Laboratory of Human Carcinogenesis and Liver Cancer Program, Center for Cancer Research, National Cancer Institute, NIH, 37 Convent Drive, MSC 4258, Building 37, Room 3044A, Bethesda, MD, 20892, USA.
Department of Surgery, Division of Surgical Research, Thomas Jefferson University, Philadelphia, PA, USA.
J Exp Clin Cancer Res. 2020 Jun 3;39(1):99. doi: 10.1186/s13046-020-01605-y.
Therapeutic options for patients with hepatocellular carcinoma (HCC) are limited. Transarterial chemoembolization (TACE) is an interventional procedure used to deliver chemotherapy and embolizing agents directly to the tumor and is the procedure of choice for patients with intermediate stage HCC. While effective, more than 40% of patients do not respond to therapy, highlighting the need to investigate possible mechanisms of resistance. We sought to evaluate mechanisms of TACE resistance and evaluate a potential therapeutic target to overcome this resistance.
Using a prognostic gene signature which predicts TACE response (TACE Navigator) in a cohort of HCC patients who received TACE, patients were classified as responders and non-responders. Transcriptomic and gene pathway analysis were used to identify potential drivers of TACE resistance. Knockdown of the gene encoding rate limiting enzyme PKM2 using shRNA in HCC cell lines, as well as pharmacologic inhibition of PKM2 with shikonin using an in vitro TACE model measured response to chemotherapy under hypoxia. Finally, we replicated the TACE model with shikonin using patient derived cell line organoids (PDC). Functional studies were performed in vitro using immunoblotting, quantitative polymerase chain reaction, glycolysis and hypoxia assays.
In patient non-responders, we identified enrichment of the glycolysis pathway, specifically of the gene encoding the rate-limiting enzyme PKM2. We identified four HCC cell lines which recapitulated a TACE responder-like and non-responder-like phenotype. PKM2 knockdown in HCC cell lines demonstrated a less proliferative and aggressive phenotype as well as improved drug sensitivity to both doxorubicin and cisplatin. In vitro TACE model demonstrated that TACE non-responder-like cells overcame therapeutic resistance and rendered them susceptible to therapy through PKM2 knockdown. Lastly, we obtained similar results using a pharmacologic PKM2 inhibitor, shikonin in both cell lines, and PDC organoids.
Elevated PKM2 is associated with treatment resistance and abbreviated survival in patients receiving TACE. Elevated PKM2 in vitro is associated with increased utilization of the glycolysis pathway, resulting in oxygen independent cell metabolism. Through PKM2 knockdown as well as with pharmacologic inhibition with shikonin, non-responder cells can be reprogrammed to act as responders and could improve TACE efficacy in patients.
肝细胞癌（HCC）患者的治疗选择有限。经动脉化疗栓塞术（TACE）是一种将化疗药物和栓塞剂直接输送到肿瘤的介入治疗方法，是中期 HCC 患者的首选治疗方法。虽然有效，但超过 40%的患者对治疗无反应，这突出表明需要研究可能的耐药机制。我们试图评估 TACE 耐药的机制，并评估一种潜在的治疗靶点来克服这种耐药性。
我们使用了一个在接受 TACE 治疗的 HCC 患者队列中预测 TACE 反应的预后基因特征（TACE Navigator），根据该特征将患者分为应答者和无应答者。通过对转录组和基因通路分析，我们确定了 TACE 耐药的潜在驱动因素。使用 shRNA 敲低 HCC 细胞系中编码限速酶 PKM2 的基因，以及使用 shikonin 在体外 TACE 模型中抑制 PKM2 的药理作用，在缺氧条件下测量对化疗的反应。最后，我们使用患者来源的细胞系类器官（PDC）用 shikonin 复制 TACE 模型。在体外使用免疫印迹、定量聚合酶链反应、糖酵解和缺氧测定进行功能研究。
在患者无应答者中，我们发现糖酵解途径富集，特别是限速酶 PKM2 的基因。我们鉴定了四种 HCC 细胞系，它们再现了 TACE 应答者样和非应答者样表型。在 HCC 细胞系中敲低 PKM2 可表现出增殖能力降低、侵袭性降低，以及对多柔比星和顺铂的药物敏感性提高。体外 TACE 模型表明，TACE 无应答者样细胞通过 PKM2 敲低克服了治疗耐药性，使它们对治疗敏感。最后，我们在细胞系和 PDC 类器官中使用药理学 PKM2 抑制剂 shikonin 获得了类似的结果。
在接受 TACE 治疗的患者中，PKM2 升高与治疗耐药和缩短生存期相关。体外 PKM2 升高与糖酵解途径的利用增加相关，导致氧不依赖的细胞代谢。通过 PKM2 敲低以及用 shikonin 进行药理学抑制，非应答细胞可以被重新编程为应答细胞，并可以提高 TACE 对患者的疗效。
