Gastroenterology
Abstract
Pancreatic ductal adenocarcinoma (PDAC) requires mitochondrial oxidative phosphorylation (OXPHOS) to fuel its growth, however, broadly inhibiting this pathway might also disrupt essential mitochondrial functions in normal tissues. PDAC cells exhibit abnormally fragmented mitochondria that are essential to its oncogenicity, but it was unclear if this mitochondrial feature was a valid therapeutic target. Here, we present evidence that normalizing the fragmented mitochondria of pancreatic cancer via the process of mitochondrial fusion reduces OXPHOS, which correlates with suppressed tumor growth and improved survival in preclinical models. Mitochondrial fusion was achieved by genetic or pharmacologic inhibition of dynamin related protein-1 (Drp1) or through overexpression of mitofusin-2 (Mfn2). Notably, we found that oral leflunomide, an FDA-approved arthritis drug, promoted a two-fold increase in Mfn2 expression in tumors and was repurposed as a chemotherapeutic agent, improving the median survival of mice with spontaneous tumors by 50% compared to vehicle. We found that the chief tumor suppressive mechanism of mitochondrial fusion was enhanced mitophagy, which proportionally reduced mitochondrial mass and ATP production. These data suggest that mitochondrial fusion is a specific and druggable regulator of pancreatic cancer growth that could be rapidly translated to the clinic.
Authors
Meifang Yu, Nicholas D. Nguyen, Yanqing Huang, Daniel Lin, Tara N. Fujimoto, Jessica M. Molkentine, Amit Deorukhkar, Ya'an Kang, F. Anthony San Lucas, Conrad J. Fernandes, Eugene J. Koay, Sonal Gupta, Haoqiang Ying, Albert C. Koong, Joseph M. Herman, Jason B. Fleming, Anirban Maitra, Cullen M. Taniguchi
Abstract
Pancreatic ductal adenocarcinoma (PDA) is a major cause of cancer-related death with limited therapeutic options available. This highlights the need for improved understanding of the biology of PDA progression, a highly complex and dynamic process featuring changes in cancer cells and stromal cells. A comprehensive characterization of PDA cancer cell and stromal cell heterogeneity during disease progression is lacking. In this study, we aimed to profile cell populations and understand their phenotypic changes during PDA progression. To that end, we employed single-cell RNA sequencing technology to agnostically profile cell heterogeneity during different stages of PDA progression in genetically engineered mouse models. Our data indicate that an epithelial-to-mesenchymal transition of cancer cells accompanies tumor progression in addition to distinct populations of macrophages with increasing inflammatory features. We also noted the existence of three distinct molecular subtypes of fibroblasts in the normal mouse pancreas, which ultimately gave rise to two distinct populations of fibroblasts in advanced PDA, supporting recent reports on intratumoral fibroblast heterogeneity. Our data also suggest that cancer cells and fibroblasts may be dynamically regulated by epigenetic mechanisms. This study systematically describes the landscape of cellular heterogeneity during the progression of PDA and has the potential to act as a resource in the development of therapeutic strategies against specific cell populations of the disease.
Authors
Abdel Nasser Hosein, Huocong Huang, Zhaoning Wang, Kamalpreet Parmar, Wenting Du, Jonathan Huang, Anirban Maitra, Eric Olson, Udit Verma, Rolf A. Brekken
Abstract
Colorectal cancer (CRC) is the third most frequent neoplastic disorder and is a main cause of tumor-related mortality as many patients progress to stage IV metastatic CRC. Standard care consists of combination chemotherapy (FOLFIRI or FOLFOX). Patients with WT KRAS typing are eligible to receive anti-EGFR therapy combined with chemotherapy. Unfortunately, predicting efficacy of CRC anti-EGFR therapy has remained challenging. Here we uncover that the EGFR-pathway component RasGRP1 acts as CRC tumor suppressor in the context of aberrant Wnt signaling. We find that RasGRP1 suppresses EGF-driven proliferation of colonic epithelial organoids. Having established that RasGRP1 dosage levels impacts biology, we focused on CRC patients next. Mining five different data platforms, we establish that RasGRP1 expression levels decrease with CRC progression and predict poor clinical outcome of patients. Lastly, deletion of one or two Rasgrp1 alleles makes CRC spheroids more susceptible to EGFR inhibition. Retrospective analysis of the CALGB80203 clinical trial shows that addition of anti-EGFR therapy to chemotherapy significantly improves outcome for CRC patients when tumors express low RasGRP1 suppressor levels. In sum, RasGRP1 is a unique biomarker positioned in the EGFR pathway and of potential relevance to anti-EGFR therapy for CRC patients.
