Gastroenterologies
Abstract
Epidemiological and histopathological findings have raised the possibility that misfolded α-synuclein protein might spread from the gut to the brain and increase the risk of Parkinson’s disease. Although past experimental studies in mouse models have relied on gut injections of exogenous recombinant α-synuclein fibrils to study gut-to-brain α-synuclein transfer, the possible origins of misfolded α-synuclein within the gut have remained elusive. We recently demonstrated that sensory cells of intestinal mucosa express α-synuclein. Here, we employed mouse intestinal organoids expressing human α-synuclein to observe the transfer of α-synuclein protein from epithelial cells in organoids to cocultured nodose neurons devoid of α-synuclein. In mice expressing human α-synuclein, but no mouse α-synuclein, α-synuclein fibril-templating activity emerged in α-synuclein–seeded fibril aggregation assays in intestine, vagus nerve, and dorsal motor nucleus. In newly engineered transgenic mice that restrict pathological human α-synuclein expression to intestinal epithelial cells, α-synuclein fibril-templating activity transfered to the vagus nerve and dorsal motor nucleus. Subdiaphragmatic vagotomy prior to induction of α-synuclein expression in intestinal epithelial cells effectively protected the hindbrain from emergence of α-synuclein fibril-templating activity. Overall, these findings highlight a potential non-neuronal source of fibrillar α-synuclein protein that might arise in gut mucosal cells.
Authors
Rashmi Chandra, Arpine Sokratian, Katherine R. Chavez, Stephanie King, Sandip M. Swain, Joshua C. Snyder, Andrew B. West, Rodger A. Liddle
Abstract
Patients with cholangiocarcinoma have poor clinical outcomes due to late diagnoses, poor prognoses, and limited treatment strategies. To identify drug combinations for this disease, we have conducted a genome-wide CRISPR screen anchored on the bromodomain and extraterminal domain (BET) PROTAC degrader ARV825, from which we identified anti-cancer synergy when combined with genetic ablation of members of the mTOR pathway. This combination effect was validated using multiple pharmacological BET and mTOR inhibitors, accompanied by increased levels of apoptosis and cell cycle arrest. In a xenograft model, combined BET degradation and mTOR inhibition induced tumor regression. Mechanistically, the two inhibitor classes converged on H3K27ac-marked epigenetic suppression of the serine glycine one carbon (SGOC) metabolism pathway, including the key regulators PHGDH and PSAT1. Knockdown of PSAT1 was sufficient to replicate synergy with single agent inhibition of either BET or mTOR. Our results tie together epigenetic regulation, metabolism, and apoptosis induction as key therapeutic targets for further exploration in this underserved disease.
Authors
Yan Zhu, Dengyong Zhang, Pooja Shukla, Young-Ho Jung, Prit Benny Malgulwar, Sharmeen Chagani, Medina Colic, Sarah Benjamin, John A. Copland III, Lin Tan, Philip L. Lorenzi, Milind Javle, Jason T. Huse, Jason Roszik, Traver Hart, Lawrence N. Kwong
Abstract
Germline APC mutation in familial adenomatous polyposis (FAP) patients promotes gastrointestinal polyposis, including the formation of frequent gastric fundic gland polyps (FGPs). In this study, we investigated how dysregulated Wnt signaling promotes FGPs and why they localize to the corpus region of the stomach. We developed a biobank of FGP and surrounding non-polyp corpus biopsies and organoids from FAP patients for comparative studies. Polyp biopsies and polyp-derived organoids exhibited enhanced Wnt target gene expression. Polyp-derived organoids with intrinsically upregulated Wnt signaling showed poor tolerance to further induction, suggesting that high Wnt restricts growth. Targeted genomic sequencing revealed that most gastric polyps did not arise via APC loss-of-heterozygosity. Studies in genetic mouse models demonstrated that heterozygous Apc loss increased epithelial cell proliferation in the corpus but not the antrum, while homozygous Apc loss was not maintained in the corpus yet induced hyperproliferation in antrum. Our findings suggest that heterozygous APC mutation in FAP patients may be sufficient to drive polyp formation in the corpus region while subsequent loss-of-heterozygosity to further enhance Wnt signaling is not tolerated. This finding contextualizes the abundant yet benign nature of gastric polyps in FAP patient corpus compared to the rare, yet adenomatous polyps in the antrum.
