LMW-PTP specific antibodies. Western blot analysis were LMW-PTP and not to the presence of the mammalian expres performed on normalized cells lysates and immunoprecipita sion vector. tions as detailed (Zantek, N. D., Azimi, M., Fedor-Chaiken, Overexpression of the HPTP-B in the MCF-10A (Neo) cell M., Wang, B., Brackenbury, R. & Kinch, M. S. (1999) Cell line resulted in decreased tyrosine phosphorylation levels of Growth & Differentiation 10, 629-638). Antibody binding EphA2. No noticeable difference in EphA2s tyrosine phos 55 was detected by enhanced chemiluminescence, (ECL: Pierce, phorylation was seen when HPTP-A was overexpressed in Rockford, Ill.), and visualized by autoradiography (Kodak the same cell line. From this information, it might be con X-OMAT: Kodak, Rochester, N.Y.). cluded that the interaction of EphA2 the LMW-PTP is isoen EGTA and Pervanadate Treatments. “Calcium Switch' Zyme specific, which is not an unreasonable possibility. Dif experiments were performed as described previously ferences in the amino acid sequence of the isoenzymes could 60 (Zantek, N. D., Azimi, M., Fedor-Chaiken, M., Wang, B., be the underlying reason why only one isoenzyme appears to Brackenbury, R. & Kinch, M. S. (1999) Cell Growth & Dif interact preferentially with EphA2. However, there are many ferentiation 10, 629-638) using MCF-10A cells grown to other reasons that explain the difference as well. 70% confluence and medium containing a final concentration Discussion of 4 mMEGTA. Pervanadate was added to MDA-MB-231 in In transformed breast epithelia such as MDA-MB-231, 65 monolayer culture at a final concentration of 0, 1, 10 or 100 EphA2 is not tyrosine phosphorylated. However, restoration mM and the treatment was allowed to incubate for 10 minutes of tyrosine phosphorylation of EphA2 occurs when these at 37° C., 5% CO. For the combined EGTA-Pervanadate US 7,662,770 B2 23 24 Treatment, MDA-MB-231 cells were first treated with 100 MDBK cells. Based on these findings, we asked whether mM Pervanadate and were then subjected to the EGTA treat tyrosine phosphatase inhibitors could prevent the loss of ment. EphA2 phosphotyrosine content in response to EGTA treat In Vitro Kinase and Phosphatase Assays. To evaluate ment. Indeed, inhibitors such as Sodium orthovanadate pre LMW-PTP activity against EphA2, EphA2 was immunopre 5 vented the decrease in EphA2 phosphotyrosine following cipitated from MCF-10A cells and incubated with purified treatment of MCF-10A cells with EGTA (FIG. 2B). LMW-PTP protein at a concentration of 0.45, 7.8, or 26.5 Previous studies by our laboratory have shown that the mg/mL for 0, 5, 15, or 30 minutes. The assay was terminated phosphotyrosine content of EphA2 is greatly reduced in through the addition of Laemmli sample buffer. The phos malignant epithelial cells as compared with non-transformed photyrosine content of the EphA2 in the treatments was then 10 epithelia (Zelinski, D. P. Zantek, N. D., Stewart, J., Irizarry, observed using Western blot analysis with antibodies specific A. & Kinch, M. S. (2001) Cancer Res 61,2301-2306; Zantek, to phosphotyrosine. To determine in vitro autophosphoryla N. D., Azimi, M., Fedor-Chaiken, M., Wang, B., Bracken tion activity, immunoprecipitated EphA2 was evaluated using bury, R. & Kinch, M. S. (1999) Cell Growth & Differentiation in vitro kinase assays as detailed previously (Zantek, N. D., 10, 629-638). Thus, we asked if tyrosine phosphatase activity Azimi, M., Fedor-Chaiken, M., Wang, B., Brackenbury, R. & 15 could contribute to the reduced phosphotyrosine content of Kinch, M. S. (1999) Cell Growth & Differentiation 10, 629 EphA2 in malignant cells. Whereas EphA2 was not tyrosine 638). phosphorylated in malignant breast cancer cells (MDA-MB Transfection and Selection. Monolayers of MCF-10A cells 231, MDA-435, MCFneoST, or PC-3 cells), incubation with were grown to 30-50% confluence and were transfected with increasing concentrations of Sodium orthovanadate induced pcDNA3.1-LMW-PTP or pcDNA3.1-D 129A-LMW-PTP rapid and vigorous tyrosine phosphorylation of EphA2 (FIG. using Lipofectamine PLUS (Life Technologies, Inc., Grand 2C). AS Vanadate treatment of cells can often lead to exag Island, N.Y.). As a control for the transfection procedure, gerated phosphorylation of physiologically irrelevant sites, empty pcDNA3.1 vector was transfected into the same cell we performed phosphopeptide-mapping studies using line in parallel. Transient transfections were allowed to grow EphA2 that had been labeled with 32P-ATP either in vitro or for 48 hours post-transfection. For stable lines, neomycin 25 in vivo. These studies revealed identical patterns of tyrosine resistant cells were selected