Authors
Oghenekevwe M. Gbenedio, Caroline Bonnans, Delphine Grun, Chih-Yang Wang, Ace J. Hatch, Michelle R. Mahoney, David Barras, Mary Matli, Yi Miao, K. Christopher Garcia, Sabine Tejpar, Mauro Delorenzi, Alan P. Venook, Andrew B. Nixon, Robert S. Warren, Jeroen P. Roose, Philippe Depeille
Abstract
Genetic susceptibility to chronic pancreatitis in humans is frequently associated with mutations that increase activation of the digestive protease trypsin. Intrapancreatic trypsin activation is an early event in experimental acute pancreatitis in rodents, suggesting that trypsin is a key driver of pathology. In contrast to trypsin, the pancreatic protease chymotrypsin serves a protective function by mitigating trypsin activation through degradation. In humans, loss-of-function mutations in chymotrypsin C (CTRC) are common risk factors for chronic pancreatitis; however, the pathogenic effect of CTRC deficiency has not been corroborated in animal models yet. Here we report that C57BL/6 mice that are widely used for genetic manipulations do not express functional CTRC due to a single-nucleotide deletion in exon 2 of the Ctrc gene. We restored a functional Ctrc locus in C57BL/6N mice and demonstrated that in the novel Ctrc+ strain the severity of cerulein-induced experimental acute and chronic pancreatitis was significantly ameliorated. Improved disease parameters were associated with reduced intrapancreatic trypsin activation suggesting a causal link between CTRC-mediated trypsinogen degradation and protection against pancreatitis. Taken together with prior human genetic and biochemical studies, the observations provide conclusive evidence for the protective role of CTRC against pancreatitis.
Authors
Andrea Geisz, Zsanett Jancsó, Balázs Csaba Németh, Eszter Hegyi, Miklós Sahin-Tóth
Abstract
The physiological process of defecation is directly controlled by colorectal motility. The transient receptor potential ankyrin 1 (TRPA1) channel is expressed in small intestine enterochromaffin cells and is involved in gastrointestinal motility via serotonin release. In the colorectum, however, enterochromaffin cell localization is largely distinct from that in the small intestine. Here, we investigated the role of lower gastrointestinal tract TRPA1 in modulating colorectal motility. We found that in colonic tissue, TRPA1 is predominantly expressed in mesenchymal cells of the lamina propria, which are clearly distinct from those in the small intestine. These cells coexpressed COX1 and microsomal prostaglandin E synthase-1. Intracolonic administration of TRPA1 agonists induced colonic contraction, which was suppressed by a prostaglandin E2 (PGE2) receptor 1 antagonist. TRPA1 activation induced calcium influx and PGE2 release from cultured human fibroblastic cells. In dextran sulfate sodium–treated animals, both TRPA1 and its endogenous agonist were dramatically increased in the colonic lamina propria, accompanied by abnormal colorectal contractions. Abnormal colorectal contractions were significantly prevented by pharmacological and genetic inhibition of TRPA1. In conclusion, in the lower gastrointestinal tract, mesenchymal TRPA1 activation results in PGE2 release and consequently promotes colorectal contraction, representing what we believe is a novel physiological and inflammatory bowel disease–associated mechanism of gastrointestinal motility.
