Cell-autonomous circadian clocks exist in nearly every organ and function to maintain homeostasis through a complex series of transcriptional-translational feedback loops. The response of these peripheral clocks to external perturbations, such as chronic jetlag and shiftwork, has been extensively investigated. However, an evaluation of the effects of chronic jetlag on the mouse pancreatic transcriptome is still lacking. Herein we report an evaluation of the diurnal variations encountered in the pancreatic transcriptome following exposure to an established chronic jetlag protocol. We found approximately 5.4% of the pancreatic transcriptome was rhythmic. Following chronic jetlag, we found the number of rhythmic transcripts decreased to approximately 3.6% of the transcriptome. Analysis of the core clock genes, which orchestrate circadian physiology, revealed that nearly all exhibited a shift in the timing of peak gene expression - known as a phase shift. Similarly, over 95% of the rhythmically expressed genes in the pancreatic transcriptome exhibited a phase shift, many of which were found to be important for metabolism. Evaluation of the genes involved in pancreatic exocrine secretion and insulin signaling revealed many pancreas-specific genes were also rhythmically expressed and several displayed a concomitant phase shift with chronic jetlag. Phase differences were found 9 days after normalization, indicating a persistent failure to reentrain to the new light-dark cycle. This study is the first to evaluate the endogenous pancreatic clock and rhythmic gene expression in whole pancreas over 48 hours, and how the external perturbation of chronic jetlag affects the rhythmic expression of genes in the pancreatic

Anal squamous cell carcinoma (SCC) will be diagnosed in an estimated 9,080 adults in the United States this year, and rates have been rising over the last several decades. Most people that develop anal SCC have associated human papillomavirus (HPV) infection (~85–95%), with approximately 5–15% of anal SCC cases occurring in HPV-negative patients from unknown etiology. This study identified and characterized the Kras-driven, female sex hormone-dependent development of anal squamous cell carcinoma (SCC) in the LSL-KrasG12D; Pdx1-Cre (KC) mouse model that is not dependent on papillomavirus infection. One hundred percent of female KC mice develop anal SCC, while no male KC mice develop tumors. Both male and female KC anal tissue express Pdx1 and Cre-recombinase mRNA, and the activated mutant KrasG12D gene. Although the driver gene mutation KrasG12D is present in anus of both sexes, only female KC mice develop Kras-mutant induced anal SCC. To understand the sex-dependent differences, KC male mice were castrated and KC female mice were ovariectomized. Castrated KC males displayed an unchanged phenotype with no anal tumor formation. In contrast, ovariectomized KC females demonstrated a marked reduction in anal SCC development, with only 15% developing anal SCC. Finally, exogenous administration of estrogen rescued the tumor development in ovariectomized KC female mice and induced tumor development in castrated KC males. These results confirm that the anal SCC is estrogen mediated. The delineation of the role of female sex hormones in mediating mutant Kras to drive anal SCC pathogenesis highlights a subtype of anal SCC that is independent of papillomavirus infection. These findings may have clinical applicability for the papillomavirus-negative subset of anal SCC patients that typically respond poorly to standard of care chemoradiation.

No abstract

Anal squamous cell carcinoma (SCC) will be diagnosed in an estimated 9,080 adults in the United States this year, and rates have been rising over the last several decades. Most people that develop anal SCC have associated human papillomavirus (HPV) infection (∼85-95%), with approximately 5-15% of anal SCC cases occurring in HPV-negative patients from unknown etiology. This study identified and characterized a Kras-driven, female sex hormone-dependent development of anal squamous cell carcinoma (SCC) in the LSL-KrasG12D ; Pdx1-Cre (KC) mouse model that is not dependent on papillomavirus infection. One hundred percent of female KC mice develop anal SCC, while no male KC mice develop tumors. Both male and female KC anal tissue express Pdx1 and Cre-recombinase mRNA, and the activated mutant KrasG12D gene. Although the driver gene mutation KrasG12D is present in anus of both sexes, only female KC mice develop Kras-mutant induced anal SCC. To understand the sex-dependent differences, KC male mice were castrated and KC female mice were ovariectomized. Castrated KC males displayed an unchanged phenotype with no anal tumor formation. In contrast, ovariectomized KC females demonstrated a marked reduction in anal SCC development, with only 15% developing anal SCC. Finally, exogenous administration of estrogen rescued the tumor development in ovariectomized KC female mice and induced tumor development in castrated KC males. These results confirm that the anal SCC is estrogen mediated. The delineation of the role of female sex hormones in mediating mutant Kras to drive anal SCC pathogenesis highlights a subtype of anal SCC that is independent of papillomavirus infection. These findings may have clinical applicability for the papillomavirus-negative subset of anal SCC patients that typically respond poorly to standard of care chemoradiation.

