An Integrated Genomic and Proteomic Analysis to Elucidate the Expression Level, Regulatory Mechanism, Signaling Pathways, and Effector Proteins of Ptp4a3 Oncogene in Gastrointestinal Stromal Tumors: Prognostic, Biological, and Therapeutic Implication

KIT or PDGFRA receptor tyrosine kinase (RTK) gene mutations are primary oncogenic events in most gastrointestinal stromal tumor (GIST), a neoplasm with a wide spectrum of clinical behavior. Despite RTK genotypes conferring prognostic impact, chromosomal imbalances, such as -9p/9q and +8q, critically affect clinical aggressiveness in GISTs. Identification of cancer-associated genes by array comparative genomic hybridization (aCGH) may elucidate tumorigenesis to develop prognostic adjuncts or derived targeted therapies. Using 385K aCGH to profile GIST samples and cell lines (GIST48, GIST882), we map amplicon cores to 8q24 harboring candidate FAK and PTP4A3 oncogenes that display preferential DNA gains in non-low-risk cases, including 2 with high-level PTP4A3 amplification. As preliminary validation, RT-PCR andWestern blotting assays show similar PTP4A3 upregulation in two GIST cell lines, while the expression fold is overtly higher in GIST882 cells without DNA gain at PTAP4A3, suggesting operation of an amplification-independent mechanism. As a 22-kD prenylated phosphatase, PTP4A3 overexpression is partly driven by gene amplification, which not only enhances cell migration and invasion but also correlates with tumor aggressiveness in colorectal and other carcinomas. Promisingly, immunostain of 321 GISTs shows that PTP4A3 overexpression significantly correlates with higher risk category, unfavorable RTK genotypes, and disease-free survival. By suppressing C-terminal SRC kinase (CSK), PTP4A3 inactivates FAK/SRC complex via inhibition of SRC. The expression of CSK and CHK (CSK homologue associating with KIT) is decreased in colorectal cancers, making SRC activity unrestrained. Accordingly, we hypothesize PTP4A3 as a novel prognosticator in GISTs by deregulating associated signaling pathways via dephosphorylation of its as yet unknown substrates. This proposal is indented to elucidate the following specific aims in 3 years: year 1, (1) complete aCGH profiling for a total of 30 GIST samples with attention given to PTP4A3 and FAK, (2) quantify gene dosage and transcripts of PTP4A3 and FAK oncogenes by real-time PCR and RT-PCR for laser capture-microdissected tumor cells in 370 GISTs, and (3) assess the correlations of branched-chain DNA assay-determined PTP4A3 mRNA expression with clincopathological factors, patient survival, and immunoexpression of potential PTP4A3-modulating proteins, with Western blotting validation in selected cases; year 2, In vitro, (1) clarify the biological function of PTP4A3 in GISTs using transfection of shPTP4A3 and catalytic inactive PTP4A3C104S mutant, (2) decipher transcriptional regulatory basis of PTP4A3 expression to identify its regulators in GISTs by deletion mapping, reporter assay, and quantitative chromatin immunoprecipitation, and (3) analyze the relevance of PTP4A3-CSK(CHK)-FAK/SRC regulatory axis in GIST tumorigenesis and therapeutic efficacy of PTA4A3 inhibitors, alone or in combination with imatinib; year 3, In xenografts, study (1) the functional effect of PTP4A3 on tumorigenicity in vivo, (2) anticancer potency of PTP4A3 inhibitors with in vivo imaging system, and (3) the potential substrates and effector proteins of PTP4A3 in GISTs by proteomic profiling with 2D-DIGE, Pro-Q stain, and MALDI-TOF-MS.

Project ID:PC10101-1788
External Project ID:NSC99-2628-B182-003-MY3