in growth medium containing 16 phosphorylation in non-transformed MCF-10A cells and mg/mL neomycin (Mediatech, Inc., Herndon, Va.). To con vanadate treated MDA-MB-231 cells. Although the cytoplas firm LMW-PTP overexpression, Western blot analysis was mic domain contains multiple sites that could have been performed using LMW-PTP specific antibodies. Parental phosphorylated promiscuously, these were not phosphory cells and cells transfected with empty pcDNA3.1 vector were 30 lated under the conditions utilized here, Suggesting that vana used as negative controls. date had not increased the phosphorylation of irrelevant sites. Growth Assay. To evaluate cell growth using monolayer Altogether, these results indicate that EphA2 is regulated by assays, 1x10 cells were seeded into tissue-culture treated an associated phosphatase that Suppresses EphA2 phospho multi-well dishes for 1, 2, 4 or 6 days in triplicate experi tyrosine content in malignant cells. ments. Cell numbers were evaluated by trypsin Suspension of 35 the samples followed by microscopic evaluation using a LMW-PTP Interacts with and Dephosphorylates Eph A2 hemacytometer. Soft agar colony formation was performed To identify tyrosine phosphatases that might regulate and quantified as detailed (Zelinski, D. P. Zantek, N. D., EphA2 in malignant cells, we considered a recent report that Stewart, J., Irizarry, A. & Kinch, M. S. (2001) Cancer Res 61, LMW-PTP regulates a related molecule, EphB4 (Jacob, A. 2301-2306); Clark, G.J., Kinch, M. S., Gilmer, T. M., Burr 40 N. Kalapurakal, J., Davidson, W. R. Kandpal, G., Dunson, idge, K. & Der, C. J. (1996) Oncogene 12, 169-176). For N., Prashar, Y., and Kandpal, R. P. (1999) Cancer Detect. experiments with EphA2 antisense, cells were incubated with Prev. 23, 325-33). Our initial experiments began to catalog oligonucleotides prior to Suspension in Soft agar. The data the expression and function of LMW-PTP in non-trans shown is representative of at least three different experiments. formed (MCF-10A Neo) and malignant (MCF-7, SK-BR-3, Antisense Treatment. Monolayers of MCF-10A Neo cells 45 MDA-MB435, MDA-MB-231) mammary epithelial cells and MCF-10A cells stably overexpressing LMW-PTP were (FIG.3). Western blot analyses of whole cell lysates revealed grown to 30% confluence and were transfected with Eph A2 relatively high levels of LMW-PTP in tumor-derived breast antisense oligonucleotides as detailed. Samples that had been cancer cells as compared with non-transformed MCF-10A transfected with an inverted EphA2 antisense oligonucleotide mammary epithelial cells. To confirm equal sample loading, 50 the membranes were stripped and re-probed with antibodies or with the transfection reagent alone provided negative con against a control protein (Vinculin), Verifying that the high trols. levels of LMW-PTP did not reflect a loading error or a gen Results eralized increase in protein levels in the malignant cells. A EphA2 is Regulated by an Associated Tyrosine Phos malignant variant of MCF-10A, MCFneoST, also demon phatase Several independent lines of investigation Suggested 55 strated elevated LMW-PTP expression, which was intriguing that EphA2 is regulated by an associated tyrosine phos based on a recent report that EphA2 is not tyrosine phospho phatase. First, EphA2 could be rapidly dephosphorylated in rylated in those cells (Zantek, N. D., Walker-Daniels, J., non-transformed epithelial cells. Western blot analysis with Stewart, J. C., Hansen, R. K. Robinson, D., Miao, H. Wang, phosphotyrosine antibodies (PY20 or 4G10) indicated lower B., Kung, H. J., Bissell, M. J. & Kinch, M. S. (2001) Clin levels of EphA2 phosphotyrosine content within 5 minutes 60 Cancer Res 7,3640-3648). The use of a genetically-matched following EGTA-mediated disruption of EphA2-ligand bind system also precluded potential differences due to cell origin ing (FIG. 2A). Similarly, tyrosine phosphorylation of Eph A2 or culture conditions. Thus, the highest levels of LMW-PTP decreased following incubation of non-transformed epithelial were consistently found in malignant epithelial cells and cells with dominant-negative inhibitors of EphA2-ligand inversely related to EphA2 phosphotyrosine content. binding (e.g., EphA2-Fc). Identical results were obtained 65 The results above provided suggestive, but indirect, evi using multiple non-transformed epithelial cell systems, dence that LMW-PTP might negatively regulate the phospho including MCF-12A, MCF10-2, HEK293, MDCK and tyrosine content of EphA2 in tumor cells. To explore this US 7,662,770 B2 25 26 hypothesis further, we first asked if the two molecules inter transfected MCF-10A cells were largely incapable of colo