Authors
Yanjing Yang, Shenglan Wang, Kimiko Kobayashi, Yongbiao Hao, Hirosato Kanda, Takashi Kondo, Yoko Kogure, Hiroki Yamanaka, Satoshi Yamamoto, Junxiang Li, Hiroto Miwa, Koichi Noguchi, Yi Dai
Abstract
Plasma calcium (Ca2+) is maintained by amending the release of parathyroid hormone and through direct effects of the Ca2+ sensing receptor (CaSR) in the renal tubule. Combined, these mechanisms alter intestinal Ca2+ absorption by modulating 1,25-dihydroxy vitamin D3 production, bone resorption, and renal Ca2+ excretion. The CaSR is a therapeutic target in the treatment of secondary hyperparathyroidism and hypocalcemia a common complication of calcimimetic therapy. The CaSR is also expressed in intestinal epithelium, however, a direct role in regulating local intestinal Ca2+ absorption is unknown. Chronic CaSR activation decreased expression of genes involved in Ca2+ absorption. In Ussing chambers, increasing extracellular Ca2+ or basolateral application of the calcimimetic cinacalcet decreased net Ca2+ absorption across intestinal preparations acutely. Conversely, Ca2+ absorption increased with decreasing extracellular Ca2+ concentration. These responses were absent in mice expressing a non-functional TRPV6, TRPV6D541A. Cinacalcet also attenuated Ca2+ fluxes through TRPV6 in Xenopus oocytes when co-expressed with the CaSR. Moreover, the phospholipase C inhibitor, U73122, prevented cinacalcet-mediated inhibition of Ca2+ flux. These results reveal a regulatory pathway whereby activation of the CaSR in the basolateral membrane of the intestine directly attenuates local Ca2+ absorption via TRPV6 to prevent hypercalcemia and help explain how calcimimetics induce hypocalcemia.
Authors
Justin J. Lee, Xiong Liu, Debbie O'Neil, Megan R. Beggs, Petra Weissgerber, Veit Flockerzi, Xing-Zhen Chen, Henrik Dimke, R. Todd Alexander
Abstract
Children with trisomy 21 (Down syndrome [DS]) have a 130-fold increased incidence of Hirschsprung Disease (HSCR), a developmental defect where the enteric nervous system (ENS) is missing from distal bowel (i.e., distal bowel is aganglionic). Treatment for HSCR is surgical resection of aganglionic bowel, but many children have bowel problems after surgery. Post-surgical problems like enterocolitis and soiling are especially common in children with DS. To determine how trisomy 21 affects ENS development, we evaluated the ENS in two DS mouse models, Ts65Dn and Tc1. These mice are trisomic for many chromosome 21 homologous genes, including Dscam and Dyrk1a, which are hypothesized to contribute to HSCR risk. Ts65Dn and Tc1 mice have normal ENS precursor migration at E12.5 and almost normal myenteric plexus structure as adults. However, Ts65Dn and Tc1 mice have markedly reduced submucosal plexus neuron density throughout the bowel. Surprisingly, the submucosal neuron defect in Ts65Dn mice is not due to excess Dscam or Dyrk1a, since normalizing copy number for these genes does not rescue the defect. These findings suggest the possibility that the high frequency of bowel problems in children with DS and HSCR may occur because of additional unrecognized problems with ENS structure.
Authors
Ellen M. Schill, Christina M. Wright, Alisha Jamil, Jonathan M. LaCombe, Randall J. Roper, Robert O. Heuckeroth
Abstract
Chemoresistance in cancer is linked to a subset of cancer cells termed “cancer stem cells” (CSCs), and in particular, those expressing the CD44 variant appear to represent a more aggressive disease phenotype. Herein, we demonstrate that CD44v6 represents a CSC population with increased resistance to chemotherapeutic agents, and its high expression is frequently associated with poor overall survival (OS) and disease-free survival (DFS) in patients with colorectal cancer (CRC). CD44v6+ cells showed elevated resistance to chemotherapeutic drugs and significantly high tumor initiation capacity. Inhibition of CD44v6 resulted in the attenuation of self-renewal capacity and resensitization to chemotherapeutic agents. Of note, miRNA profiling of CD44v6+ spheroid-derived CSCs identified a unique panel of miRNAs indicative of high self-renewal capacity. In particular, miR-1246 was overexpressed in CD44v6+ cells, and associated with poor OS and DFS in CRC patients. We demonstrate that CD44v6+ CSCs induced chemoresistance and enhance tumorigenicity in CRC cells, and this was in part orchestrated by a distinct panel of miRNAs with dysregulated profiles. These findings suggest that specific miRNAs could serve as therapeutic targets as well as promising prognostic biomarkers in patients with colorectal neoplasia.
