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.