分析Contactin 4於大腸直腸癌所調控之抑癌分子機制

Abstract

在我國，大腸直腸癌之發生率與死亡率歷年以來皆位居於前位，其主要源於致癌基因或抑癌基因之基因變異，導致腸黏膜上皮細胞的過度增生。本實驗室先前於人類第三號染色體3p26.3發現Contactin 4 (CNTN4)在大腸直腸癌可能作為抑癌基因。於HCT116細胞表現CNTN4得以抑制細胞增生、固著與非固著依賴性胞落形成能力，且CNTN4表現可以抑制裸鼠皮下腫瘤生長及減少血管新生，並假設CNTN4會與Protein tyrosine phosphatase receptor type gamma (PTPRG)相互作用後，抑制Erk1/2及其下游CREB和c-Jun之磷酸化。本論文欲藉由RNA-Sequencing (RNA-seq)結果，以不同生物資訊資料庫分析，探討CNTN4於大腸直腸癌細胞可能調控的分子機制。初步，利用Gene Set Enrichment Analysis (GSEA)做分析，指出CNTN4的表現與Epidermal growth factor receptor (EGFR)及NOTCH訊號傳遞路徑呈現顯著負相關。接續利用即時定量聚合酶連鎖反應與西方墨點法，驗證在大腸癌細胞株表現CNTN4後，EGFR之mRNA與蛋白質表現量皆有下降。目前已知，c-MYC的表現量會受上游EGFR透過Erk1/2所調控，並影響細胞增生，因此檢測而知c-MYC表現量與CNTN4表現明顯呈現負相關。另一方面，RNA-seq結果顯示WNT/β-catenin訊號傳遞路徑與CNTN4表現呈負相關，而β-catenin已知具有調控c-MYC轉錄的功能，於是進一步研究其表現與CNTN4之關聯，在HCT116及SW620細胞株表現CNTN4確實可降低-catenin之mRNA及蛋白表現量，且活化態之β-catenin也隨之減少，並將β-catenin滯留於細胞膜附近，減少其進入細胞核內，以降低下游基因轉錄。對於NOTCH相關路徑，初步已利用即時定量聚合酶連鎖反應檢測NOTCH1-NOTCH4於HCT116及HCT15單一穩定表現CNTN4細胞株之mRNA表現量，結果顯示，當CNTN4表現時，NOTCH1-NOTCH4 mRNA表現皆有下降趨勢。後續偵測Notch intracellular domain (NICD)及其下游目標基因之表現量，結果發現CNTN4會影響NOTCH訊號傳遞之活性。藉由RNA-seq基因富集分析指出CNTN4可能參與細胞週期之調控，也從文獻得知c-MYC會調控下游目標基因以影響細胞週期，實驗結果也發現於HCT116細胞株表現CNTN4，p21表現量上升並減少Rb磷酸化，進一步使細胞週期停滯於G0/G1時期。綜合而論，CNTN4透過調控細胞週期，並影響EGFR與NOTCH下游相關訊息路徑以減緩大腸直腸癌細胞生長。

Colorectal cancer incidence has been rising worldwide. In our previous study, we delineated a minimal deletion region (MDR) at 3p26.2-p26.3 by using loss of heterozygosity study, followed by clinical relevance assessment of patients with colorectal cancer. Within the MDR, we proposed Contactin 4 (CNTN4), a cell adhesion protein, as a candidate tumor suppressor gene. CNTN4-expressing single stable clones were established in different CRC cell lines, and exhibited a decreased capability of cell proliferation and anchorage-dependent and independent colony formation, as well as reduced xenograft tumor growth in nude mice. Then, we purposed CNTN4 may mediate the tumor suppression through decreased the phosphorylation of CREB, c-Jun and ERK1/2. In the study, the CNTN4-mediated molecular mechanisms in HCT116 cells were explored by using RNA-sequencing. Furthermore, the differential genes expression between HCT116/Mock and HCT116/CNTN4-c7 cells was analyzed by RNA-sequencing to identify CNTN4-regulated genes and pathways. The enrichment analysis indicated that CNTN4 expression is negatively correlated to EGFR and NOTCH signaling pathway related genes. Consequently, we verified the inhibition of mRNA and protein levels with EGFR, c-MYC, β-catenin and NOTCH in CNTN4-expressing CRC cells. Furthermore, CNTN4 may inhibit β-catenin translocation into nucleus to decrease the activity and decrease the NOTCH intracellular domain expression to inactivate the signal transduction. According to the gene enrichment analysis, we also monitored the DNA content to identify the cell cycle progression. The results indicated that CNTN4 may retard cell cycle into G0/G1 phase by mediating p21 expression and Rb phosphorylation level. Taken together, CNTN4 is a novel tumor suppressor through modulating EGFR-associated signaling pathway to reduce downstream molecules phosphorylation level and thus decrease related-transcription factor expression, in addition retardant cell cycle to decrease cell proliferation in colorectal cancer.