Vessel co-option has been demonstrated to mediate colorectal cancer liver metastasis (CRCLM) resistance to anti-angiogenic therapy. The current mechanisms underlying vessel co-option have mainly focused on the "hijacker" tumor cells, whereas the function of the “hijackee” sinusoidal blood vessels has not been explored. Here, we found that the occurrence of vessel co-option in bevacizumab-resistant CRCLM xenografts was associated with increased expression of fibroblast activation protein alpha (FAPα) in the co-opted hepatic stellate cells (HSCs), which was dramatically attenuated in HSC-specific conditional Fap-knockout mice bearing CRCLM allografts. Mechanistically, bevacizumab treatment induced hypoxia to upregulate the expression of fibroblast growth factor-binding protein 1 (FGFBP1) in tumor cells. Gain- or loss-of-function experiments revealed that the bevacizumab-resistant tumor cell-derived FGFBP1 induced FAPα expression by enhancing the paracrine FGF2-FGFR1-ERK1/2-EGR1 signaling pathway in HSCs. FAPα promoted CXCL5 secretion in HSCs, which activated CXCR2 to promote the epithelial-mesenchymal transition of tumor cells and the recruitment of myeloid-derived suppressor cells. These findings were further validated in CRCLM patient-derived tumor tissues. Targeting FAPα+ HSCs effectively disrupted the co-opted sinusoidal blood vessels and overcame bevacizumab resistance. Our study highlights the role of FAPα+ HSCs in vessel co-option and provides an effective strategy to overcome the vessel co-option-mediated bevacizumab resistance.
Ming Qi, Shuran Fan, Maohua Huang, Jinghua Pan, Yong Li, Qun Miao, Wenyu Lyu, Xiaobo Li, Lijuan Deng, Shenghui Qiu, Tongzheng Liu, Weiqing Deng, Xiaodong Chu, Chang Jiang, Wenzhuo He, Liangping Xia, Yunlong Yang, Jian Hong, Qi Qi, Wenqian Yin, Xiangning Liu, Changzheng Shi, Minfeng Chen, Wencai Ye, Dongmei Zhang
Intrahepatic neutrophil infiltration has been implicated in severe alcoholic hepatitis (SAH) pathogenesis; however, the mechanism underlying neutrophil-induced injury in SAH remains obscure. This translational study aims to describe the patterns of intrahepatic neutrophil infiltration and its involvement in SAH pathogenesis. Immunohistochemistry analyses of explanted livers identified two SAH phenotypes despite a similar clinical presentation, one with high intrahepatic neutrophils (Neuhi), but low levels of CD8+ T cells, and vice versa. RNA-Seq analyses demonstrated that neutrophil cytosolic factor 1 (NCF1), a key factor in controlling neutrophilic ROS production, was upregulated and correlated with hepatic inflammation and disease progression. To study specifically the mechanisms related to Neuhi in AH patients and liver injury, we used the mouse model of chronic-plus-binge ethanol feeding and found that myeloid-specific deletion of the Ncf1 gene abolished ethanol-induced hepatic inflammation and steatosis. RNA-Seq analysis and the data from experimental models revealed that neutrophilic NCF1-dependent ROS promoted alcoholic hepatitis (AH) by inhibiting AMP-activated protein kinase (a key regulator of lipid metabolism) and microRNA-223 (a key antiinflammatory and antifibrotic microRNA). In conclusion, two distinct histopathological phenotypes based on liver immune phenotyping are observed in SAH patients, suggesting a separate mechanism driving liver injury and/or failure in these patients.
Jing Ma, Adrien Guillot, Zhihong Yang, Bryan Mackowiak, Seonghwan Hwang, Ogyi Park, Brandon J. Peiffer, Ali Reza Ahmadi, Luma Melo, Praveen Kusumanchi, Nazmul Huda, Romil Saxena, Yong He, Yukun Guan, Dechun Feng, Pau Sancho-Bru, Mengwei Zang, Andrew MacGregor Cameron, Ramon Bataller, Frank Tacke, Zhaoli Sun, Suthat Liangpunsakul, Bin Gao
Epithelial cells lining mucosal surfaces of the gastrointestinal and respiratory tracts uniquely express ERN2/IRE1β, a paralogue of the most evolutionarily conserved endoplasmic reticulum stress sensor ERN1/IRE1α. How ERN2 functions at the host-environment interface and why a second paralogue evolved remain incompletely understood. Using conventionally raised and germ-free Ern2-/- mice, we found that ERN2 was required for microbiota-induced goblet cell maturation and mucus barrier assembly in the colon. This occurred only after colonization of the alimentary tract with normal gut microflora, which induced Ern2 expression. ERN2 acted by splicing Xbp1 mRNA to expand ER function and prevent ER stress in goblet cells. Although ERN1 can also splice Xbp1 mRNA, it did not act redundantly to ERN2 in this context. By regulating assembly of the colon mucus layer, ERN2 further shaped the composition of the gut microbiota. Mice lacking Ern2 had a dysbiotic microbial community that failed to induce goblet cell development and increased susceptibility to colitis when transferred into germ-free wild type mice. These results show that ERN2 evolved at mucosal surfaces to mediate crosstalk between gut microbes and the colonic epithelium required for normal homeostasis and host defense.
