TCF7L1 regulates colorectal cancer cell migration by repressing GAS1 expression.

Dysregulated Wnt/ß-catenin signaling is a common feature of colorectal cancer (CRC). The T-cell factor/lymphoid enhancer factor (TCF/LEF; hereafter, TCF) family of transcription factors are critical regulators of Wnt/ß-catenin target gene expression. Of the four TCF family members, TCF7L1 predominantly functions as a transcriptional repressor. Although TCF7L1 has been ascribed an oncogenic role in CRC, only a few target genes whose expression it regulates have been characterized in this cancer. Through transcriptome analyses of TCF7L1 regulated genes, we noted enrichment for those associated with cellular migration. By silencing and overexpressing TCF7L1 in CRC cell lines, we demonstrated that TCF7L1 promoted migration, invasion, and adhesion. We localized TCF7L1 binding across the CRC genome and overlapped enriched regions with transcriptome data to identify candidate target genes. The growth arrest-specific 1 (GAS1) gene was among these and we demonstrated that GAS1 is a critical mediator of TCF7L1-dependent CRC cell migratory phenotypes. Together, these findings uncover a novel role for TCF7L1 in repressing GAS1 expression to enhance migration and invasion of CRC cells.

Identification of differentially expressed genes and splicing events in early-onset colorectal cancer.

Background: The incidence of colorectal cancer (CRC) has been steadily increasing in younger individuals over the past several decades for reasons that are incompletely defined. Identifying differences in gene expression profiles, or transcriptomes, in early-onset colorectal cancer (EOCRC, < 50 years old) patients versus later-onset colorectal cancer (LOCRC, > 50 years old) patients is one approach to understanding molecular and genetic features that distinguish EOCRC. Methods: We performed RNA-sequencing (RNA-seq) to characterize the transcriptomes of patient-matched tumors and adjacent, uninvolved (normal) colonic segments from EOCRC (n=21) and LOCRC (n=22) patients. The EOCRC and LOCRC cohorts were matched for demographic and clinical characteristics. We used The Cancer Genome Atlas Colon Adenocarcinoma (TCGA-COAD) database for validation. We used a series of computational and bioinformatic tools to identify EOCRC-specific differentially expressed genes, molecular pathways, predicted cell populations, differential gene splicing events, and predicted neoantigens. Results: We identified an eight-gene signature in EOCRC comprised of ALDOB, FBXL16, IL1RN, MSLN, RAC3, SLC38A11, WBSCR27 and WNT11, from which we developed a score predictive of overall CRC patient survival. On the entire set of genes identified in normal tissues and tumors, cell type deconvolution analysis predicted a differential abundance of immune and non-immune populations in EOCRC versus LOCRC. Gene set enrichment analysis identified increased expression of splicing machinery in EOCRC. We further found differences in alternative splicing (AS) events, including one within the long non-coding RNA, HOTAIRM1. Additional analysis of AS found seven events specific to EOCRC that encode potential neoantigens. Conclusion: Our transcriptome analyses identified genetic and molecular features specific to EOCRC which may inform future screening, development of prognostic indicators, and novel drug targets.

Loss of STIM2 in colorectal cancer drives growth and metastasis through metabolic reprogramming and PERK-ATF4 endoplasmic reticulum stress pathway.

The endoplasmic reticulum (ER) stores large amounts of calcium (Ca2+), and the controlled release of ER Ca2+ regulates a myriad of cellular functions. Although altered ER Ca2+ homeostasis is known to induce ER stress, the mechanisms by which ER Ca2+ imbalance activate ER stress pathways are poorly understood. Stromal-interacting molecules STIM1 and STIM2 are two structurally homologous ER-resident Ca2+ sensors that synergistically regulate Ca2+ influx into the cytosol through Orai Ca2+ channels for subsequent signaling to transcription and ER Ca2+ refilling. Here, we demonstrate that reduced STIM2, but not STIM1, in colorectal cancer (CRC) is associated with poor patient prognosis. Loss of STIM2 causes SERCA2-dependent increase in ER Ca2+, increased protein translation and transcriptional and metabolic rewiring supporting increased tumor size, invasion, and metastasis. Mechanistically, STIM2 loss activates cMyc and the PERK/ATF4 branch of ER stress in an Orai-independent manner. Therefore, STIM2 and PERK/ATF4 could be exploited for prognosis or in targeted therapies to inhibit CRC tumor growth and metastasis.

