ADCK4靜默透過增加幹性促進肺腺癌的生成

Abstract

肺腺癌病人的存活期因為基因檢驗及新藥進步而延長，因此轉移癌的預防及治療更為重要。在肺腺癌好發的轉移中，顱內轉移對病患生活品質及壽命影響最嚴重，鑑定腦轉移有關的分子調控機制以協助評估病患腦轉移的風險、甚至選擇最佳的治療方式尤為重要。 在本研究中,我們利用慢性病毒(Lentivirus)將6000多組抑制人類Kinase及phosphatase的shRNA 基因組送入肺腺癌A549細胞中，使每一顆肺腺癌A549細胞隨機的帶有一種shRNA。將這群細胞由小鼠的內頸動脈注射，並於小鼠腦部長出腫瘤後取出腫瘤經培養種到下一批老鼠中，總計重複三次。接著將三次實驗的腫瘤以PCR與NGS定序的方式，得到各shRNA在腫瘤中所佔的比例。經Western及qPCR驗證A549中的ADCK4確實被抑制後，我們先進行了體外實驗，分別是Boyden chamber 癌細胞侵襲實驗 和 克隆形成實驗，均觀察到差異。 我們又在免疫缺陷鼠皮下注射該細胞，在皮下長成腫瘤後進行切除，模擬臨床上病人治療後的情況，觀察小鼠復發及轉移的情形。發現在shADCK4組別的腫瘤，除了生長速度較快，病理切片上可以看出分化較不完全，腫瘤細胞的核質比較高。在體外和體內的實驗中，都發現當A549細胞中的ADCK4被抑制後，與對照組相比有了更惡性的變化，這證明ADCK4靜默確實促進癌生成。另一方面，我們亦發現A549細胞被抑制掉ADCK4後，會擁有幹細胞特性，或許,ADCK4是透過影響肺癌細胞的幹性促進癌生成。 在之後的實驗中,我們選取不同的肺癌細胞,抑制掉ADCK4後進行相同的實驗,證明不僅僅是在A549細胞上才有此現象。最後，未來還需測量病人檢體上ADCK4的表現量高低，並與病人的轉移狀況與存活相比較，觀察ADCK4是否具有臨床應用價值。

With the advances of gene testing and drug discovery, the survival of lung adenocarcinoma is significantly prolonged. The prevention and treatment of metastatic cancer become one of the most challenging subjects clinically. Brain metastasis occurs frequently in advanced lung adenocarcinoma, which affects patients’ life seriously. We would like to identify the underlying molecular mechanism of brain metastasis for new treatment development. In this study, A549 cells were infected with a lentivirus library containing more than 6, 000 independent shRNA clones that target human kinases and phosphatases. After antibiotics selection, the resulting stable cell lines were injected into the brain of SCID mice through internal carotid artery. The tumor cells of metastatic loci were harvested from brain and cultured for next injection. The in vivo selections were repeated three times. Next, the shRNA clones of brain tumor lesions in the three times injections were amplified by PCR, and the proportion of individual shRNA in the tumor was calculated after NGS AmpliSeq. We found that ADCK4, a mitochondria protein, can inhibit cell invasion and tumorigenesis in vitro assessed by Boyden chamber invasion assay and colony formation assay. We confirmed the finding by an in vivo subcutaneous tumorigenesis assay. The tumor growth of A549 cells with shADCK4 was higher than the control, A549 cells with shLacZ. On the other hand, we found that knockdown of ADCK4 increases stemness in A549 cells. It is possible ADCK4 silencing increases tumorigenesis via stemness acquisition. In the following experiments, we selected different lung cancer cells and conducted the same experiment after inhibiting ADCK4, proving that this phenomenon was not only found on A549 cells. Finally, the further experiments should be performed to measure the level of ADCK4 expression on the patients’ lung cancer samples and to correlate with the patients’ metastasis status and survival, so as to observe whether it has the potential for clinical applications.