Michael J. Grey, Heidi De Luca, Doyle V. Ward, Irini A.M. Kreulen, Katlynn Bugda Gwilt, Sage E. Foley, Jay R. Thiagarajah, Beth A. McCormick, Jerrold R. Turner, Wayne I. Lencer
Functional constipation (FC) with intractable nature is the most severe form of constipation, but its etiology has long been unknown. In light of the intractable nature, we hypothesized that such intractable FC (IFC) sufferers were caused by intractable infection of a pathogenic bacterium. Here, we isolated a bacterium of Shigella sp. PIB from IFC patients that significantly inhibited the peristaltic contraction of colon by production of docosapentaenoic acid (DPA). PIB could colonize mice at least for six months. Oral administration of PIB was sufficient to induce constipation, which was reversed by PIB-specific phages. The mutated PIB with reduced DPA was incapable of inhibiting colonic function and inducing constipation, suggesting that DPA produced by PIB was the key mediator for the genesis of constipation. The PIB were detected in stools of 56% (38/68) of the IFC patients, but not in non-IFC or healthy populations (0/180). DPA levels in stools were elevated in 44.12% (30/68) of the IFC patients, but none of the healthy volunteers (0/97). Our results suggest Shigella sp. PIB may be the critical causative pathogen for IFC, and detections of fecal PIB bacteria plus DPA may be reliable methods for IFC diagnosis and classification.
Xin Chen, Tian-Tian Qiu, Ye Wang, Li-Yang Xu, Jie Sun, Zhi-Hui Jiang, Wei Zhao, Tao Tao, Yu-Wei Zhou, Lisha Wei, Yeqiong Li, Yanyan Zheng, Guo-Hua Zhou, Huaqun Chen, Jian Zhang, Xiao-Bo Feng, Fangyu Wang, Ning Li, Xue-Na Zhang, Jun Jiang, Min-Sheng Zhu
Variants in the UNC45A cochaperone have been recently associated with a syndrome combining diarrhea, cholestasis, deafness, and bone fragility. Yet the mechanism underlying intestinal failure in UNC45A deficiency remains unclear. Here, biallelic variants in UNC45A were identified by next-generation sequencing in 6 patients with congenital diarrhea. Corroborating in silico prediction, variants either abolished UNC45A expression or altered protein conformation. Myosin VB was identified by mass spectrometry as client of the UNC45A chaperone and was found misfolded in UNC45AKO Caco-2 cells. In keeping with impaired myosin VB function, UNC45AKO Caco-2 cells showed abnormal epithelial morphogenesis that was restored by full-length UNC45A, but not by mutant alleles. Patients and UNC45AKO 3D organoids displayed altered luminal development and microvillus inclusions, while 2D cultures revealed Rab11 and apical transporter mislocalization as well as sparse and disorganized microvilli. All those features resembled the subcellular abnormalities observed in duodenal biopsies from patients with microvillus inclusion disease. Finally, microvillus inclusions and shortened microvilli were evidenced in enterocytes from unc45a-deficient zebrafish. Taken together, our results provide evidence that UNC45A plays an essential role in epithelial morphogenesis through its cochaperone function of myosin VB and that UNC45A loss causes a variant of microvillus inclusion disease.