Pyoderma Gangrenosum Is Associated With Increased Risk of Inflammatory Pouch-Related Complications: A Retrospective Cohort Study.

Background: Pyoderma gangrenosum (PG) is a rare, neutrophilic dermatosis that is a well-established extraintestinal manifestation (EIM) of inflammatory bowel disease. The clinical implications of developing PG in patients with ulcerative colitis (UC) who undergo total proctocolectomy colectomy and ileal pouch anal anastomosis (TPC-IPAA) surgery remain unknown. Methods: Study participants were selected from patients enrolled in the Carlino Family Inflammatory Bowel and Colorectal Disease Biobank between 1998 and 2021 with a pre-colectomy diagnosis of UC and who underwent TPC-IPAA surgery. A retrospective study comparing patients with PG and those without PG was performed. The outcomes measured included the development of pouchitis, pouchitis classification, presence of pouch fistula, anal fistula, anal stenosis, and pouch failure. Results: In this study, 357 IPAA patients were included, 10 of whom suffered PG. Patients with PG and without PG had similar demographics and clinical characteristics. Both groups had similar rates of pouchitis (80% in PG patients and 64% in patients without PG, P = .504). However, IPAA patients with PG had a higher risk of developing pouch fistula (50% vs 10%, P = .002), anal fistula (40% vs 12%, P = .031), and Crohn's-like disease of the pouch (70% vs 15%, P = .003) compared to patients without PG. Patients who developed PG prior to their first episode of pouchitis were more likely to eventually experience pouch failure (odds ratio: 20.7, 95% confidence interval: 3.9, 110.7, q = 0.003 after false discovery rate adjustment). Conclusions: Among UC patients who undergo TPC-IPAA surgery, the development of PG portends poor pouch outcomes and is predictive of pouch failure.

Control of Paneth cell function by HuR regulates gut mucosal growth by altering stem cell activity.

Rapid self-renewal of the intestinal epithelium requires the activity of intestinal stem cells (ISCs) that are intermingled with Paneth cells (PCs) at the crypt base. PCs provide multiple secreted and surface-bound niche signals and play an important role in the regulation of ISC proliferation. Here, we show that control of PC function by RNA-binding protein HuR via mitochondria affects intestinal mucosal growth by altering ISC activity. Targeted deletion of HuR in mice disrupted PC gene expression profiles, reduced PC-derived niche factors, and impaired ISC function, leading to inhibited renewal of the intestinal epithelium. Human intestinal mucosa from patients with critical surgical disorders exhibited decreased levels of tissue HuR and PC/ISC niche dysfunction, along with disrupted mucosal growth. HuR deletion led to mitochondrial impairment by decreasing the levels of several mitochondrial-associated proteins including prohibitin 1 (PHB1) in the intestinal epithelium, whereas HuR enhanced PHB1 expression by preventing microRNA-195 binding to the Phb1 mRNA. These results indicate that HuR is essential for maintaining the integrity of the PC/ISC niche and highlight a novel role for a defective PC/ISC niche in the pathogenesis of intestinal mucosa atrophy.

Creating a Surgical Biobank: The Hershey Medical Center Experience.

BACKGROUND: Tissue harvesting at the time of surgery offers surgeons and scientists a unique opportunity to discover and better understand disease pathophysiology. Tissue biobanking presents challenges in patient consents, specimen collection, preparation, and storage, but the potential for scientific discovery justifies the effort. Although the number of tissue biobanks is increasing worldwide, information regarding necessary infrastructure, process flow, and management of expected obstacles is lacking. OBJECTIVE: To provide a framework and motivation for clinician scientists intending to start an intestinal tissue biobank under their direction. DATA SOURCES: The Carlino Family Inflammatory Bowel and Colorectal Diseases Biobank is housed at the Milton S. Hershey Medical Center. STUDY SELECTION: Review. INTERVENTION: Implementation of a surgical tissue biobank at a large tertiary care institution. MAIN OUTCOME MEASURES: Assess critical challenges and obstacles over the years as well as keys to the success of the program. RESULTS: Over 2 decades, the institutional biobank grew from an IBD biobank to one which now incorporates thousands of surgical specimens representing numerous colorectal diseases. This was done through a process of refinement focusing on patient recruitment and an efficient consenting and specimen management process. The biobank's success is further insured by institutional, external, and philanthropic support; scientific collaborations; and sharing of biological specimens with other groups of dedicated researchers. LIMITATIONS: This is a single-center experience in collecting surgically resected colorectal specimens. CONCLUSIONS: Surgical specimen biobanks are essential in studying disease cause using genomics, transcriptomics, and proteomic technologies. Therefore, surgeons, clinicians, and scientists should build biobanks at their institutions to promote further scientific discovery and improve specimen diversity.