acted in vivo. EphA2 was immunoprecipitated from MDA nizing soft agar, LMW-PTP-overexpressing cells formed an MB-231 cells using specific antibodies (clone D7) and these average of 4.9 colonies per high-powered microscope field complexes were resolved by SDS-PAGE. Subsequent West (P<0.01; FIG.7B). Based on recent findings with other three ern blot analyses revealed that LMW-PTP was prominently dimensional assay systems, we also evaluated cell behavior found within EphA2 immune complexes (FIG. 4A). The using three-dimensional, reconstituted basement mem inverse experiment confirmed that EphA2 could similarly be branes. Consistent with a more aggressive phenotype, micro detected in complexes of immunoprecipitated LMW-PTP scopic assessment of cell behavior in Matrigel confirmed the (FIG. 4B). Control immunoprecipitations with irrelevant malignant character of LMW-PTP overexpressing cells. antibodies confirmed the specificity of the interactions of the 10 When plated atop or within Matrigel, LMW-PTP-overex two molecules. pressing cells formed larger colonies than vector-transfected The co-immunoprecipitation studies did not clarify cells. Altogether, consistent results with multiple and differ whether EphA2 can serve as a substrate for LMW-PTP. To ent systems suggest that overexpression of LMW-PTP is suf address this directly, EphA2 was immunoprecipitated from ficient to induce malignant transformation. MCF-10A cells, where it is normally tyrosine phosphory 15 The Oncogenic Phenotype of LMW-PTP-Overexpressing lated. The purified EphA2 was then incubated with different concentrations of purified LMW-PTP before Western blot Cells is Related to EphA2 Expression analyses of EphA2 with phosphotyrosine-specific antibodies Tyrosine phosphorylation of EphA2 induces its internal (PY20 and 4G 10). These experiments demonstrated that puri ization and degradation. Thus, we postulated that overexpres fied LMW-PTP could dephosphorylate EphA2 in a dose and sion of LMW-PTP might increase the protein levels of time-dependent manner (FIG. 5A). EphA2. Indeed, Western blot analyses of whole cell lysates Although in vitro studies indicated that EphA2 could be revealed higher levels of EphA2 in MCF-10A cells that over phosphorylated by LMW-PTP in vitro, we recognized that in express LMW-PTP as compared with vector-transfected con vitro studies are not always be representative of the analogous trols (FIG. 6A). Moreover, this EphA2 was not tyrosine phos situation in vivo. Thus, LMW-PTP was ectopically overex 25 phorylated (FIG. 6B). However, Western blot analyses pressed in MCF-10A cells. This particular cell system was revealed that the reduced phosphotyrosine content was selec selected because non-transformed MCF-10A cells have low tive for EphA2, as the general levels of phosphotyrosine were levels of endogenous LMW-PTP and because the EphA2 in not altered in LMW-PTP transformed cells (FIG. 6C). these non-transformed epithelial cells is normally tyrosine The finding that overexpression of LMW-PTP increased phosphorylated. Ectopic overexpression of LMW-PTP was 30 EphA2 expression and decreased its phosphotyrosine content achieved by stable transfection, as determined by Western was intriguing since this phenotype was reminiscent of highly blot analyses with specific antibodies (FIG. 6A). Importantly, aggressive tumor cells (Zelinski. D. P. Zantek, N. D., Stew overexpression of LMW-PTP was sufficient to reduce the art, J., Irizarry, A. & Kinch, M. S. (2001) Cancer Res 61, phosphotyrosine content of EphA2 as compared with vector 2301-2306; Zantek, N. D., Azimi, M., Fedor-Chaiken, M., transfected negative controls (FIG. 6A). Identical results 35 Wang, B., Brackenbury, R. & Kinch, M. S. (1999) Cell were obtained using different experiments, with different Growth & Differentiation 10, 629-638)). Thus, we asked transfectants and in both stably and transiently-transfected whether selective targeting of LMW-PTP in malignant cells samples, thus eliminating potential concerns about clonal would impact EphA2. To accomplish this, an enzymatic variation. Moreover, the decreased phosphotyrosine content mutant of LMW-PTP (D129A) that is catalytically inactive was specific for EphA2 as the phosphotyrosine content 40 (Zhang, Z., Harms, E. & Van Etten, R. L. (1994) Journal of LMW-PTP overexpressing cells was not generally decreased Biological Chemistry 269,25947-25950) was overexpressed in MDA-MB-231 cells, which have high levels of wild-type (FIG. 6B). LMW-PTP (FIG. 3) and overexpress unphosphorylated LMW-PTP Overexpression Causes Malignant Transforma EphA2. Ectopic overexpression of LMW-PTPD129A was tion of Epithelial Cells 45 found to decrease the levels of