Summary Microbiome dysbiosis is a feature of diabetes, but how microbial products influence insulin production is poorly understood. Here we report the mechanism of BefA, a microbiome-derived protein that increases proliferation of insulin-producing β-cells during pancreatic development in gnotobiotic zebrafish and mice. BefA disseminates systemically via multiple anatomic routes to act directly on pancreatic islets. We report the structure of BefA, containing a lipid-binding SYLF domain, and demonstrate that it permeabilizes synthetic liposomes and bacterial membranes. A BefA mutant impaired in membrane disruption fails to expand β-cells whereas the pore-forming host defense protein, Reg3, stimulates β-cell proliferation. Our work demonstrates that membrane permeabilization by microbiome-derived and host defense proteins is necessary and sufficient for β-cell expansion during pancreas development, thereby connecting microbiome composition with diabetes risk.
Abstract The primary goal of this study is to characterize interactions between the master acinar transcription factor, Ptf1a, and signal transduction pathways implicated in acinar reprogramming. Animal models suggest that acinar-ductal metaplasia (ADM) is one of the first steps in the initiation of pancreatic cancer. It is now established that acinar-specific activation of the Kras oncogene is sufficient at the whole-organ level to cause ADM and PanIN formation in mice, although it does so only inefficiently at the level of individual cells. Recently, our lab demonstrated that deletion of the master acinar transcriptional regulator, Ptf1a, facilitates ADM. Additionally, loss of Ptf1a dramatically accelerates PanIN formation when compounded with an oncogenic Kras mutation. Mechanistically, loss of Ptf1a activates transcriptional signatures of several signaling pathways that have been implicated in promoting acinar to ductal metaplasia (ADM) as well as PDAC progression. Prime among these candidate pathways is epidermal growth factor receptor (EGFR) signaling, which promotes acinar to ductal metaplasia (ADM) via the Ras-Raf-MEK-ERK pathway. In this study we test whether Ptf1a negatively regulates EGFR signaling to restrain acinar reprogramming and acinar-to-ductal metaplasia. To test this hypothesis, we performed in vitro culture assays on acinar clusters isolated from mice containing activated oncogenic KRAS alone (KrasG12D) or KrasG12D-Ptf1a cKO double mutant mice. We find that acinar clusters from KrasG12D-Ptf1a cKO double mutant mice undergo reprogramming, giving rise to duct-like cysts, both more rapidly and at a greater frequency than KrasG12D mice. In congruence with previous work, stimulation with the EGFR ligand, TGFα, increases the size and frequency of cyst formation in both KrasG12D and KrasG12D-Ptf1a cKO mice, suggesting that EGFR activation increases the transforming ability of oncogenic KRAS. Consistent with previous studies, blocking EGFR signaling using the pharmacological inhibitor Erlotinib, prevented ADM in acinar clusters from mice expressing oncogenic KRAS alone. Surprisingly, however, Erlotinib does not prevent ADM and cyst formation in KrasG12D-Ptf1a cKO mice, suggesting that acinar differentiation programs are required for the inhibitory effects of this drug. These results additionally indicate that EGFR is dispensable for ADM when Ptf1a is deleted. On the other hand, restriction of downstream signaling using the MEK inhibitor PD-0325901 inhibits reprogramming in acinar clusters, despite deletion of Ptf1a in KrasG12D mice. Since Ptf1a is downregulated in both mouse and human PDAC, these results highlight the potential of MEK inhibitors as feasible therapeutics, and the possibility that varying differentiation competence of tumor cells underlies the variable clinical effectiveness of Erlotinib and other EGFR inhibitors. Going forward, we will investigate whether reintroducing Ptf1a into dedifferentiated cells is sufficient to reprogram them back to an acinar-like phenotype. Using in vitro culture, we will determine if cells re-differentiate and regain sensitivity to Erlotinib with ectopic expression of Ptf1a. We will also characterize target genes of Ptf1a whose dysregulation leads to EGFR independence. Ultimately, these studies will lead us to investigate if reactivation of Ptf1a expression could represent a novel therapeutic avenue for PDAC. Citation Format: Shuba M. Narayanan, Nathan M. Krah, L. Charles Murtaugh.{Authors}. The acinar differentiation factor PTF1A negatively regulates EGFR-mediated acinar cell reprogramming. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr A47.