Rémi Duclaux-Loras, Corinne Lebreton, Jérémy Berthelet, Fabienne Charbit-Henrion, Ophelie Nicolle, Céline Revenu de Courtils, Stephanie Waich, Taras Valovka, Anis Khiat, Marion Rabant, Caroline Racine, Ida Chiara Guerrera, Júlia Baptista, Maxime M. Mahe, Michael W. Hess, Béatrice Durel, Nathalie Lefort, Céline Banal, Mélanie Parisot, Cecile Talbotec, Florence Lacaille, Emmanuelle Ecochard-Dugelay, Arzu Meltem Demir, Georg F. Vogel, Laurence Faivre, Astor Rodrigues, Darren Fowler, Andreas R. Janecke, Thomas Müller, Lukas A. Huber, Fernando Rodrigues-Lima, Frank M. Ruemmele, Holm H. Uhlig, Filippo Del Bene, Grégoire Michaux, Nadine Cerf-Bensussan, Marianna Parlato
SMAD4, a mediator of TGF-β signaling, plays an important role in T cells to prevent inflammatory bowel disease (IBD). However, the precise mechanisms underlying this control remain elusive. Using both genetic and epigenetic approaches, we revealed an unexpected mechanism by which SMAD4 prevents naive CD8+ T cells from becoming pathogenic for the gut. Prior to the engagement of the TGF-β receptor, SMAD4 restrains the epigenetic, transcriptional, and functional landscape of the TGF-β signature in naive CD8+ T cells. Mechanistically, prior to TGF-β signaling, SMAD4 binds to promoters and enhancers of several TGF-β target genes, and by regulating histone deacetylation, suppresses their expression. Consequently, regardless of a TGF-β signal, SMAD4 limits the expression of TGF-β negative feedback loop genes, such as Smad7 and Ski, and likely conditions CD8+ T cells for the immunoregulatory effects of TGF-β. In addition, SMAD4 ablation conferred naive CD8+ T cells with both a superior survival capacity, by enhancing their response to IL-7, as well as an enhanced capacity to be retained within the intestinal epithelium, by promoting the expression of Itgae, which encodes the integrin CD103. Accumulation, epithelial retention, and escape from TGF-β control elicited chronic microbiota-driven CD8+ T cell activation in the gut. Hence, in a TGF-β–independent manner, SMAD4 imprints a program that preconditions naive CD8+ T cell fate, preventing IBD.
Ramdane Igalouzene, Hector Hernandez-Vargas, Nicolas Benech, Alexandre Guyennon, David Bauché, Célia Barrachina, Emeric Dubois, Julien C. Marie, Saïdi M’Homa Soudja
Enhanced de novo lipogenesis mediated by sterol regulatory element-binding proteins (SREBPs) is thought to be involved in nonalcoholic steatohepatitis (NASH) pathogenesis. In this study, we assessed the impact of SREBP inhibition on NASH and liver cancer development in murine models. Unexpectedly, SREBP inhibition via deletion of the SREBP cleavage-activating protein (SCAP) in the liver exacerbated liver injury, fibrosis, and carcinogenesis, despite markedly reduced hepatic steatosis. These phenotypes were ameliorated by restoring SREBP function. Transcriptome and lipidome analyses revealed that SCAP–SREBP pathway inhibition altered the fatty acid (FA) composition of phosphatidylcholines due to both impaired FA synthesis and disorganized FA incorporation into phosphatidylcholine via lysophosphatidylcholine acyltransferase 3 (LPCAT3) downregulation, which led to endoplasmic reticulum (ER) stress and hepatocyte injury. Supplementation of phosphatidylcholines significantly improved liver injury and ER stress induced by SCAP deletion. The activity of SCAP-SREBP-LPCAT3 axis was found inversely associated with liver fibrosis severity in human NASH. SREBP inhibition also cooperated with impaired autophagy to trigger liver injury. Thus, excessively strong and broad lipogenesis inhibition was counterproductive for NASH therapy, which will have important clinical implications in NASH treatment.
Satoshi Kawamura, Yuki Matsushita, Shigeyuki Kurosaki, Mizuki Tange, Naoto Fujiwara, Yuki Hayata, Yoku Hayakawa, Nobumi Suzuki, Masahiro Hata, Mayo Tsuboi, Takahiro Kishikawa, Hiroto Kinoshita, Takuma Nakatsuka, Masaya Sato, Yotaro Kudo, Yujin Hoshida, Atsushi Umemura, Akiko Eguchi, Tsuneo Ikenoue, Yoshihiro Hirata, Motonari Uesugi, Ryosuke Tateishi, Keisuke Tateishi, Mitsuhiro Fujishiro, Kazuhiko Koike, Hayato Nakagawa
Gastric carcinogenesis is mediated by complex interactions among Helicobacter pylori, host, and environmental factors. We now demonstrate that H. pylori augments gastric injury in INS-GAS mice under iron deficient conditions. Mechanistically, these phenotypes were not driven by alterations in the gastric microbiota; however, discovery-based and targeted metabolomics revealed that bile acids were significantly altered in H. pylori-infected mice with iron deficiency, with significant upregulation of deoxycholic acid (DCA), a carcinogenic bile acid. Severity of gastric injury was further augmented when H. pylori-infected mice were treated with DCA, and, in vitro, DCA increased translocation of the H. pylori oncoprotein CagA into host cells. Conversely, bile acid sequestration attenuated H. pylori-induced injury under conditions of iron deficiency. To translate these findings into human populations, the association between bile acid-sequestrant use and gastric cancer risk was evaluated in a large human cohort. Among 416,885 individuals, a significant dose-dependent reduction in risk was associated with cumulative bile acid-sequestrant use. Further, expression of the bile acid receptor TGR5 paralleled the severity of carcinogenic lesions in humans. These data demonstrate that increased H. pylori-induced injury within the context of iron deficiency is tightly linked to altered bile acid metabolism, which may promote gastric carcinogenesis.