Authors
Kevin P. McGowan, Elizabeth Delgado, Theresa M. Keeley, Elise S. Hibdon, Danielle Kim Turgeon, Elena M. Stoffel, Linda C. Samuelson
Abstract
Emerging evidence shows that KRAS-mutant colorectal cancer (CRC) depends on glutamine (Gln) for survival and progression, indicating that targeting Gln metabolism may be a promising therapeutic strategy for KRAS-mutant CRC. However, the precise mechanism by which Gln metabolism reprogramming promotes and coordinates KRAS-mutant CRC progression remains to be fully investigated. Here, we discovered that solute carrier 25 member 21 (SLC25A21) expression was downregulated in KRAS-mutant CRC, and that SLC25A21 downregulation was correlated with poor survival of KRAS-mutant CRC patients. SLC25A21 depletion selectively accelerated the growth, invasion, migration, and metastasis of KRAS-mutant CRC cells in vitro and in vivo, and inhibited Gln-derived α-ketoglutarate (α-KG) efflux from mitochondria, thereby potentiating Gln replenishment, accompanied by increased GTP availability for persistent KRAS activation in KRAS-mutant CRC. The restoration of SLC25A21 expression impaired the KRAS-mutation-mediated resistance to cetuximab in KRAS-mutant CRC. Moreover, the arrested α-KG efflux that occurred in response to SLC25A21 depletion inhibited the activity of α-KG–dependent DNA demethylases, resulting in a further decrease in SLC25A21 expression. Our studies demonstrate that SLC25A21 plays a significant role as a tumor suppressor in KRAS-mutant CRC by antagonizing Gln-dependent anaplerosis to limit GTP availability for KRAS activation, which suggests potential alternative therapeutic strategies for KRAS-mutant CRC.
Authors
Sha-Sha Hu, Yue Han, Tian-Yuan Tan, Hui Chen, Jia-Wen Gao, Lan Wang, Min-Hui Yang, Li Zhao, Yi-Qing Wang, Yan-Qing Ding, Shuang Wang
Abstract
Intestinal epithelial transit amplifying cells are essential stem progenitors required for intestinal homeostasis, but their rapid proliferation renders them vulnerable to DNA damage from radiation and chemotherapy. Despite their critical roles in intestinal homeostasis and disease, few studies have described genes that are essential to transit amplifying cell function. We report that the RNA methyltransferase, METTL3, is required for survival of transit amplifying cells in the murine small intestine. Transit amplifying cell death after METTL3 deletion was associated with crypt and villus atrophy, loss of absorptive enterocytes, and uniform wasting and death in METTL3-depleted mice. Sequencing of polysome-bound and methylated RNAs in enteroids and in vivo demonstrated decreased translation of hundreds of unique methylated transcripts after METTL3 deletion, particularly transcripts involved in growth factor signal transduction such as Kras. Further investigation confirmed a relationship between METTL3 and Kras methylation and protein levels in vivo. Our study identifies METTL3 as an essential factor supporting the homeostasis of small intestinal tissue via direct maintenance of transit amplifying cell survival. We highlight the crucial role of RNA modifications in regulating growth factor signaling in the intestine with important implications for both homeostatic tissue renewal and epithelial regeneration.
