Discovery of Hippo signaling as a regulator of CSPG4 expression and as a therapeutic target for Clostridioides difficile disease

The signaling pathways and networks regulating expression of chondroitin sulfate proteoglycan 4 (CSPG4), a cancer-related protein that serves as a receptor for Clostridiodes difficile TcdB, are poorly defined. In this study, TcdB-resistant/CSPG4-negative HeLa cells were generated by exposure to increasing concentrations of the toxin. The cells that emerged (HeLa R5) lost expression of CSPG4 mRNA and were resistant to binding by TcdB. mRNA expression profiles paired with integrated pathway analysis correlated changes in the Hippo and estrogen signaling pathways with a CSPG4 decrease in HeLa R5 cells. Both signaling pathways altered CSPG4 expression when modulated chemically or through CRISPR-mediated deletion of key transcriptional regulators in the Hippo pathway. Based on the in vitro findings, we predicted and experimentally confirmed that a Hippo pathway inactivating drug (XMU-MP-1) provides protection from C. difficile disease in a mouse model. These results provide insights into key regulators of CSPG4 expression and identify a therapeutic for C. difficile disease. Author summaryCSPG4 is a cancer-associated protein and a receptor for C. difficile TcdB. Though CSPG4 is important in both cancer and infection, little is known about the signaling networks regulating its expression. In this study, estrogen and Hippo signaling were found to independently impact CSPG4 transcription and the detectable levels of CSPG4 in cells. Based on this information, we used a small molecule inhibitor of Hippo signaling to block CSPG4 expression during C. difficile infection in a rodent model. Doing so protected against C. difficile disease by reducing tissue pathologies, inflammation, and weight loss following infection. Overall, results from this study provides a significant advance in both understanding how CSPG4 expression is regulated and the prospect of targeting CSPG4 regulators to reduce toxin effects during C. difficile disease.