Autophagy induction results in enhanced anoikis resistance in models of peritoneal disease

Peritoneal carcinomatosis and peritoneal sarcomatosis is a potential complication of nearly all solid tumors and results in profoundly increased morbidity and mortality. Despite the ubiquity of peritoneal carcinomatosis/peritoneal sarcomatosis, there are no clinically relevant targeted therapies for either its treatment or prevention. To identify potential therapies, we developed in vitro models of peritoneal carcinomatosis/peritoneal sarcomatosis using tumor cell lines and patient-derived spheroids (PDS) that recapitulate anoikis resistance and spheroid proliferation across multiple cancer types. Epithelial-and mesenchymalderived cancer cell lines (YOU, PANC1, HEYA8, CHLA10, and TC71) were used to generate spheroids and establish growth characteristics. Differential gene expression analyses of these spheroids to matched adherent cells revealed a consensus spheroid signature. This spheroid signature discriminates primary tumor specimens from tumor cells found in ascites of ovarian cancer patients and in ourPDSmodels. Key in this gene expression signature is BNIP3 and BNIP3L, known regulators of autophagy and apoptosis. Elevated BNIP3 mRNA expression is associated with poor survival in ovarian cancer patients and elevated BNIP3 protein, as measured by IHC, and is also associated with higher grade tumors and shorter survival. Pharmacologic induction of autophagy with rapamycin significantly increased spheroid formation and survival while decreasing the induction of apoptosis. In contrast, the autophagy inhibitor hydroxychloroquine abrogated spheroid formation with a clear increase in apoptosis. Modulation of BNIP3 and the critical autophagy gene Beclin-1 (BECN1) also caused a significant decrease in spheroid formation. Combined, these data demonstrate how modulation of BNIP3-related autophagy, in PDS and in vitro spheroid models, alters the survival and morphology of spheroids. Implications: Development of BNIP3/BNIP3L-targeting agents or autophagy-targeting agents may reduce morbidity and mortality associated with peritoneal carcinomatosis and sarcomatosis.