EphA2. Moreover, Western Tyrosine phosphorylated EphA2 negatively regulates blot analyses of immunoprecipitated material revealed that tumor cell growth whereas unphosphorylated EphA2 acts as this EphA2 was tyrosine phosphorylated (FIG. 6C). Thus, a powerful oncoprotein (Zelinski, D. P. Zantek, N. D., Stew consistent results indicate that overexpression of wild type art, J., Irizarry, A. & Kinch, M. S. (2001) Cancer Res 61, LMW-PTP is necessary and sufficient to confer the overex 2301-2306; Zantek, N. D., Azimi, M., Fedor-Chaiken, M., 50 pression and functional alterations of EphA2 that have been Wang, B., Brackenbury, R. & Kinch, M. S. (1999) Cell observed in tumor-derived cells. Growth & Differentiation 10, 629-638). Thus, we asked Although the EphA2 in the LMW-PTP overexpressing whether overexpression of LMW-PTP would be sufficient to MCF-10A cells was not tyrosine phosphorylated, it retained induce malignant transformation. To address this question, enzymatic activity. In vitro kinase assays verified that the we utilized the MCF-10A cells, described above, which had 55 EphA2 from LMW-PTP-transformed MCF-10A cells had been transfected with either wild-type LMW-PTP or a vector levels of enzymatic activity that were comparable to vector control. Our initial studies evaluated the growth rates of con transfected controls (FIG. 8A). To verify equal sample load trol and LMW-PTP-overexpressing cells in monolayer cul ing, two controls were performed. Equal amounts of input ture. When evaluated using standard, two-dimensional cul lysate were verified by Western blot analyses with B-catenin ture conditions, the growth rates of LMW-PTP 60 antibodies. In addition, the immunoprecipitated EphA2 was overexpressing MCF-10A cells were significantly lower than divided and half of the material was resolved by SDS-PAGE the growth rates of matched controls (P<0.05) (FIG. 7A). and analyzed by Western blot analyses with EphA2 and phos Two-dimensional assessments of growth often do not photyrosine-specific antibodies (FIG. 8B). Thus phosphory reflect the malignant character of tumor cells. Instead, three lated and unphosphorylated EphA2 were both capable of dimensional analyses of cell behavior using soft agar and 65 enzymatic activity. reconstituted basement membranes can provide a more rel Since the levels of EphA2 were elevated in LMW-PTP evant way of assessing malignant behavior. Whereas vector transformed cells, we asked whether the oncogenic activity of US 7,662,770 B2 27 28 EphA2 might have contributed to this phenotype. To address transformation, our findings Support a very different conclu this, we utilized our experience with antisense strategies to sion. Consistent with this, recent studies by our laboratory selectively decrease EphA2 expression in LMW-PTP trans and others have shown that malignant transformation of formed cells (Hess A. R., Seftor, E. A., Gardner, L. M., MCF-10A cells is often accompanied by decreased mono Carles-Kinch, K., Schneider, G. B., Seftor, R.E., Kinch, M.S. layer growth rates and that the most aggressive variants of & Hendrix, M. J. C. (2001) Cancer Res 61, 3250-3255). We verified the success of these strategies by Western blot analy MCF-10A in vivo demonstrate the slowest growth in mono ses (FIG.9A) and then asked if decreased EphA2 expression layer culture. These findings have important implications for would alter Soft agar colonization. Indeed, transfection with the design and interpretation of oncogene function when EphA2 antisense oligonucleotides decreased the soft agar 10 using non-transformed epithelial cell systems. colonization of LMW-PTP-transformed MCF-10A cells by at The biochemical consequences of EphA2 tyrosine phos least 87% (P<0.01; FIG.9B). In contrast, transfection of these phorylation remain largely unclear. Unlike other receptor cells with an inverted antisense control nucleotide control did tyrosine kinases, where autophosphorylation is necessary for not significantly alter soft agar colonization. Thus, we were enzymatic activity, tyrosine phosphorylation of EphA2 is not able to exclude that the results with the antisense oligonucle 15 required for its enzymatic activity. Consistent with our otides had resulted from non-specific toxicities caused by the present results, EphA2 retains comparable levels of enzy transfection procedure. Altogether, our results indicate that, matic activity in non-transformed and tumor-derived cells, in cells that express EphA2, the oncogenic actions of overex despite dramatic differences in its phosphotyrosine content pressed LMW-PTP require high levels of EphA2. (Zantek, N. D., Azimi, M., Fedor-Chaiken, M., Wang, B., Discussion Brackenbury, R. & Kinch, M. S. (1999) Cell