The Microbiome of Complicated Diverticulitis: An Imbalance of Sulfur-Metabolizing Bacteria.

BACKGROUND: The progression to acute diverticulitis from the relatively benign condition of colonic diverticulosis is not well characterized. A smaller subset may even develop complicated (perforated) diverticulitis resulting in sepsis and/or death. Characterizing the differences between recurrent, uncomplicated diverticulitis, and the more virulent, complicated diverticulitis is necessary to guide clinical decision-making. Alterations to the microbiome offer a possible explanation for local inflammation and the pathophysiology of diverticular disease. OBJECTIVE: This study aimed to characterize the mucosal-associated microbiome in patients with recurrent uncomplicated diverticulitis and complicated (perforated) diverticulitis. DESIGN: Microbial DNA was extracted from full-thickness surgical specimens for 16S rRNA gene sequencing, targeting the V4 hypervariable region. Sequences were analyzed and a quantitative characterization based on taxonomic classification was performed. SETTING: A tertiary care academic medical center. PATIENTS: This study compared 48 patients with recurrent, uncomplicated diverticulitis and 35 patients with radiographically confirmed perforated (complicated) diverticulitis. Tissues were harvested from surgical resection specimens to include both diseased regions and nondiseased (adjacent normal) regions. MAIN OUTCOME MEASURES: We assessed differences in relative abundance and taxonomic classification of mucosal-associated microbes in surgical resection specimens from diverticular disease. RESULTS: When analyzing the tissue of diverticular resection specimens, the complicated diseased segments demonstrated an increased abundance of sulfur-reducing and sulfur-oxidizing bacteria compared to nondiseased, adjacent normal regions. When comparing diseased segments, tissues of patients with complicated diverticulitis had a marked increase in sulfur-reducing microbes. LIMITATIONS: We characterized the mucosal-associated microbiome present at the time of surgical resection, limiting conclusions on its role in pathophysiology. Furthermore, antibiotic usage and bowel preparation before surgery may result in perturbations to microbial flora. CONCLUSIONS: The microbiome of complicated diverticulitis is marked by a localized imbalance of sulfur-metabolizing microbes. The abundance of sulfur-reducing microbes may lead to an excess of hydrogen sulfide and subsequent inflammation. See Video Abstract at http://links.lww.com/DCR/C175 . LA MICROBIOMA DE LA DIVERTICULITIS COMPLICADA UN DESEQUILIBRIO DE LAS BACTERIAS METABOLIZADORAS DE AZUFRE: ANTECEDENTES: La progresión a diverticulitis aguda de la condición relativamente benigna de diverticulosis colónica no está bien caracterizada. Un subgrupo más pequeño puede incluso desarrollar diverticulitis complicada (perforada) que resulta en sepsis y/o muerte. Es necesario caracterizar las diferencias entre la diverticulitis recurrente no complicada y la diverticulitis complicada más virulenta para guiar la toma de decisiones clínicas. Las alteraciones del microbioma ofrecen una posible explicación de la inflamación local y la fisiopatología de la enfermedad diverticular.OBJETIVO: Caracterizar el microbioma asociado a la mucosa en pacientes con diverticulitis no complicada recurrente y diverticulitis complicada (perforada).DISEÑO: El ADN microbiano se extrajo de especímenes quirúrgicos de espesor completo para la secuenciación del gen 16S rRNA, dirigido a la región hipervariable V4. Se analizaron las secuencias y se realizó una caracterización cuantitativa basada en la clasificación taxonómica.AJUSTE: Un centro médico académico de atención terciaria.PACIENTES: Este estudio comparó 48 pacientes con diverticulitis recurrente no complicada y 35 pacientes con diverticulitis perforada (complicada) confirmada radiográficamente. Se recogieron tejidos de especímenes de resección quirúrgica para incluir tanto regiones enfermas como regiones no enfermas (normales adyacentes).PRINCIPALES MEDIDAS DE RESULTADO: Evaluamos las diferencias en la abundancia relativa y la clasificación taxonómica de los microbios asociados a la mucosa en muestras de resección quirúrgica de enfermedad diverticular.RESULTADOS: Al analizar el tejido de las muestras de resección diverticular, los segmentos enfermos complicados demostraron una mayor abundancia de bacterias reductoras de azufre y oxidantes de azufre en comparación con las regiones normales adyacentes no enfermas. Al comparar segmentos enfermos, los tejidos de pacientes complicados tenían un marcado aumento de microbios reductores de azufre.LIMITACIONES: Caracterizamos el microbioma asociado a la mucosa presente en el momento de la resección quirúrgica, lo que limita las conclusiones sobre su papel en la fisiopatología. Además, el uso de antibióticos y la preparación intestinal antes de la cirugía pueden provocar alteraciones en la flora microbiana.CONCLUSIONES: El microbioma de la diverticulitis complicada está marcado por un desequilibrio localizado de microbios metabolizadores de azufre. La abundancia de microbios reductores de azufre puede provocar un exceso de sulfuro de hidrógeno y la consiguiente inflamación. Consulte Video Resumen en http://links.lww.com/DCR/C175 . (Traducción-Dr. Ingrid Melo ).

