Valproic Acid improves efficacy of oxaliplatin/fluoropyrimidine-based chemotherapy in colorectal adenocarcinoma models by targeting cancer stem cell compartment via β-Catenin modulation.

Metastatic colorectal cancer (mCRC) patients despite the advances in systemic therapy approach have poor outcome in cases harboring BRAF or RAS mutations. Cancer stem cells (CSCs) play a central role in drug resistance and CRC recurrence. In this scenario targeting the epigenetic mechanisms that sustain CSC properties represents a promising therapeutic approach. In this study, we report the efficacy and the potential to overcome chemotherapy resistance of a treatment strategy based on the addition of the epigenetic agent valproic acid (VPA), a well-known antiepileptic drug, to the standard chemotherapy oxaliplatin/fluoropyrimidine doublet, in wild-type as well as BRAF- and RAS-muted CSCs enriched primary spheroid CRC cultures. Specifically, we demonstrated that VPA, combined with chemotherapy, more effectively than other epigenetic drug-chemotherapy combinations, induced inhibition of cell proliferation and clonogenic growth as well as apoptosis and DNA damage. Mechanistically, proteomic analysis demonstrated that VPA induced CSC differentiation and that β-Catenin represent critical target of VPA. Indeed VPA promotes proteasome-dependent degradation of β-Catenin by enhancing its binding to the E2 ubiquitin-conjugating enzyme UBE2a, leading to a marked reduction in nuclear and cytoplasmic β-Catenin levels, subsequently diminishing the activation of β-Catenin/TCF-LEF target promoters. These data were confirmed in three in vivo CRC xenograft models, including syngeneic CT26 immunocompetent mouse model, where VPA combined with oxaliplatin/capecitabine chemotherapy plus anti-VEGF treatment, a standard first line treatment for mCRC, significantly suppressed tumor growth and prolonged survival with minimal toxicity. Proteomic analysis of tumor tissue samples from the in vivo CRC models confirmed the VPA-mediated downmodulation of both CSC markers and β-Catenin.尽管全身治疗方案已有进展，但携带BRAF或RAS突变的转移性结直肠癌（metastatic colorectal cancer, mCRC）患者预后仍然不佳。癌症干细胞（cancer stem cells, CSCs）在结直肠癌耐药与复发过程中发挥核心作用。在此背景下，靶向维持CSCs干性的表观遗传机制，是极具前景的治疗策略。本研究报道了一种治疗策略的疗效及其克服化疗耐药的潜力：在标准奥沙利铂/氟嘧啶双药联合化疗方案中加入表观遗传药物丙戊酸（valproic acid, VPA）——一种经典抗癫痫药物，该方案可应用于野生型、BRAF突变及RAS突变的、富集CSCs的原代结直肠癌球状体培养体系。 具体而言，我们证实，与其他表观遗传药物联合化疗方案相比，VPA联合化疗可更有效地抑制细胞增殖与克隆形成能力，同时诱导细胞凋亡与DNA损伤。从机制上看，蛋白质组学分析显示，VPA可诱导CSCs分化，且β-连环蛋白（β-Catenin）是VPA的关键靶点。实际上，VPA可通过增强β-连环蛋白与E2泛素结合酶UBE2a的结合，促进其蛋白酶体依赖性降解，进而显著降低细胞核与细胞质中的β-连环蛋白水平，最终削弱β-连环蛋白/TCF-LEF靶基因启动子的激活活性。 上述结果在三种结直肠癌异种移植模型中得到验证，其中包括同源CT26免疫健全小鼠模型：在该模型中，VPA联合奥沙利铂/卡培他滨化疗联合抗血管内皮生长因子（VEGF）治疗——这是mCRC的标准一线治疗方案——可显著抑制肿瘤生长并延长小鼠生存期，且毒性极低。对体内结直肠癌模型的肿瘤组织样本进行蛋白质组学分析证实，VPA可下调CSCs标志物与β-连环蛋白的表达水平。