Growth & Dif The major finding of our present study is that EphA2 is ferentiation 10, 629-638). Similarly, antibody-mediated regulated by an associated tyrosine phosphatase and we iden stimulation of EphA2 autophosphorylation does not change tify LMW-PTP as a critical regulator of EphA2 tyrosine phos the levels of EphA2 enzymatic activity. Phosphopeptide phorylation. We also demonstrate that LMW-PTP is overex 25 analyses of the EphA2 cytoplasmic domain provide one pressed in metastatic cancer cells and that LMW-PTP potential explanation. Although EphA2 has a predicted acti overexpression is sufficient to confer malignant transforma vation loop tyrosine at residue 772 (Lindberg, R.A.& Hunter, tion upon non-transformed epithelial cell models. Finally, we T. (1990) Molecular & Cellular Biology 10, 6316-6324), demonstrate that LMW-PTP upregulates the expression of neither in vitro nor in vivo phosphopeptide analyses found EphA2 and that the oncogenic activities of LMW-PTP require 30 that this site is not phosphorylated either in normal cell mod this overexpression of EphA2. els or in response to exogenous ligands in malignant cell Recent reports from our laboratory and others have shown models. Thus, the lack of a consensus activation loop tyrosine that many malignant epithelial cells express high levels of may account for the retention of EphA2 enzymatic activity in EphA2 that is not tyrosine phosphorylated. Previously, we cells where it is not tyrosine phosphorylated. had related these depressed levels of EphA2 tyrosine phos 35 phorylation with decreased ligand binding. Malignant cells Whereas tyrosine phosphorylation of EphA2 does not often have unstable cell-cell contacts and we postulated that appear to be necessary for its intrinsic enzymatic activity, this decreases the ability of EphA2 to stably interact with its ligand-mediated tyrosine phosphorylation regulates EphA2 ligands, which are anchored to the membrane of adjacent cell. protein stability. Specifically, tyrosine phosphorylation fates In part, our present data Suggests a new paradigm in which the 40 EphA2 to interact with the c-Cbl adapter protein and to sub phosphotyrosine content of EphA2 is also negatively regu sequently be internalized and degraded within proteosomes lated by an associated tyrosine phosphatase that is overex (J. Walker-Daniels et al., Mol. Cancer Res. 2002 November; pressed in malignant cells. Given the relationship between 1(1):79-87). Consequently, the phosphatase activity of EphA2 phosphorylation and cell-cell adhesion, we cannot LMW-PTP would be predicted to increase EphA2 protein exclude that cell-cell contacts could also regulate the expres 45 stability. Indeed, the highest levels of EphA2 are consistently sion or function of LMW-PTP and future investigation should found in cells with high levels of LMW-PTP. One interesting address this possibility. implication of this finding is that it provides a mechanism, The fact that high levels of LMW-PTP were observed in independent of genetic regulation of the EphA2 gene, to several different cell models of metastatic cancer is notable explain why high levels of EphA2 are found in many different given that LMW-PTP overexpression is sufficient to confer 50 tumors. An alternative possibility is that LMW-PTP upregu malignant transformation. LMW-PTP overexpressing cells lates EphA2 gene expression and our present findings do not gain the ability to colonize soft agar and acquire a malignant formally eliminate this possibility. The fact that EphA2 phenotype when cultured in three-dimensional basement inhibitors reversed the malignant character of LMW-PTP membranes, such as Matrigel. Notably, however, LMW-PTP overexpressing cells Suggests that the upregulation of EphA2 overexpressing MCF-10A epithelial cells displayed reduced 55 is relevant to the cellular behaviors of LMW-PTP-mediated rates of cell growth as measured using two-dimensional transformation. assays of cell growth. This latter observation is consistent with recent reports that high levels of LMW-PTP similarly In Summary, our present studies, as described in this decrease the monolayer growth rates of other cell types example and in Kikawa et al., J. Biol. Chem. 277 (42): 39274 (Shimizu, H., Shiota, M., Yamada, N., Miyazaki, K., Ishida, 60 39279 (2002)) identify LMW-PTP as a new oncogene that is N. Kim, S. & Miyazaki, H. (2001) Biochemical & Biophysi overexpressed in tumor-derived carcinoma cells. We also link cal Research Communications 289, 602-607; Fiaschi, T., the biochemical and biological actions of overexpressed Chiarugi, P., Buricchi, F., Giannoni, E., Taddei, M. L., Talini, LMW-PTP as with EphA2. These