p38-p14ARF-CtBP2 axis as a novel regulator of CSC phenotype and tumor cell dormancy

Abstract Introduction: Cancer cell dormancy and the cancer stem cell (CSC) phenotype are interlinked and play a crucial role in disease relapse. The pp38/pERK signaling ratio positively regulates cellular dormancy. We observed that heparin hexasaccharide (HS06) inhibited colon CSC self-renewal through isoform specific induction of pp38α/β, increasing the pp38/pERK ratio, and downregulation of C-terminal binding protein-2 (CtBP2) levels which is known to play a critical role in CSC growth. Hence, we hypothesized that HS06 inhibits CSCs self-renewal and prevents dormancy escape through regulation of CtBP2 in a manner dependent on p38 activation. Method: Spheroid culture of HT29 and HCT116 colon cancer cells was used for CSC self-renewal monitoring. Transient transfection with p38α, p14ARF, CtBP2 shRNA and/or expression vector with appropriate scrambled or vector controls was used to modulate cell signaling through this pathway. Standard western blotting, Q-PCR, flow cytometry, and immunoprecipitation were used to examine protein levels, their phosphorylation status and protein-protein interactions. Short term exposure of colon cancer cells (HT29) to chemotherapy (5-Fluorouracil (125μM) and oxaliplatin (5μM) (5-FUOX)) was used to study a transient dormancy-like state followed by growth escape. HT29 CSC (1x105 CD133+/CXCR4+ cells) induced xenografts were established s.c. in NCr nude mice were treated with HS06 (150mg/kg 3xwk., 3 weeks) and monitored for tumor growth, and xenografts were analyzed for CSC makers and p38-p14ARF-CtBP2 axis status. Results: HS06 inhibited CtBP2 levels but increased the expression of its negative regulator p14 ARF in vitro and in vivo in a manner dependent on p38 activation, as SB203580, a selective p38α/β inhibitor, as well as p38α shRNA, both near-completely reversed the effects of HS06 on p14ARF, CtBP2, and CSC self-renewal. This regulation of p14ARF and CtBP2 was post-transcriptional in nature. Moreover, reciprocal coimmunoprecipitation studies revealed a novel physical interaction between endogenous pp38 and p14ARF, which resulted in serine phosphorylation of p14ARF in a p38 activation-dependent manner that was critical for regulation of CtBP2 levels and CSC self-renewal. Using a chemotherapy induced cellular dormancy model, we observed that activation/upregulation of p38 and p14ARF prevented escape from dormancy in vitro, whereas increased CtBP2 had the opposite effect. Conclusion: We have discovered a novel p38-p14ARF-CtBP2 axis that plays a dual role in colon cancer CSC self-renewal and cellular dormancy. Agents targeting this pathway e.g., HS06 and CtBP2 inhibitors, represent novel therapeutic strategies to inhibit CSCs and prevent dormancy escape leading to reduced tumor relapse and/or cure. Citation Format: Rio S. Boothello, Nirmita J. Patel, Priyadarshan K. Damle, Chetna Sharon, Kranthi Kumar Chougoni, Umesh R. Desai, Steven R. Grossman, Bhaumik B. Patel. p38-p14ARF-CtBP2 axis as a novel regulator of CSC phenotype and tumor cell dormancy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3460.