TCF7L1 Regulates LGR5 Expression in Colorectal Cancer Cells.

Mutations in components of the Wnt/ß-catenin signaling pathway drive colorectal cancer (CRC), in part, by deregulating expression of genes controlled by the T-cell factor (TCF) family of transcription factors. TCFs contain a conserved DNA binding domain that mediates association with TCF binding elements (TBEs) within Wnt-responsive DNA elements (WREs). Intestinal stem cell marker, leucine-rich-repeat containing G-protein-coupled receptor 5 (LGR5), is a Wnt target gene that has been implicated in CRC stem cell plasticity. However, the WREs at the LGR5 gene locus and how TCF factors directly regulate LGR5 gene expression in CRC have not been fully defined. Here, we report that TCF family member, TCF7L1, plays a significant role in regulating LGR5 expression in CRC cells. We demonstrate that TCF7L1 binds to a novel promoter-proximal WRE through association with a consensus TBE at the LGR5 locus to repress LGR5 expression. Using CRISPR activation and interference (CRISPRa/i) technologies to direct epigenetic modulation, we demonstrate that this WRE is a critical regulator of LGR5 expression and spheroid formation capacity of CRC cells. Furthermore, we found that restoring LGR5 expression rescues the TCF7L1-mediated reduction in spheroid formation efficiency. These results demonstrate a role for TCF7L1 in repressing LGR5 gene expression to govern the spheroid formation potential of CRC cells.

Th17 Cell-Derived Amphiregulin Promotes Colitis-Associated Intestinal Fibrosis Through Activation of mTOR and MEK in Intestinal Myofibroblasts.

BACKGROUND & AIMS: Intestinal fibrosis is a significant complication of Crohn's disease (CD). Gut microbiota reactive Th17 cells are crucial in the pathogenesis of CD; however, how Th17 cells induce intestinal fibrosis is still not completely understood. METHODS: In this study, T-cell transfer model with wild-type (WT) and Areg-/- Th17 cells and dextran sulfate sodium (DSS)-induced chronic colitis model in WT and Areg-/- mice were used. CD4+ T-cell expression of AREG was determined by quantitative reverse-transcriptase polymerase chain reaction and enzyme-linked immunosorbent assay. The effect of AREG on proliferation/migration/collagen expression in human intestinal myofibroblasts was determined. AREG expression was assessed in healthy controls and patients with CD with or without intestinal fibrosis. RESULTS: Although Th1 and Th17 cells induced intestinal inflammation at similar levels when transferred into Tcrßxδ-/- mice, Th17 cells induced more severe intestinal fibrosis. Th17 cells expressed higher levels of AREG than Th1 cells. Areg-/- mice developed less severe intestinal fibrosis compared with WT mice on DSS insults. Transfer of Areg-/- Th17 cells induced less severe fibrosis in Tcrßxδ-/- mice compared with WT Th17 cells. Interleukin (IL)6 and IL21 promoted AREG expression in Th17 cells by activating Stat3. Stat3 inhibitor suppressed Th17-induced intestinal fibrosis. AREG promoted human intestinal myofibroblast proliferation, motility, and collagen I expression, which was mediated by activating mammalian target of rapamycin and MEK. AREG expression was increased in intestinal CD4+ T cells in fibrotic sites compared with nonfibrotic sites from patients with CD. CONCLUSIONS: These findings reveal that Th17-derived AREG promotes intestinal fibrotic responses in experimental colitis and human patients with CD. Thereby, AREG might serve as a potential therapeutic target for fibrosis in CD.