findings have important D., Cozzi, G. Zecchi-Orlandini, S., Raugei. G. & Ramponi, implications for understanding the biochemical and biologi G. (2001) Journal of Biological Chemistry 276, 49156 65 cal mechanisms that contribute to the metastatic progression 49163). Although such a finding had been interpreted to sug of epithelial cells. Moreover, our present studies identify an gest that LMW-PTP might negatively regulate malignant important signaling system that could ultimately provide an US 7,662,770 B2 29 30 opportunity to target the large number of cancer cells that EphA2 from binding its membrane-anchored ligands. This overexpress EphA2 or LMW-PTP. suggests that D129A prevents EphA2 from being dephospho rylated even after loss of ligand binding. Example III FIG. 15 is a table that summarizes evidence from immun ofluorescence microscopic studies using LMW-PTP trans Effects of LMW-PTP Overexpression fected MCF-10A cells and D129A-transfected MDA-MB 231 (transformed) cells. The altered morphology and markers Cell lines and reagents were as described in Example II of LMW-PTP-transfected MCF-10A cells is consistent with (Kikawa et al., J. Biol. Chem. 277 (42): 39274-39279 (2002)). malignant transformation. Moreover, the morphology of Methods for making cell lysates and for performing immu 10 D129A overexpressing cells is consistent with a less aggres noprecipitation and western blot (immunoblot) analyses, sive (more differentiated) phenotype. EGTA and pervanadate treatments, transfection and selec tion, and growth assays were also as described in Example II Co-Localization of EphA2 and LMW-PTP in Transformed ((Kikawa et al., J. Biol. Chem. 277 (42): 39274-39279 and Nontransformed Cells (2002)). 15 Subcellular localization of EphA2 (using D7 antibodies) and LMW-PTP (using rabbit polyclonal sera) in control and Morphological Effects of LMW-PTPOverexpression in Non LMW-transfected MCF-10A cells was evaluated informalin Transformed Cells fixed (3.7%, 2 minutes), detergent permeabilized (PBS con Monolayer cultures of MCF-10A cells that had been stably taining 0.5% Triton-X-100) monolayers, cultured on glass transfected with either wild-type human LMW-PTP, or a coverslips. The images (FIG. 16A) were viewed on a Nikon matching vector control, were subjected to microphotogra microscope (600X) and images captured using Nikon digital phy (600X) (FIG. 10). Whereas the non-transformed (vector) cameras and software. cells retained a characteristic epithelial morphology, LMW Subcellular localization of EphA2 (using D7 antibodies) PTP-transfected cells adopted a mesenchymal phenotype that and LMW-PTP (using rabbit polyclonal sera) was likewise is characteristic of malignant epithelial cells. Overexpression evaluated in control and D129A overexpressing MDA-MB of LMW-PTP was thus observed to alter two-dimensional 25 morphology of the cells. 231 cells (FIG. 16B). The LMW-PTP transfected MCF-10A cells were further Effects of LMW-PTP Overexpression on Actin Organization observed to form three-dimensional foci, a hallmark of malig in Transformed and Nontransformed Cells nant transformation, when cultured at high cell density (FIG. The organization of the actin cytoskeleton was evaluated in 11). 30 a MDA-MB-231 cell line stably expressing the D129A LMW-PTP mutation (Bisoform), the MCF-10A cell line, and Effects of LMW-PTP Inactivation in Transformed Cells the MCF-10A cell line stably expressing the wild-type (WT) To evaluate the biological outcomes of inhibiting LMW LMW-PTP molecule (B isoform) by immunofluorescence PTP in tumor cells, highly invasive MDA-MB-231 cells were localization of fluorescein-conjugated phalloidin (Molecular stably transfected with a mutant of LMW-PTP (D129A). 35 Probes, Eugene, Oreg.). The subcellular localization of actin D129A functions as a Substrate trapping mutant and thereby (phalloidin staining) was evaluated informalin-fixed (3.7%, 2 competes away the activity of endogenous LMW-PTP in the minutes), detergent permeabilized (PBS containing 0.5% Tri tumor cells. It effectively inactivates LMW-PTP in the trans ton-X-100) monolayers, cultured on glass coverslips. The formed cells. images (FIG. 17) were viewed on a Nikon microscope (600X) D129A transfected cells showed reduced colony formation 40 and images captured using Nikon digital cameras and soft in soft agar relative to matched (vector) controls (FIG. 12). Wa. Thus LMW-PTP inactivation in transformed cells results in Overexpression of wild-type LMW-PTP was found to decreased soft agar colonization. This indicates that LMW cause the formation of stress fibers (as opposed to the adhe PTP is necessary for anchorage-independent cell growth and/ sion belts that predominate in control cells). In the converse or Survival, which are hallmarks of malignant cells. 