Purpose: This research investigates the association between benzodiazepines (BZDs) and cancer patient survival outcomes. Due to the high prevalence of BZD use in pancreatic cancer patients, we evaluated the effect of commonly prescribed BZDs on the pancreatic cancer tumor microenvironment and cancer-associated fibroblast (CAF) signaling. Experimental Design: Multivariate Cox regression modeling was used to retrospectively measure associations between Roswell Park cancer patient survival outcomes and BZD prescription records. Immunohistochemistry, H&E, Massons trichrome, in situ hybridization, and RNA sequencing were used to evaluate the impact of lorazepam (LOR) on the PDAC tumor microenvironment, using murine pancreatic cancer models. ELISA and qPCR were used to determine the impact of BZDs on IL-6 expression/secretion by human immortalized pancreatic CAFs. PRESTO-Tango assays, reanalysis of PDAC single cell sequencing/TCGA datasets, and GPR68 CRISPRi knockdown CAF cells were used to mechanistically determine the impact of BZDs on CAF-specific GPR68 signaling. Results: LOR is associated with worse progression-free survival (PFS) while alprazolam (ALP) is associated with improved PFS, in pancreatic cancer patients receiving chemotherapy. LOR promotes desmoplasia (fibrosis and extracellular matrix protein deposition), inflammatory signaling, IL-6 expression/secretion in CAFs, and ischemic necrosis. LOR promotes inflammatory signaling and IL-6 secretion by CAFs through activation of GPR68. GPR68 is preferentially expressed on human PDAC CAFs, and n-unsubstituted BZDs significantly increase GPR68 activation under acidic conditions. LOR increases IL-6 expression and secretion in CAFs in a pH and GPR68-dependent manner. Conversely, ALP, and other GPR68 non-activator BZDs decrease IL-6 in human CAFs in a pH and GPR68-independent manner. Across many cancer types, LOR is associated with worse survival outcomes relative to ALP and patients not receiving BZDs. Conclusion: We demonstrate that LOR stimulates fibrosis and inflammatory signaling, promotes ischemic necrosis, and is associated with decreased pancreatic cancer patient survival.

The authors have declared no competing interest.

Research reported in this publication was supported by Roswell Park Comprehensive Cancer Center and the National Cancer Institute (NCI) of the National Institutes of Health (NIH) under Award Numbers P30CA016056 and F31CA260942, and by seed funding from the Roswell Park Alliance Foundation. NCI grant P30CA016056 supported the use of Roswell Park Comprehensive Cancer Center Pathology Network, Biomedical Research Informatics, Biostatistics and Statistical Genomics, Genome Modulation Services, Translational Imaging, Experimental Tumor Models, Drug Discovery Core, and Bioanalytics, Metabolomics, and Pharmacokinetics Shared Resources. Additional support from NIH (R01EY028580, to M.P) and the 5th AHEPA Cancer Research Foundation Inc, as well as R01CA269660, S10ODO23666 and P30CA06927 (Core Microscopy Facility) to J.F.B and E.C. C.F receives funding from the National Comprehensive Cancer Network Foundation, National Comprehensive Cancer Network Oncology Research Program, Taiho Oncology, and Pfizer Inc (all to RPCC).

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