Transcriptome Analyses Identify Deregulated MYC in Early Onset Colorectal Cancer.

Despite a global decrease in colorectal cancer (CRC) incidence, the prevalence of early-onset colorectal cancer (EOCRC), or those occurring in individuals before the age of 50, has steadily increased over the past several decades. When compared to later onset colorectal cancer (LOCRC) in individuals over 50, our understanding of the genetic and molecular underpinnings of EOCRCs is limited. Here, we conducted transcriptomic analyses of patient-matched normal colonic segments and tumors to identify gene expression programs involved in carcinogenesis. Amongst differentially expressed genes, we found increased expression of the c-MYC proto-oncogene (MYC) and its downstream targets in tumor samples. We identified tumors with high and low differential MYC expression and found patients with high-MYC tumors were older and overweight or obese. We also detected elevated expression of the PVT1 long-non-coding RNA (lncRNA) in most tumors and found gains in copy number for both MYC and PVT1 gene loci in 35% of tumors evaluated. Our transcriptome analyses indicate that EOCRC can be sub-classified into groups based on differential MYC expression and suggest that deregulated MYC contributes to CRCs that develop in younger patients.

PIGN spatiotemporally regulates the spindle assembly checkpoint proteins in leukemia transformation and progression.

Phosphatidylinositol glycan anchor biosynthesis class N (PIGN) has been linked to the suppression of chromosomal instability. The spindle assembly checkpoint complex is responsible for proper chromosome segregation during mitosis to prevent chromosomal instability. In this study, the novel role of PIGN as a regulator of the spindle assembly checkpoint was unveiled in leukemic patient cells and cell lines. Transient downregulation or ablation of PIGN resulted in impaired mitotic checkpoint activation due to the dysregulated expression of spindle assembly checkpoint-related proteins including MAD1, MAD2, BUBR1, and MPS1. Moreover, ectopic overexpression of PIGN restored the expression of MAD2. PIGN regulated the spindle assembly checkpoint by forming a complex with the spindle assembly checkpoint proteins MAD1, MAD2, and the mitotic kinase MPS1. Thus, PIGN could play a vital role in the spindle assembly checkpoint to suppress chromosomal instability associated with leukemic transformation and progression.

COLQ and ARHGAP15 are Associated with Diverticular Disease and are Expressed in the Colon.

BACKGROUND: Diverticular disease is a common but poorly understood disease of the gastrointestinal tract. Recent studies have identified several single nucleotide polymorphisms (SNPs) that are associated with diverticular disease. MATERIALS AND METHODS: The genotypes of three SNPs (rs4662344 in ARHGAP15, rs7609897 in COLQ, and rs67153654 in FAM155A) were identified by Taqman assay in 204 patients with diverticular disease. Clinical characteristics were obtained from the medical record to study association with genotype. To evaluate gene expression in colon tissue, qPCR was performed on 24 patients with diverticulitis, and COLQ was localized using immunohistochemistry. RESULTS: The ARHGAP15 and COLQ SNPs were significantly associated with both diverticular disease and specifically diverticulitis, while the FAM155A was not associated with either. No association was found with clinical disease characteristics. Heterozygous genotypes at the ARHGAP15 SNP was associated with lower ARHGAP15 expression in colon tissues. COLQ protein localized to the myenteric plexus in the colon. CONCLUSIONS: This study confirmed association of the ARHGAP15 and COLQ SNPs with diverticular disease in our patients but could not confirm FAM155A SNP association. Neither of these SNPs appeared to associate with more severe disease, but genotype at the ARHGAP15 SNP did impact expression of ARHGAP15 in the colon. Additionally, this study is the first to localize COLQ in the colon. Its presence in the myenteric nervous system suggests COLQ SNP variants may contribute to diverticular disease by altering motility.

CD163L1+CXCL10+ Macrophages are Enriched Within Colonic Lamina Propria of Diverticulitis Patients.