Introduction: Emerging data suggest a role for circadian disruption in tumor formation and progression. Yet little is known regarding the effects of circadian disruption on the oncogenesis of pancreatic ductal adenocarcinoma (PDAC). We sought to determine if circadian disruption enhances PDAC development through a genetically engineered mouse model, and evaluate possible mechanisms with transcriptomic analysis. Methods: KrasG12D/+ mice (K) were crossed with Pdx-1 Cre (C) mice on a C57BL/6 background to generate KC mice. At 4 weeks, mice were subjected to standard lighting conditions (KC normal circadian [KCNC]) or a chronic jet-lag protocol known to induce circadian disruption (KCCD), with light/dark cycles shifted 8 hours every 2-3 days. Mice were sacrificed at 9 months for histologic analysis, examining for PDAC and its precursor lesions (PanINs). Comparisons were made with Fischer's Exact Test. For pancreatic transcriptomic profiling, 144 4-week-old wild-type (WT) C57BL/6 mice underwent disruption (WTCD, n = 72) or standard lighting conditions (WTNC, n = 72) for 4 weeks to identify cycling genes over a 48 hour period. Four weeks was chosen to understand the effects of disruption on gene expression impacting tumor initiation. Standard gene cycling analysis was performed with meta2d utilizing a false discovery rate of q = 0.1, and comparisons were made with the Audic-Claverie Distribution tool. Results: All KC mice (27 KCNC & 7 KCCD) exhibited chronic pancreatitis and PanIN-1 (p = 1). However, KCCD mice developed higher rates of acute pancreatitis (86% vs 4%; p < 0.01) and PanIN-2 (43% vs 7%; p = 0.04) versus KCNC mice. PanIN-3 (14% vs 7% p = 0.48) and PDAC (29% vs 16% p = 0.6) were also increased but did not reach statistical significance. Given this phenotype, we sought a mechanism through transcriptomic analysis. In total, we found 12.8% of the protein-coding pancreatic transcriptome was cycling in either WTNC or WTCD. Pathway enrichment analysis demonstrated genes important for circadian rhythm were cycling in WTNC, while those important for catabolism and protein localization were cycling in WTCD. Only 129 genes, shared between male and female mice, exclusively cycled following circadian disruption. Of those, roughly 15% have been implicated in PDAC development, reported as prognostic biomarkers in PDAC or possibly contribute to chemotherapeutic resistance in PDAC. Conclusion: Little is known about the effects of circadian disruption on PDAC. We have demonstrated histopathologic evidence of an increased incidence of pancreatic inflammation and pathogenic lesions following circadian disruption. Transcriptomic analysis of wild-type pancreas revealed an increased number of cycling genes involved in energy production following disruption. Future work will investigate the candidate genes identified to ascertain how circadian disruption leads to increased development of pancreatic lesions. Citation Format: Patrick Beard Schwartz, Morgan Walcheck, Noah D. Carrillo, Kristina A. Matkowskyj, Christopher A. Bradfield, Sean Ronnekleiv-Kelly. Circadian disruption enhances development of pancreatic inflammation and pancreatic cancer precursors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3811.

Pancreatic ductal adenocarcinoma (PDAC) is a devastating cancer with an 80% five-year mortality rate - reflecting an inadequate understanding of pertinent drivers of cancer development. One potential driver of PDAC development is circadian disruption, known to control a myriad of pancreatic processes. Clock dysfunction is associated with multiple risk factors for PDAC, including obesity, diabetes, and heightened inflammation, and has been shown to accelerate the development of other solid-organ cancers. Using a well-characterized model of chronic jetlag (CJ) to mimic irregular sleep patterns, we subjected mice harboring a pancreas-specific activated Kras mutation (KC mice) to either a normal lighting pattern or CJ and observed the resultant histopathologic changes. We found that CJ accelerated the development of fibroinflammatory infiltrate/pancreatic intraepithelial neoplasia and increased the grade of the pancreatic lesions. Applying single-cell RNA sequencing over multiple time points, we further identified fibroblasts in the developing pancreatic tumor microenvironment as a putative target of CJ-induced changes. Our results suggest a role for clock dysfunction in PDAC initiation which may hold relevance for preventative efforts.

Objectives: The pathogenesis of pancreas cancer (PDAC) remains poorly understood, hindering efforts to develop a more effective therapy for PDAC. Recent discoveries show the aryl hydrocarbon receptor (AHR) plays a crucial role in the pathogenesis of several cancers, and can be targeted for therapeutic effect. However, its involvement in PDAC remains unclear. Therefore, we evaluated the role of AHR in the development of PDAC in vivo. Methods: We created a global AHR-null, mutant Kras-driven PDAC mouse model (A-/-KC) and evaluated the changes in PDAC precursor lesion formation (Pan-IN 1, 2, and 3) and associated fibro-inflammation between KC and A-/-KC at 5 months of age. We then examined the changes in the immune microenvironment followed by single-cell RNA-sequencing analysis to evaluate concomitant transcriptomic changes. Results: We found a significant increase in PanIN-1 lesion formation and PanIN-1 associated fibro-inflammatory infiltrate in A-/-KC vs KC mice. This was associated with significant changes in the adaptive immune system, particularly a decrease in the CD4+/CD8+ T-cell ratio, as well as a decrease in the T-regulatory/Th17 T-cell ratio suggesting unregulated inflammation. Conclusion: These findings show the loss of AHR results in heightened Kras-induced PanIN formation, through modulation of immune cells within the pancreatic tumor microenvironment.

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