Chronic jetlag-induced alterations in pancreatic diurnal gene expression.

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 shift work, 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 h, and how the external perturbation of chronic jetlag affects the rhythmic expression of genes in the pancreatic transcriptome.

Aryl Hydrocarbon Receptor Knockout Accelerates PanIN Formation and Fibro-Inflammation in a Mutant Kras -Driven Pancreatic Cancer Model.

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 development of several cancers and can be targeted for therapeutic effect. However, its involvement in the pathogenesis of PDAC remains unclear. To address this gap, we evaluated the role of AHR in the development of PDAC precancerous lesions in vivo . MATERIALS AND METHODS: We created a global AHR-null, mutant Kras -driven PDAC mouse model (A -/- KC) and evaluated the changes in PDAC precursor lesion formation (PanIN-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 identified a significant increase in PanIN-1 lesion formation and PanIN-1 associated fibro-inflammatory infiltrate in A -/- KC versus 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. CONCLUSIONS: These findings show the loss of AHR results in heightened Kras -induced PanIN formation, through modulation of immune cells within the pancreatic tumor microenvironment.

Pdx1 expression in hematopoietic cells activates Kras-mutation to drive leukemia in KC (Pdx1-Cre; LSL-KrasG12D/+) mice.

The highly utilized KC model has a reported lethality rate of about 30%, which has been attributed to pancreas cancer. However, a competing cause of lethality in KC mice is due to the activation of mutant-Kras gene (KrasG12D/+) in the multipotent progenitor cells (MPP), and subsequent development of Kras-mutant T-cell acute lymphoblastic leukemia (T-ALL). Overall, 20% (5/25) of KC mice developed T-ALL by 9 months of age. Transplantation of pooled bone marrow from KC mice into CD45 congenic mice caused T-ALL in 100% of recipient mice, confirming that mutant-Kras expression in the hematologic compartment is driving the development of T-ALL in the KC mouse model. These results are an essential consideration for investigators using this model. Further, the lower penetrance of T-ALL in KC mice (versus existing leukemia models) suggests this model could be considered as an alternative research model to evaluate onset and factors that exacerbate the development of T-ALL.

Chronic jetlag accelerates pancreatic neoplasia in conditional Kras-mutant mice.

Misalignment of the circadian clock compared to environmental cues causes circadian desynchrony, which is pervasive in humans. Clock misalignment can lead to various pathologies including obesity and diabetes, both of which are associated with pancreatic ductal adenocarcinoma - a devastating cancer with an 80% five-year mortality rate. Although circadian desynchrony is associated with an increased risk of several solid-organ cancers, the correlation between clock misalignment and pancreas cancer is unclear. Using a chronic jetlag model, we investigated the impact of clock misalignment on pancreas cancer initiation in mice harboring a pancreas-specific activated Kras mutation. We found that chronic jetlag accelerated the development of pancreatic cancer precursor lesions, with a concomitant increase in precursor lesion grade. Cell-autonomous knock-out of the clock in pancreatic epithelial cells of Kras-mutant mice demonstrated no acceleration of precursor lesion formation, indicating non-cell-autonomous clock dysfunction was responsible for the expedited tumor development. Therefore, we applied single-cell RNA sequencing over time and identified fibroblasts as the cell population manifesting the greatest clock-dependent changes, with enrichment of specific cancer-associated fibroblast pathways due to circadian misalignment.

Aryl hydrocarbon receptor knockout accelerates PanIN formation and fibro-inflammation in a mutant Kras-driven pancreatic cancer model.

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.

Sex-dependent development of Kras-induced anal squamous cell carcinoma in mice.

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.