Jennifer M. Noto, M. Blanca Piazuelo, Shailja C. Shah, Judith Romero-Gallo, Jessica L. Hart, Chao Di, James D. Carmichael, Alberto G. Delgado, Alese E. Halvorson, Robert A. Greevy, Jr., Lydia E. Wroblewski, Ayushi Sharma, Annabelle B. Newton, Margaret M. Allaman, Keith T. Wilson, M. Kay Washington, M. Wade Calcutt, Kevin L. Schey, Bethany P. Cummings, Charles R. Flynn, Joseph P. Zackular, Richard M. Peek, Jr.
De novo and acquired resistance are major impediments to the efficacy of conventional and targeted cancer therapy. In unselected gastric cancer (GC) patients with advanced disease, trials combining chemotherapy and an anti-EGFR monoclonal antibody have been largely unsuccessful. In an effort to identify biomarkers of resistance so as to better select patients for such trials, we screened the secretome of chemotherapy-treated human GC cell lines. We found that levels of CGA, the α-subunit of glycoprotein hormones, were markedly increased in the conditioned media of chemoresistant GC cells, and CGA immunoreactivity was enhanced in GC tissues that progressed on chemotherapy. CGA levels in plasma increased in GC patients who received chemotherapy, and this increase was correlated with reduced responsiveness to chemotherapy and poor survival. Mechanistically, secreted CGA was found to bind to EGFR and activate EGFR signaling, thereby conferring a survival advantage to GC cells. N-glycosylation of CGA at Asn52 and Asn78 is required for its stability, secretion, and interaction with EGFR. GATA2 was found to activate CGA transcription, whose increase, in turn, induced the expression and phosphorylation of GATA2 in an EGFR-dependent manner, forming a positive feedback circuit that was initiated by GATA2 autoregulation upon sublethal exposure to chemotherapy. Based on this circuit, combination strategies involving anti-EGFR therapies or targeting CGA with microRNAs (miR-708-3p and miR-761) restored chemotherapy sensitivity. These findings identify a clinically actionable CGA/EGFR/GATA2 circuit and highlight CGA as a predictive biomarker and therapeutic target in chemoresistant GC.
Tianyu Cao, Yuanyuan Lu, Qi Wang, Hongqiang Qin, Hongwei Li, Hao Guo, Minghui Ge, Sarah E. Glass, Bhuminder Singh, Wenyao Zhang, Jiaqiang Dong, Feng Du, Airong Qian, Ye Tian, Xin Wang, Cunxi Li, Kaichun Wu, Daiming Fan, Yongzhan Nie, Robert J. Coffey, Xiaodi Zhao
The striatin-interacting phosphatase and kinase (STRIPAK) complexes integrate extracellular stimuli to result in intracellular activities. Previously, we discovered STRIPAK to be a key machinery responsible for loss of the Hippo tumor suppressor signal in cancer. Here, we identified the Hippo-STRIPAK complex to be an essential player for the control of DNA double-strand break (DSB) repair and genomic stability. Specifically, the MST1/2 kinases were found, independent of the classical Hippo signaling, to directly phosphorylate ZMYND8 and hence result in suppression of DNA repair in the nucleus. In response to genotoxic stress, the cGAS-STING pathway was determined to relay nuclear DNA damage signals to the dynamic assembly of Hippo-STRIPAK via a TBK1-induced structural stabilization of the SIKE1-SLMAP arm. As such, STRIPAK-mediated MST1/2 inactivation was found to increase the DSB repair capacity of cancer cells and to endow these cells with resistance to radio/chemotherapy and PARP inhibition. Importantly, targeting the STRIPAK assembly with each of three distinct peptide inhibitors efficiently recovered the kinase activity of MST1/2 to suppress DNA repair and re-sensitize cancer cells to PARPi in both animal and patient-derived tumor models. Overall, our findings not only uncovered a previously unrecognized role for STRIPAK in modulating DSB repair, but also provided translational implications of co-targeting STRIPAK and PARP for a new type of synthetic lethality anti-cancer therapy.
Liwei An, Zhifa Cao, Pingping Nie, Hui Zhang, Zhenzhu Tong, Fan Chen, Yang Tang, Yi Han, Wenjia Wang, Zhangting Zhao, Qingya Zhao, Yuqin Yang, Yuanzhi Xu, Gemin Fang, Lei Shi, Huixiong Xu, Haiqing Ma, Shi Jiao, Zhaocai Zhou