| 摘要: | 肝細胞癌是全世界最常見的原發性惡性腫瘤。Sorafenib為multi-target kinase inhibitor並且有效抑制癌細胞增生與血管新生作用，是晚期肝細胞癌患者第一線口服抗癌藥物，然而長期使用sorafenib被證實產生抗藥性。近年來，許多研究指出糖解作用導致乳酸堆積促進抗藥性的生成，並且進一步影響肝細胞癌的治療效能。3-Hydroxymethyl glutaryl-CoA synthase 2 (HMGCS2)是酮體生成的限速?，其協助合成酮體β-hydroxybutyrate (β-HB)與acetoacetate (AcAc)。β-HB是體內最多的酮體，其與AcAc以四比一的比例存在於人體中。近年來，酮體被證實會透過改變癌細胞代謝途徑與促進癌細胞凋亡的機轉協助癌症治療。我們先前的研究指出HMGCS2基因的表現被抑制會導致酮體的表現量下降，進而調控c-Myc/cyclin D1與caspase-dependent signaling促進肝細胞癌的進程。然而，HMGCS2調控之酮體生成是否會改變肝細胞癌對於sorafenib的感受性仍未知，並且給予酮體是否會對sorafenib抗藥性肝癌細胞代謝轉變產生影響仍未知。此研究中，我們發現當HMGCS2表現下降會透過活化磷酸化的ERK、p38與AKT相關路徑以降低sorafenib所誘導的抗細胞增生現象，相反地，過度表現HMGCS2會抑制ERK活化以提高sorafenib對於癌細胞的細胞毒殺效果。除此之外，我們發現在Huh7與HepG2細胞中同時給予sorafenib治療並抑制HMGCS2基因會透過降低ZO-1與提高c-Myc表現以促進癌細胞轉移能力。然而，在癌細胞過度表現HMGCS2基因並給予sorafenib治療並未改變轉移能力，也不降低ZO-1、c-Myc與N-cadherin的表現。最後，我們想確認HMGCS2的表現是否會藉由酮體依賴方式影響癌細胞對於sorafenib的感受性。我們發現β-HB預處理有效提高Huh7與HepG2中sorafenib所誘導的抗增生能力。此外，我們發現HMGCS2與酮體β-HB在四種不同的sorafenib抗藥性肝癌細胞中表現差異。低HMGCS2與β-HB表現的抗藥性肝癌細胞與高糖解作用與乳酸生成有相關。給予β-HB可以有效提高抗藥性肝癌細胞中PDH的表現，並且同時降低LDH與乳酸的堆積。除此之外，同時給予β-HB與sorafenib或regorafenib會透過抑制B-raf/MAPK與N-cadherin-vimentin路徑減緩抗藥性肝癌細胞的增生與轉移的能力。在小鼠實驗模型中，腹腔注射β-HB可以抑制抗藥性肝腫瘤增生蛋白與糖解作用蛋白的表現。本研究發現HMGCS2基因表現與酮體不但能減緩肝癌細胞增生與遠端轉移，還能透過降低無氧糖解作用與乳酸堆積逆轉抗藥性，並且協同第一線與第二線抗癌藥物治療肝癌。
Hepatocellular carcinoma (HCC) is the most common primary malignant tumor worldwide. Sorafenib, which is a multi-target kinase inhibitor that blocks tumor cell proliferation and angiogenesis, is a first-line drug for advanced-stage HCC patient, however, long-term treatment often results in acquired resistance. Recently, glycolysis-mediated lactate production was reported to contribute to drug resistance and disturb the HCC treatment efficacy. 3-Hydroxymethyl glutaryl-CoA synthase 2 (HMGCS2) is the rate-limiting enzyme for ketogenesis, which synthesizes the ketone bodies, β-hydroxybutyrate (β-HB) and acetoacetate (AcAc). β-HB is the most abundant ketone body which is present in a 4:1 ratio compared to AcAc. Recently, ketone body treatment has been shown to have therapeutic effects against many cancers by inducing metabolic alterations and cancer cell apoptosis. Our previous publication showed that HMGCS2 downregulation-mediated ketone body reduction promoted HCC clinicopathological progression through regulating c-Myc/cyclin D1 and caspase-dependent signaling. However, it remains unclear whether HMGCS2-regulated ketone body production alters the sensitivity of human HCC to sorafenib treatment and whether ketone body treatment alters the metabolic shift in sorafenib-resistant HCC cells. Herein, we showed that HMGCS2 downregulation attenuated the anti-proliferative effects of sorafenib by activating expressions of phosphorylated (p)-extracellular signal-regulated kinase (ERK), p-p38, and p-AKT. In contrast, HMGCS2 overexpression enhanced the cytotoxic effects of sorafenib in HCC cells by inhibiting ERK activation. Furthermore, we showed that knockdown of HMGCS2 promoted migratory ability by inhibiting zonula occludens protein (ZO)-1 and increasing c-Myc expression in both sorafenib-treated Huh7 and HepG2 cells. Although HMGCS2 overexpression did not alter the migratory effect, expressions of ZO-1, c-Myc, and N-cadherin were decreased in sorafenib-treated HMGCS2-overexpressing HCC cells. Finally, we investigated whether HMGCS2 expression influenced sorafenib sensitivity in a ketone-dependent manner. We showed that β-HB pretreatment significantly enhanced the anti-proliferative effects of sorafenib in both Huh7 and HepG2 cells. Furthermore, we found differential expressions of HMGCS2 and the ketone body, β-HB, in four different sorafenib-resistant HCC cell lines. Lower levels of HMGCS2 and β-HB correlated with higher glycolytic alterations and lactate production in sorafenib-resistant HCC cells. β-HB treatment enhanced the expression of pyruvate dehydrogenase (PDH) and decreased lactate dehydrogenase (LDH) and lactate production in sorafenib-resistant HCC cells. Additionally, β-HB combined with sorafenib or regorafenib promoted the anti-proliferative and anti-migratory abilities of sorafenib-resistant HCC cells by inhibiting the B-raf/mitogen-activated protein kinase (MAPK) pathway and mesenchymal N-cadherin-vimentin axis. In vivo β-HB administration ameliorated subcutaneous sorafenib-resistant tumors by inhibiting expressions of proliferative and glycolytic proteins. Collectively, this study supported the positive therapeutic effect of HMGCS2 and β-HB through limiting cell proliferation and migration in both parental and sorafenib-resistant HCC cells, moreover, and may serve as another energy source that downregulated lactate production and reversed sorafenib resistance by inducing a glycolytic shift, which was also found to possess a synergetic ability with second-line drug treatment in sorafenib-resistant HCC cells. |