45 situation, dominant-negative inhibitors (D129A) of LMW It was also found that inactivation of LMW-PTP alters PTP decrease the number of stress fibers in MDA-MB-231. two-dimensional morphology and EphA2 distribution in These observations are consistent with the hypothesis that transformed cells. The morphology of MDA-MB-231 cells wild-type LMW-PTP promotes a malignant (migratory and that express dominant-negative LMW-PTP (D129A) or a invasive) phenotype whereas inhibition of LMW-PTP is suf matched vector control was evaluated by immunofluores 50 cence microphotography of labeled EphA2 (FIG. 13). Con ficient to reverse an aggressive phenotype. trol cultures MDA-MB-231 normally adopt a mesenchymal Effects of LMW-PTP Overexpression on Focal Adhesion morphology with EphA2 diffusely distributed or enriched The organization of focal adhesion, as determined using with membrane ruffles. In contrast, D129A-transfected cells paxillin-specific antibodies, was also evaluated in MDA-MB display a characteristic epithelial morphology, with Eph A2 55 231 cells by immunofluorescence microscopy. The subcellu enriched within sites of cell-cell contact. lar localization of paxillin was evaluated in formalin-fixed D129A LMW-PTPMDA-MB-231 cells were treated with (3.7%, 2 minutes), detergent permeabilized (PBS containing EGTA to determine its effect on the phosphorylation status of 0.5% Triton-X-100) monolayers, cultured on glass cover EphA2. Detergent extracts from 5x10 control and D129A slips. The images (FIG. 18) were viewed on a Nikon micro transfected MDA-MB-231 cells were harvested as described 60 Scope (600X) and images captured using Nikon digital cam in Examples I and II. After immunoprecipitating EphA2 with eras and Software. D7 antibodies, the samples were resolved by SDS-PAGE and Overexpression of wild-type LMW-PTP was found to subjected to Western blot analyses with phosphotyrosine increase the prominence of focal adhesion, particularly at the specific (4G10) antibodies. The EphA2 in D129A-trans leading edge of cell migration and invasion, which is consis fected cells was found to be more highly tyrosine phospho 65 tent with a more aggressive phenotype. In the converse situ rylated, even following treatment with EGTA (FIG. 14). ation, dominant-negative inhibitors (D129A) of LMW-PTP EGTA destabilizes cell-cell contacts and thereby prevent decrease the predominance of focal adhesion in MDA-MB US 7,662,770 B2 31 32 231 cells, resulting in a diffuse (rather than polarized) distri were observed 20 days after injection, and the size of the bution of focal adhesions, which is not consistent with cell tumor (if present) was measured. migration or invasion. FIG. 21 shows tumor measurement data for mice injected Pathological Markers of Malignant Character with 5x10 cells, observed 20 days post-injection. None of The expression of cytokeratin (FIG. 19) and vimentin the mice injected with the parental MCF-10A cells or the (FIG. 20) was evaluated using immunofluorescence micros control vector exhibited tumorogenesis at the injection site. copy. The staining of cytokeratin and vimentin was evaluated Mice injected with the MCF-10A cells stably overexpressing in formalin-fixed (3.7%, 2 minutes), detergent permeabilized WTLMW-PTP, however, exhibited significant growth in all3 (PBS containing 0.5% Triton-X-100) monolayers, cultured of the mice injected with 5 million cells, and 2 of the 3 mice on glass coverslips. The images were viewed on a Nikon 10 injected with 1 million cells. These results suggest that LMW microscope (600X) and images captured using Nikon digital PTP overexpression is sufficient to confer tumorigenic poten cameras and software. tial upon non-transformed epithelial cells. EphA2 is the only Overexpression of wild-type LMW-PTP was found to other oncogene we are aware of that is capable of conferring decrease cytokeratin but increase vimentin expression. These 15 tumorigenic potential upon non-transformed epithelial cells. results are notable given that these changes in intermediate filament protein expression are frequently used by patholo The complete disclosure of all patents, patent applications, gists for cancer diagnosis and typing. and publications, and electronically available material (in cluding, for example, nucleotide sequence Submissions in, Example IV e.g., GenBank and RefSeq, and amino acid sequence Submis sions in, e.g., SwissProt, PIR, PRF, PDB, and translations Effect of LMW-PTP Overexpression on Tumorigenic from annotated coding regions in GenBank and RefSeq) cited Potential of Non-Transformed Epithelial Cells