Authors
Charles H. Danan, Kaitlyn E. Naughton, Katharina E. Hayer, Sangeevan Vellappan, Emily A. McMillan, Yusen Zhou, Rina Matsuda, Shaneice K. Nettleford, Kay Katada, Louis R. Parham, Xianghui Ma, Afrah Chowdhury, Benjamin J. Wilkins, Premal Shah, Matthew D. Weitzman, Kathryn E. Hamilton
Abstract
Despite being in the same pathway, mutations of KRAS and BRAF in colorectal carcinomas (CRCs) determine distinct progression courses. ZEB1 induces an epithelial-to-mesenchymal transition (EMT) and is associated with worse progression in most carcinomas. Using samples from patients with CRC, mouse models of KrasG12D and BrafV600E CRC, and a Zeb1-deficient mouse, we show that ZEB1 had opposite functions in KRAS- and BRAF-mutant CRCs. In KrasG12D CRCs, ZEB1 was correlated with a worse prognosis and a higher number of larger and undifferentiated (mesenchymal or EMT-like) tumors. Surprisingly, in BrafV600E CRC, ZEB1 was associated with better prognosis; fewer, smaller, and more differentiated (reduced EMT) primary tumors; and fewer metastases. ZEB1 was positively correlated in KRAS-mutant CRC cells and negatively in BRAF-mutant CRC cells with gene signatures for EMT, cell proliferation and survival, and ERK signaling. On a mechanistic level, ZEB1 knockdown in KRAS-mutant CRC cells increased apoptosis and reduced clonogenicity and anchorage-independent growth; the reverse occurred in BRAFV600E CRC cells. ZEB1 is associated with better prognosis and reduced EMT signature in patients harboring BRAF CRCs. These data suggest that ZEB1 can function as a tumor suppressor in BRAF-mutant CRCs, highlighting the importance of considering the KRAS/BRAF mutational background of CRCs in therapeutic strategies targeting ZEB1/EMT.
Authors
Ester Sánchez-Tilló, Leire Pedrosa, Ingrid Vila, Yongxu Chen, Balázs Győrffy, Lidia Sánchez-Moral, Laura Siles, Juan J. Lozano, Anna Esteve-Codina, Douglas S. Darling, Miriam Cuatrecasas, Antoni Castells, Joan Maurel, Antonio Postigo
Abstract
Cellular senescence and biliary fibrosis are prototypical features of obliterative cholangiopathies, such as Primary Sclerosing Cholangitis (PSC). Telomere dysfunction can lead to senescence either through telomere erosion or damaged telomeres. Our goal was to investigate a mechanistic relationship between telomere damage and biliary fibrosis in PSC. Telomere attrition was observed in the bile ducts of PSC patients along with a reduction in telomerase reverse transcriptase (TERT) expression compared to normal livers. Similarly, liver tissue from mice models of biliary fibrosis showed telomere attrition with increased damage at telomeres measured as telomere-associated foci (TAF). Cellular models of senescence induction increased the TAFs in cholangiocytes. This coincided with decreased TERT expression and increased senescence, which was rescued by modulating TERT levels. Epigenetic analysis revealed increased acquisition of repressive histone methylation at the TERT promoter which correlated with decreased TERT transcription. Cholangiocyte-selective deletion of TERT in mice exacerbated fibrosis whereas androgen therapy towards telomerase rescued liver fibrosis and liver function in genetic mouse model of PSC. Our results demonstrate a mechanistic role for telomere dysfunction in cellular senescence and fibrosis that characterize PSC. This suggests that PSC may be, in part, a telomere biology disorder, and identifies TERT as a potential therapeutic target.
Authors
Nidhi Jalan-Sakrikar, Abid Anwar, Usman Yaqoob, Can Gan, Anthony B. Lagnado, Alexander Q. Wixom, Diana Jurk, Robert C. Huebert
Abstract
Eosinophilic esophagitis (EoE) is an esophageal immune-mediated disease characterized by eosinophilic inflammation and epithelial remodeling, including basal cell hyperplasia (BCH). Although BCH is known to correlate with disease severity and with persistent symptoms in patients in histological remission, the molecular processes driving BCH remain poorly defined. Here, we demonstrated that BCH is predominantly characterized by an expansion of non-proliferative suprabasal cells that are still committed to early differentiation. Furthermore, we discovered that suprabasal and superficial esophageal epithelial cells retain progenitor identity programs in EoE, which was evidenced by increased quiescent cell identity scoring and the enrichment of signaling pathways regulating stem cell pluripotency. Enrichment and trajectory analyses identified SOX2 and KLF5 as potential drivers of the increased quiescent identity and epithelial remodeling observed in EoE. Notably, these alterations were not observed in GERD. These findings provide additional insights into the differentiation process in EoE and highlight the distinct characteristics of suprabasal and superficial esophageal epithelial cells in the disease.