Pancreatic ductal adenocarcinoma (PDAC) is among the worst diagnoses in medicine, with a 5-year survival rate of 4%, in part because most cases are detected well after metastasis has occurred. Models of PDAC evolutionary history have emerged from DNA sequencing studies of metastases and primary tumours and reveal a progressive elaboration of malignant and metastatic cells that takes 20 or more years.1 Interestingly, at least half of this time is spent in a premalignant state, termed pancreatic intraepithelial neoplasia (PanIN), suggesting that a decade-long window exists for prediction, detection and prevention of pancreatic cancer. Two papers published in Gut use mouse models to derive new insights into human-relevant genetic and environmental influences that drive PanIN formation and progression, and highlight the importance of differentiation as a barrier to tumourigenesis.2 ,3
The first genetic event on the road to invasive pancreatic cancer is mutational activation of KRAS , which occurs in the earliest precancerous PanIN-1 lesions. Experimental research on PDAC initiation was transformed 10 years ago, when a mouse model of PanIN–PDAC progression was developed based on pancreas-specific, Cre recombinase-mediated activation of endogenous mouse Kras .4 This model has been used to validate and dissect both rare genetic risk factors for PDAC, such as Ink4a/Arf deficiency,5 and environmental risk factors …
Activating mutations in the KRAS proto-oncogene occur almost ubiquitously in pancreatic ductal adenocarcinoma (PDAC) and in its putative precursor lesions, pancreatic intraepithelial neoplasia (PanIN). Conditional expression of an activated Kras allele in the mouse pancreas produces a model that faithfully recapitulates PanIN formation and progression to PDAC. Importantly, although nearly every cell in the pancreata of these mice express activated Kras, only a very small minority of cells give rise to PanINs. How the transforming activity of Kras is constrained in the pancreas remains unknown, and the cell types from which PanINs and PDAC arise are similarly unknown. Here, we describe our recent results demonstrating that acinar cells are competent to form Kras-induced PanINs, and that active Notch signaling can synergize with Kras in PanIN initiation and progression. Further efforts to understand how Notch and Kras synergize, as well as experiments to determine how other pancreatic cell types contribute to PDAC development, should aid in the development of new therapies and early detection techniques that are desperately needed for this cancer.
Pancreatitis is caused by inflammatory injury to the exocrine pancreas, from which both humans and animal models appear to recover via regeneration of digestive enzyme-producing acinar cells. This regenerative process involves transient phases of inflammation, metaplasia, and redifferentiation, driven by cell-cell interactions between acinar cells, leukocytes, and resident fibroblasts. The NFκB signaling pathway is a critical determinant of pancreatic inflammation and metaplasia, whereas a number of developmental signals and transcription factors are devoted to promoting acinar redifferentiation after injury. Imbalances between these proinflammatory and prodifferentiation pathways contribute to chronic pancreatitis, characterized by persistent inflammation, fibrosis, and acinar dedifferentiation. Loss of acinar cell differentiation also drives pancreatic cancer initiation, providing a mechanistic link between pancreatitis and cancer risk. Unraveling the molecular bases of exocrine regeneration may identify new therapeutic targets for treatment and prevention of both of these deadly diseases.
The formation of epithelial tubes underlies the development of diverse organs. In the skin, hair follicles resemble tube-like structures with lumens that are generated through poorly understood cellular rearrangements. Here, we show that creation of the hair follicle lumen is mediated by early outward movement of keratinocytes from within the cores of developing hair buds. These migratory keratinocytes express keratin 79 (K79) and stream out of the hair germ and into the epidermis prior to lumen formation in the embryo. Remarkably, this process is recapitulated during hair regeneration in the adult mouse, when K79(+) cells migrate out of the reactivated secondary hair germ prior to formation of a new hair canal. During homeostasis, K79(+) cells line the hair follicle infundibulum, a domain we show to be multilayered, biochemically distinct and maintained by Lrig1(+) stem cell-derived progeny. Upward movement of these cells sustains the infundibulum, while perturbation of this domain during acne progression is often accompanied by loss of K79. Our findings uncover previously unappreciated long-distance cell movements throughout the life cycle of the hair follicle, and suggest a novel mechanism by which the follicle generates its hollow core through outward cell migration.
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