抗失巢凋亡在胃癌中的表觀改變與其潛在機制

Abstract

胃癌是第六種最普及的癌症，由於胃癌常常伴隨著快速發展的腹腔轉移，此現象被視為導致癌症死亡的其中一個最普遍的原因，腹腔轉移在胃癌末期的病人中是相當普見的，大概百分之八十到九十的胃癌末期病患都伴隨著腫瘤轉移與不好的預後。然而，腹腔轉移的機制還未被清楚釐清。 失巢凋亡(Anoikis)是一種細胞脫離細胞外基質而導致的一種細胞凋亡，不過惡性腫瘤有能力逃脫失巢凋亡，進而促進他們侵犯與轉移的能力，這樣的現象叫做”抵抗失巢凋亡”，因此，我們推測抵抗失巢凋亡可能與腹腔轉移有關。 本篇中，我們首先利用不同細胞株包括肺癌細胞LU65與LU99, 胰臟癌細胞MIA-PaCa2與BxPC3, 乳癌細胞4T1, MCF7與T47D, 以及 胃癌細胞AGS與NCI-N87來建立具有抵抗失巢凋亡能力的細胞，我們發現胃癌細胞NCI-N87在24天後有抵抗失巢凋亡的能力。我們分析該細胞株(在本篇用NCI-N87-AR表示) 並發現其侵犯與增生能力更好。透過RNA定序結果發現，NCI-N87-AR 與其懸浮態NCI-N87-AR(s) 和不飽和脂肪酸的生化合成、脂肪酸代謝、glycine, serine與threonine代謝、黏著型連接、cysteine與methionine代謝、 胺基酸的生化合成、調控幹細胞全能性的訊號途徑、局部黏著、Wnt 訊號途徑、癌症的蛋白聚醣與可能的乙型轉化生長因子途徑有關。我們決定選擇黏著型連接途徑做後續的研究。和黏著型連接相關的7個基因在即時聚合酶連鎖反應實驗中被測出在AR與AR(s) 當中有高度的表現，其中一個基因是Smad4, 它是TGF-β其中一個下游基因。我們因此認為TGF-β可能是導致抵抗失巢凋亡能力的重要因素之一。因此，我們在細胞中加入TGF-β與 TGF-β抑制劑，結果顯示TGF-β抑制劑Galunisertib以及LY2109761會抑制AR(s)，促進其細胞凋亡。再者，我們還發現了Src, Bim與Smad2/3在NCI-N87-AR與NCI-N87-AR(s) 中表現量較高，不過其機制與原因還不明確。 綜觀上述，我們的結果顯示抵抗失巢凋亡在胃癌中可能促進腫瘤侵犯與增生，且其可能被TGF-β所誘導，因此抑制TGF-β可能導致AR(s)的細胞凋亡。Src也在具抵抗失巢凋亡能力的細胞株與其懸浮態有較高的表現。

Gastric cancer is the sixth most common cancer and it represents one of the most common causes of cancer death worldwide because of its tendency of rapid peritoneal metastasis, which is a common form of metastasis among advanced gastric cancer patients. About 80-90% of gastric cancer patients are diagnosed at advanced stage with metastasis and poor prognosis. However, the mechanism of peritoneal metastasis is poorly understood. Anoikis is a form of cell death induced by detachment of cells from the extracellular matrix (ECM). Yet, malignant tumor cells are able to develop specific mechanism to escape from anoikis to promote their invasion ability and metastasis, which is called “anoikis resistance”. Thus, we suggested that anoikis resistance might be involved in peritoneal metastasis. In this study, we first built up anoikis-resistant cell lines with different cell lines including lung cancer cell lines LU65 and LU99, pancreatic cell lines MIA-PaCa2 and BxPC3, breast cancer cell lines 4T1, MCF7 and T47D, and gastric cancer cell line AGS and NCI-N87. We found that gastric cancer cell line NCI-N87 had the tendency of anoikis resistance (AR) after 24 days. We analyzed the AR subclone and found that it has much better invasion and proliferation ability. Through RNA sequencing data, NCI-N87-AR and its suspension type NCI-N87-AR(s) had alteration in biosynthesis of unsaturated fatty acids, fatty acid metabolism, glycine, serine and threonine metabolism, adherens junction, cysteine and methionine metabolism, biosynthesis of amino acids, signaling pathways regulating pluripotency of stem cells, focal adhesion, Wnt signaling pathway, proteoglycans in cancer and TGF-β signaling pathway. We decided to choose adherens junction pathway for further study. 7 genes related to adherens junction pathway were highly expressed in AR and AR(s) cell lines, confirmed by qPCR. One of the genes was Smad4, which is a downstream gene of TGF-β. We thought that TGF-β might be an important factor of anoikis resistance. Therefore, recombinant human TGF-β and TGF-β inhibitor were added. Galunisertib and LY2109761 were shown to have inhibition effect on AR(s) cell line. In addition, we found that Src, Bim and Smad2/3 were upregulated in NCI-N87-AR and NCI-N87-AR(s) cell lines but the mechanism and the reason are still unclear. In conclusion, our results indicated that anoikis resistance could promote invasion and proliferation in gastric cancer. Anoikis resistance might be triggered by TGF-β and thus TGF-β inhibitor might lead to apoptosis in AR(s) cell line. Src was also shown to have higher expression in anoikis-resistant cell line in both adhesion and suspension states.