herein are incorporated by reference. The foregoing detailed description and examples have been given for clarity of Cells (MCF-10A, MCF-10A Neo (control) and transfected understanding only. No unnecessary limitations are to be MCF-10A cells stably overexpressing wild-type LMW-PTP) 25 understood therefrom. The invention is not limited to the were introduced into mice via Subcutaneous injection. Two exact details shown and described, for variations obvious to dosage levels were used: approximately 2 million and 5 mil one skilled in the art will be included within the invention lion cells. Three mice were included in each group. The mice defined by the claims. SEQUENCE LISTING <16 Os NUMBER OF SEO ID NOS: 3 SEO ID NO 1 LENGTH: 35 TYPE: DNA ORGANISM: artificial FEATURE; OTHER INFORMATION: primer <4 OOs SEQUENCE: 1 aatttaaagc titcCatggcg galacaggct a ccaag 35 SEO ID NO 2 LENGTH: 34 TYPE: DNA ORGANISM: artificial FEATURE; OTHER INFORMATION: primer <4 OOs SEQUENCE: 2 cgttcttgga galaggcc cac taga attct tcgt. 34 SEO ID NO 3 LENGTH: 28 TYPE: DNA ORGANISM: artificial FEATURE; OTHER INFORMATION: primer <4 OOs SEQUENCE: 3 gcgc.gcggat CCtcagtggg ccttct c c 28 US 7,662,770 B2 33 34 What is claimed is: detecting the bound complex; and 1. A method for treating cancer in a mammal comprising a determining whether LMW-PTP is overexpressed in the cancer cell that expresses a low molecular weight protein biological material relative to a noncancerous biological tyrosine kinase (LMW-PTP), the method comprising admin material, wherein overexpression of LMW-PTP is istering to the mammala treatmentagent comprising a mutant 5 indicative of the presence of cancer cells in the mammal. LMW-PTP protein D129A in an amount effective to inhibit 10. A method for diagnosing cancer in a mammal compris the activity of LMW-PTP. ing: assaying a biological material comprising cells of the 2. A method for treating cancer in a mammal comprising a mammal for LMW-PTP activity; and cancer cell that expresses a low molecular weight protein determining whether LMW-PTP is overexpressed in the tyrosine kinase (LMW-PTP) and an EphA2 receptor mol 10 biological material relative to a noncancerous biological ecule, the method comprising administering to the mammala material, wherein overexpression of LMW-PTP is first treatment agent comprising a mutant LMW-PTP protein indicative of the presence of cancer cells in the mammal. D129A in an amount effective to inhibit the activity of LMW 11. The method of claim 10 wherein the biological material PTP and a second treatment agent comprising an anti-EpbA2 is in the mammal. antibody or an ephrin A1 linand in an amount effective to 15 12. The method of claims 9 or 10 wherein the biological favorably alter the biological activity of the EphA2 receptor material comprises a tissue, organ, blood, urine, saliva, or molecule. spinal fluid of the mammal. 3. The method of claim 2 wherein the first and second 13. The method of claim 12 wherein the biological material treatment agents are delivered simultaneously. is obtained from the mammal. 4. The method of claim 2 wherein the first treatment agent 14. The method of claim 13 further comprising obtaining is delivered prior to the second treatment agent. the biological material from the mammal. 5. The method of claim 2 wherein the second treatment 15. A method for evaluating the efficacy of a candidate agent is delivered prior to the first treatment agent. cancer therapeutic agent that targets the EphA2 receptor mol 6. The method of claim 2 wherein favorably altering the ecule, the method comprising: biological activity of the EpbA2 receptor molecule comprises 25 contacting a cancer cell expressing EphA2 receptor mol increasing the phosphotyrosine content of the EphA2 recep ecule and LMW-PTP with a candidate therapeutic agent tor molecule. to yield a treated cancer cell; 7. The method of claim 1 or 2 wherein at least one treatment determining the amount or activity of LMW-PTP in the agent is covalently linked to a cytotoxic agent. treated cancer cell; and 8. The method of claim 1 or 2 wherein the cancer cell is a 30 comparing the amount or activity of LMW-PTP in the metastatic carcinoma cell. treated cancer cell with the amount or activity of LMW 9. A method for diagnosing cancer in a mammal compris PTP in an analogous untreated cancer cell, wherein a 1ng: reduction in the amount or activity of LMW-PTP in the lysing cells in a biological material obtained from the treated cell is indicative of the efficacy of EphA2 target mammal to yield a cell lysate; 35 ing of the candidate therapeutic agent. contacting the cell lysate with an antibody that binds LMW-PTP to form a bound complex: k k k k k