分析Contactin 4於大腸直腸癌介導EGFR分子訊號傳遞路徑之抑癌分子機制

Abstract

長年以來，我國之大腸直腸癌發生率與死亡率在皆位居於前三位，大腸直腸癌主要起因於致癌基因或抑癌基因之基因變異，進而導致腸黏膜上皮細胞的過度增生，最終形成具侵襲性之癌症。本實驗室先前於人類第三號染色體3p26.3發現Contactin 4 (CNTN4) 在大腸直腸癌可能為抑癌基因功能。當HCT116細胞表現CNTN4可抑制細胞增生、固著與非固著依賴性胞落形成，且表現CNTN4可抑制裸鼠皮下腫瘤生長並減少血管新生。根據先前文獻研究，我們假設CNTN4透過與Protein tyrosine phosphatase receptor type gamma (PTPRG) 相互作用，進而抑制Epidermal growth factor receptor (EGFR) 之下游路徑Erk/CREB/c-Jun之磷酸化、WNT/β-catenin路徑以及NOTCH之活化；同時，CNTN4可使p21表現量上升及減少Rb磷酸化，使細胞週期停滯於G0/G1時期。本論文首先證實CNTN4可表現於細胞膜上，接續利用Proximity Ligation Assay (PLA) 驗證CNTN4與PTPRG之間具有交互作用。接續，利用RNA-seq的Gene Set Enrichment Analysis (GSEA) 分析，進而探討CNTN4於大腸直腸癌介導EGFR分子訊號傳遞路徑之影響。利用大腸直腸癌穩定表現CNTN4之細胞株，經由EGF誘導後，CNTN4可以降低EGFR表現及EGFR之磷酸化，根據GSEA結果顯示：CNTN4表現與PI3K/AKT/mTOR路徑呈現顯著負相關 (NES= -1.574, p<0.05)。於是利用即時定量聚合酶連鎖反應與西方墨點法，驗證大腸癌細胞株表現CNTN4後，PI3K之mRNA與蛋白質表現量皆有所下降，並且可降低PI3K之下游AKT、mTOR以及其活化態的蛋白質表現。藉由RNA-seq及公開資料庫之數據發現：CNTN4的表現與mTOR下游之4EBP1呈現顯著負相關，於是偵測4EBP1以及其活化態的蛋白質表現量，結果發現CNTN4確實明顯抑制4EBP1的活性，而對於mTOR另一下游分子p70S6K的活性則沒有顯著影響。另外， GSEA結果也顯示JAK/STAT相關路徑與CNTN4呈現顯著負相關 (NES= -1.328, p<0.05)，利用西方墨點法亦支持表現CNTN4之HCT116細胞株，活化態STAT3確實有下降趨勢。根據RNA-seq分析結果及先前文獻，得知EGFR下游也會調控 β-catenin活性，利用免疫螢光染色證實表現CNTN4後可降低β-catenin進入細胞核，進而降低其下游基因的轉錄。綜合而論，本論文揭露CNTN4會藉由與PTPRG交互作用，降低EGFR以及其下游相關訊號路徑之活化，以達抑制大腸直腸癌發展之功能。

The incidence and mortality rates of colorectal cancer (CRC) in Taiwan have consistently ranked among the top three over the years. In our previous study, we proposed Contactin 4 (CNTN4), a cell adhesion molecule, as a candidate tumor suppressor gene associated with CRC. CNTN4-expressing HCT116 single stable clones established in CRC cell lines exhibited attenuated malignant phenotypes, including cell proliferation, anchorage-dependent and -independent colony formation, as well as reduced xenograft tumorigenicity in nude mice. According to the literature review, we proposed that CNTN4 may interact with protein tyrosine phosphatase receptor type gamma (PTPRG), thereupon mediate the tumor suppression via decreasing the phosphorylation of different components of EGFR signaling pathways such as ERK/CREB/c-Jun, WNT/β-catenin and NOTCH signaling pathway. In addition, CNTN4 expression could retard cell cycle into G0/G1 phase by mediating p21 expression and Rb phosphorylation level. In the study, we first demonstrated that CNTN4 can be expressed on the cell membrane, and subsequently verified the interaction between CNTN4 and PTPRG by proximity ligation assay. Then, followed by a full exploration of CNTN4-modulated molecular mechanisms in CRC cells by using gene set enrichment analysis (GSEA) of RNA-sequencing data. We demonstrated CNTN4 expression decreased EGFR phosphorylation in CRC cells after EGF treatment. Based on GSEA, CNTN4 expression is negatively correlated to PI3K signaling pathway related genes. Correspondingly, the expression of PI3K, AKT, mTOR and 4EBP1 was down-regulated in CNTN4-expressing CRC cells, indicating that CNTN4 could inhibit the PI3K/AKT/mTOR signaling pathway, resulting in the modulation of gene transcription related to cancer aggressiveness, such as 4EBP1. However, there is no significant effect on the activity of another downstream molecule of mTOR, p70S6K. Then we also observed the down-regulation between CNTN4 and JAK/STAT signaling pathway according to GSEA. Subsequent Western blot analysis revealed a definite decreasing trend in the expression level of activated STAT3 in CNTN4 expressing HCT116 cells. Futhermore, CNTN4 could inhibit β-catenin translocation into nucleus to reduce its activity. Taken together, CNTN4 is a novel tumor suppressor through modulating EGFR-associated signaling pathways to reduce downstream molecules phosphorylation, and further decreasing the related tumor-promoting factors expression in CRC.