Authors
Margarette H. Clevenger, Adam L. Karami, Dustin A. Carlson, Peter J. Kahrilas, Nirmala Gonsalves, John E. Pandolfino, Deborah R. Winter, Kelly A. Whelan, Marie-Pier Tétreault
Abstract
Eosinophilic esophagitis (EoE) is a chronic gastrointestinal disorder characterized by food antigen–driven eosinophilic inflammation and hyperproliferation of esophageal mucosa. By utilizing a large-scale, proteomic screen of esophageal biopsies, we aimed to uncover molecular drivers of the disease. Proteomic analysis by liquid chromatography–tandem mass spectrometry identified 402 differentially expressed proteins (DEPs) that correlated with the EoE transcriptome. Immune cell–related proteins were among the most highly upregulated DEPs in EoE compared to controls, whereas proteins linked to epithelial differentiation were primarily downregulated. Notably, in the inflamed esophageal tissue, all six subunits of the minichromosome maintenance (MCM) complex, a DNA helicase essential for genomic DNA replication, were significantly upregulated at the gene and protein levels. Furthermore, treating esophageal epithelial cells with a known inhibitor of the MCM complex (ciprofloxacin) blocked esophageal epithelial proliferation. In a murine model of EoE driven by overexpression of IL-13, ciprofloxacin treatment decreased basal zone thickness and reduced dilated intercellular spaces by blocking the transition of the epithelial cell through the S-phase of the cell cycle. Collectively, a broad-spectrum proteomic screen has identified the involvement of the MCM complex in EoE and has highlighted MCM inhibitors as potential therapeutic agents for the disease.
Authors
Mark Rochman, Yrina Rochman, Julie M. Caldwell, Lydia Mack, John A Besse, Nathan P Manes, Sung Hwan Yoon, Tetsuo Shoda, Aleksandra Nita-Lazar, Marc Rothenberg
Abstract
Intestinal mucins play an essential role in the defense against bacterial invasion and the maintenance of gut microbiota, which is instrumental in the regulation of host immune systems; hence, its dysregulation is a hallmark of metabolic disease and intestinal inflammation. However, the mechanism by which intestinal mucins control the gut microbiota as well as disease phenotypes remains nebulous. Herein, we report that N-acetylglucosamine (GlcNAc)-6-O-sulfation of O-glycans on intestinal mucins performs a protective role against obesity and intestinal inflammation. Chst4-/- mice, lacking GlcNAc-6-O-sulfation of the mucin O-glycans, showed significant weight gain and increased susceptibility to dextran sodium sulfate-induced colitis as well as colitis-associated cancer accompanied by significantly reduced immunoglobulin A (IgA) production caused by impaired T follicular helper cell-mediated IgA response. Interestingly, the protective effects of GlcNAc-6-O-sulfation against obesity and intestinal inflammation depend on the gut microbiota, evidenced by the modulation of the gut microbiota by co-housing or microbiota transplantation reversing disease phenotypes and IgA production. Collectively, our findings provide novel insight into the significance of host glycosylation, more specifically GlcNAc-6-O-sulfation on intestinal mucins, in protecting against obesity and intestinal inflammation via regulation of the gut microbiota.
Authors
Hirohito Abo, Aoi Muraki, Akihito Harusato, Tetsuya Imura, Maki Suzuki, Kohta Takahashi, Timothy L. Denning, Hiroto Kawashima
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