Objective

Intra-tumoral expression of the serine hydrolase carboxylesterase 2 (CES2) contributes to the activation of the pro-drug irinotecan in pancreatic ductal adenocarcinoma (PDAC). Given other potential roles of CES2, we assessed its regulation, downstream effects, and contribution to tumor development in PDAC.

Methods

Association between the mRNA expression of CES2 in pancreatic tumors and overall survival was assessed using The Cancer Genome Atlas. Cell viability, clonogenic, and anchorage-independent growth assays as well as an orthotopic mouse model of PDAC were used to evaluate the biological relevance of CES2 in pancreatic cancer. CES2-driven metabolic changes were determined by untargeted and targeted metabolomic analyses.

Results

Elevated tumoral CES2 mRNA expression was a statistically significant predictor of poor overall survival in PDAC patients. Knockdown of CES2 in PDAC cells reduced cell viability, clonogenic capacity, and anchorage-independent growth in vitro and attenuated tumor growth in an orthotopic mouse model of PDAC. Mechanistically, CES2 was found to promote the catabolism of phospholipids resulting in HNF4α activation through a soluble epoxide hydrolase (sEH)-dependent pathway. Targeting of CES2 via siRNA or small molecule inhibitors attenuated HNF4α protein expression and reduced gene expression of classical/progenitor markers and increased basal-like markers. Targeting of the CES2-sEH-HNF4α axis using small molecule inhibitors of CES2 or sEH reduced cell viability.

Conclusions

We establish a novel regulatory loop between CES2 and HNF4α to sustain the progenitor subtype and promote PDAC progression and highlight the potential utility of CES2 or sEH inhibitors for the treatment of PDAC as part of non-irinotecan-containing regimens.

PURPOSE: The combination chemotherapy of fluorouracil, leucovorin, irinotecan, and oxaliplatin (FOLFIRINOX) has provided clinically meaningful improvement for pancreatic ductal adenocarcinoma (PDAC). We previously uncovered a role for the serine hydrolase carboxylesterase 2 (CES2) in mediating intratumoral activation of the prodrug irinotecan, a constituent of FOLFIRINOX. We aimed to further test the predictive value of CES2 for response to irinotecan using patient-derived xenograft (PDX) models and to elucidate the determinants of CES2 expression and response to FOLFIRINOX treatment among patients with PDAC. METHODS: PDXs were engrafted subcutaneously into nude mice and treated for 4 weeks with either saline control or irinotecan. CES2 and hepatocyte nuclear factor 4 alpha (HNF4A) expression in PDAC tissues was evaluated by immunohistochemical and Western blot analysis. Kaplan-Meier and Cox regression analyses were applied to assess the association between overall survival and hemoglobin A1C (HbA1C) levels in patients who underwent neoadjuvant FOLFIRINOX treatment. RESULTS: High CES2 activity in PDAC PDXs was associated with increased sensitivity to irinotecan. Integrated gene expression, proteomic analyses, and in vitro genetic experiments revealed that nuclear receptor HNF4A, which is upregulated in diabetes, is the upstream transcriptional regulator of CES2 expression. Elevated CES2 protein expression in PDAC tissues was positively associated with a history of type 2 diabetes (odds ratio, 4.84; P = .02). High HbA1C levels were associated with longer overall survival in patients who received neoadjuvant FOLFIRINOX treatment (P = .04). CONCLUSION: To our knowledge, we provide, for the first time, evidence that CES2 expression is associated with a history of type 2 diabetes in PDAC and that elevated HbA1C, by predicting tumor CES2 expression, may represent a novel marker for stratifying patients most likely to respond to FOLFIRINOX therapy.