Authors
Shusuke Toden, Shigeyasu Kunitoshi, Jacob Cardenas, Jinghua Gu, Elizabeth Hutchins, Kendall Van Keuren-Jensen, Hiroyuki Uetake, Yuji Toiyama, Ajay Goel
Abstract
GPR55, a lipid-sensing receptor, is implicated in cell cycle control, malignant cell mobilization, and tissue invasion in cancer. However, a physiological role for GPR55 is virtually unknown for any tissue type. Here, we localize GPR55 to self-renewing ductal epithelial cells and their terminally differentiated progeny in both human and mouse salivary glands. Moreover, we find GPR55 expression downregulated in salivary gland mucoepidermoid carcinomas and GPR55 reinstatement by antitumor irradiation, suggesting that GPR55 controls renegade proliferation. Indeed, GPR55 antagonism increases cell proliferation and function determination in quasiphysiological systems. In addition, Gpr55–/– mice present ~50% enlarged submandibular glands with many more granulated ducts, as well as disordered endoplasmic reticuli and with glycoprotein content. Next, we hypothesized that GPR55 could also modulate salivation and glycoprotein content by entraining differentiated excretory progeny. Accordingly, GPR55 activation facilitated glycoprotein release by itself, inducing low-amplitude Ca2+ oscillations, as well as enhancing acetylcholine-induced Ca2+ responses. Topical application of GPR55 agonists, which are ineffective in Gpr55–/– mice, into adult rodent submandibular glands increased salivation and saliva glycoprotein content. Overall, we propose that GPR55 signaling in epithelial cells ensures both the life-long renewal of ductal cells and the continuous availability of saliva and glycoproteins for oral health and food intake.
Authors
Solomiia Korchynska, Mirjam I. Lutz, Erzsébet Borók, Johannes Pammer, Valentina Cinquina, Nataliya Fedirko, Andrew J. Irving, Ken Mackie, Tibor Harkany, Erik Keimpema
Abstract
The circadian clock network is an evolutionally conserved system involved in the regulation of metabolic homeostasis; however, its impacts on skeletal metabolism remain largely unknown. We herein demonstrated that circadian clock network in the intestines plays pivotal roles in skeletal metabolism such that the lack of Bmal1 gene in the intestines (Bmal1Int-/- mice) caused bone loss with bone resorption being activated and bone formation suppressed. Mechanistically, Clock interaction with Vitamin D receptor (Vdr) accelerated its binding to VDR response element by enhancing histone acetylation in a circadian-dependent manner, and this was lost in Bmal1Int-/- mice because nuclear translocation of Clock required the presence of Bmal1. Accordingly, the rhythmic expression of Vdr-target genes involved in transcellular calcium (Ca) absorption was created, and this was not observed in Bmal1Int-/- mice. As a result, transcellular Ca absorption was impaired and bone resorption was activated in Bmal1Int-/- mice. Additionally, sympathetic tone, the activation of which suppresses bone formation, was elevated through afferent vagal nerves in Bmal1Int-/- mice, the blockade of which partially recovered bone loss by increasing bone formation and suppressing bone resorption in Bmal1Int-/- mice. These results demonstrate that the intestinal circadian system regulates skeletal bone homeostasis.
Authors
Masanobu Kawai, Saori Kinoshita, Miwa Yamazaki, Keiko Yamamoto, Clifford J. Rosen, Shigeki Shimba, Keiichi Ozono, Toshimi Michigami
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