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

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Project title

Code/計畫編號

NSC99-2628-B182A-064-MY3

Translated Name/計畫中文名

基因體與蛋白質體整合分析以探究PTP4A3致癌基因於胃腸道基質瘤之表現量、調節機轉、訊息路徑及受動蛋白---預後、生物及治療意涵

Funding Organization/主管機關

National Science and Technology Council

Co-Investigator(s)/共同執行人

黃玄贏(計畫主持人)
薛佑玲
李健逢

Department/Unit

Chang Gung Memorial Hospital Anatomic Pathology

Website

https://www.grb.gov.tw/search/planDetail?id=2132148

Year

2010

Start date/計畫起

01-08-2010

Expected Completion/計畫迄

31-07-2011

Co-Investigator(s)

Tai-Yuan Chen

Bugetid/研究經費

2450千元

ResearchField/研究領域

臨床醫學
生物技術(醫)

Description

Abstract

胃腸道基質瘤(GIST)為一包含廣泛不同臨床行為之腫瘤，而KIT 或PDGFRA 酪氨酸激酶(RTK)受器基因突變為其最初之致瘤變化。雖然RTK 基因型具有預後意義，但染色體的不平衡如9p/9q 減少或8q 增加，卻關鍵地影響GIST 之臨床惡性度。利用微陣列比較基因雜交(aCGH)來探尋癌症相關基因可以釐清腫瘤之生成，藉以發展預後標記或衍生之標靶治療。我們利用高解析385K 之aCGH 晶片分析GIST 樣本及細胞株之染色體變化，初步發現於8q24 有放大區段涵蓋FAK 及PTP4A3 致癌基因，其特別在非低危險度腫瘤樣本呈現DNA 倍數增加，尤其有兩標本可見顯著PTP4A3 高度基因放大。初步以反轉錄PCR 及西方點漬驗證發現於兩GIST 細胞株其PTP4A3 mRNA 及蛋白質表現量皆呈現類似上揚，但無 DNA 增加之GIST882 細胞卻有明顯更高之表現量，此顯示於GIST 另有與基因放大無關之PTP4A3 過度表現機轉。PTP4A3 為一22-kD 異戊烯化磷酸酶, 其於大腸癌中過度表現有些乃基因放大所驅動，此可增進細胞移動及侵襲力並與腫瘤惡性度有關。令人鼓舞的是，在321 例GIST 的免疫染色中，PTP4A3 過度表現與較高危險度、預後不利之RTK 基因型、及無疾病復發存活率皆顯著相關。目前已知藉由壓抑C 端SRC 激酶(CSK)，PTP4A3 可抑制SRC 而將FAK/SRC 複合蛋白去活化。在一些大腸直腸癌細胞中，CSK 及其可與KIT 結合之相似蛋白 -CHK-之表現量皆呈減少，因而令SRC 之活性不受控制。據此，吾等假設PTP4A3 為GIST 之新穎預後因子，其可藉由其尚未發現之蛋白受質去磷酸化，致使其調控之訊息路徑失調。在此計畫，我們將於三年中釐清下列特定目標: 第一年，(1)完成總數30 例 GIST 之aCGH 描繪，並特別著眼於PTP4A3 及FAK 基因之變化。(2)於雷射擷取之純粹GIST 腫瘤細胞將以同步即時PCR 及RT-PCR 分別定量PTP4A3 與FAK 基因之倍數與mRNA 之表現量。(3) 以分枝鏈DNA 原位雜交法大規模分析370 例GIST 之PTP4A3 mRNA 表現量，及其與臨床病理因子、病人存活、PTP4A3 可能調節蛋白之免疫染色的關聯性，並以部分病例作西方點漬交叉驗證。第二年，於體外實驗，吾等將(1)轉殖PTP4A3 之干擾RNA 與催化性非活化之 PTP4A3C104S 突變體以澄清PTP4A3 於GIST 之生物功能。(2)以刪除描繪法、報導螢光酶、及定量染色質免疫沉澱法，解答在PTP4A3 表現之轉錄調控機制，並尋找其在GIST 之轉錄調節子。(3) 分析在GIST 腫瘤生成中 PTP4A3-CSK(CHK)-FAK/SRC 調節路徑之切題性及單獨使用PTP4A3 抑制劑或合併imatinib 對GIST 之治療效益。第三年，於異種移植模式將確定(1) PTP4A3 表現於生物體內促進GIST 腫瘤生成之功能效應。(2)以生物內影像系統分析 PTP4A3 抑制劑於GIST 之療效。(3)以2D-DIGE、Pro-Q 染色、及MALDI-TOF 質譜儀等蛋白質體方法來確認PTP4A3 可能之下游受質及受動蛋白。 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.

Keyword(s)

胃腸道基質瘤
PTP4A3基因放大
比較基因雜交微陣
GIST
PTP4A3 amplification
array comparative CGH