BACKGROUND: Inflammation of diverticula, which are outpouchings of the colonic bowl wall, causes diverticulitis. Severe cases of diverticulitis require surgical intervention. Through RNA-seq analysis of intestinal tissues, we previously found that the innate immune response was deregulated in surgical diverticulitis patients. In that study, pro-inflammatory and macrophage markers were differentially expressed in the colons of diverticulitis versus control patients. Here we investigate CD163L1+ macrophages and the pro-inflammatory chemokine, CXCL10, in diverticulitis. MATERIALS AND METHODS: We assessed tissue from an uninvolved area adjacent to a region of the sigmoid colon chronically affected by diverticulitis and performed Spearman's correlation on transcripts associated with macrophage signaling. We identified altered CD163L1 and CXCL10 gene expression levels that we confirmed by RT-qPCR analysis on an independent cohort of diverticulitis patients and controls. We used immunofluorescence microscopy to localize CD163L1+ macrophages and CXCL10 levels in intestinal tissue and ELISA to measure CXCL10 levels in patient serum. RESULTS: We found a positive correlation between intestinal CD163L1 and CXCL10 gene expression and an increased number of CD163L1+ macrophages in the sigmoid colons of diverticulitis patients relative to controls (P = 0.036). Macrophages at the apices of colonic crypts expressed the chemokine CXCL10. Correspondingly, these diverticulitis patients also displayed heightened CXCL10 levels in their serum (P = 0.007). CONCLUSIONS: We identified a novel population of CD163L1+CXCL10+ macrophages in the colonic crypts of diverticulitis patients and demonstrated increased expression of serum CXCL10 in these patients. CXCL10 may serve as a prognostic biomarker to aid in clinical decision making for diverticulitis patients.

Impairment of Tissue-Resident Mesenchymal Stem Cells in Chronic Ulcerative Colitis and Crohn's Disease.

BACKGROUND AND AIMS: Little is known about the presence and function of tissue-resident mesenchymal stem cells [MtSCs] within the gastrointestinal mucosa in health and inflammatory bowel disease [IBD]. The contribution of MtSCs to the generation of inflammatory fibroblasts during IBD is also poorly understood. We hypothesized that IBD-MtSCs are impaired and contribute to the generation of the pathological myofibroblasts in IBD. METHODS: In a cohort of clinically and endoscopically active IBD patients and normal controls, we used quantitative RT-PCR and stem cell differentiation assays, as well as confocal microscopy, to characterize MtSCs. RESULTS: Expression of two stem cell markers, Oct4 and ALDH1A, was increased in the inflamed IBD colonic mucosa and correlated with an increase of the mesenchymal lineage marker Grem1 in ulcerative colitis [UC], but not Crohn's disease [CD]. Increased proliferation and aberrant differentiation of Oct4+Grem1+ MtSC-like cells was observed in UC, but not in CD colonic mucosa. In contrast to normal and UC-derived MtSCs, CD-MtSCs lose their clonogenic and most of their differentiation capacities. Our data also suggest that severe damage to these cells in CD may account for the pathological PD-L1low phenotype of CD myofibroblasts. In contrast, aberrant differentiation of MtSCs appears to be involved in the appearance of pathological partially differentiated PD-L1high myofibroblasts within the inflammed colonic mucosa in UC. CONCLUSION: Our data show, for the first time, that the progenitor functions of MtSCs are differentially impaired in CD vs UC, providing a scientific rationale for the use of allogeneic MSC therapy in IBD, and particularly in CD.

Clinical and Genetic Factors Impact Time to Surgical Recurrence After Ileocolectomy for Crohn's Disease.

OBJECTIVE: The aim of this study was to evaluate factors associated with time to surgical recurrence after Crohn's ileocolectomy. SUMMARY BACKGROUND DATA: The most common surgery performed for Crohn's disease is ileocolectomy. Identifying patients at high risk for surgical recurrence may assist with medical and surgical decision-making. METHODS: Data were obtained from 409 patients with Crohn's disease (CD) who had undergone ≥1 ileocolectomies at Penn State Hershey Medical Center. Six single-nucleotide polymorphisms (SNPs) associated with CD were evaluated in these patients: rs2076756, rs2066844, and rs2066845 in NOD2, rs4958847 and rs13361189 in IRGM, and rs2241880 in ATG16L1. Genotype and clinical factors were analyzed to determine associations with time to recurrent ileocolectomy. A subgroup analysis was performed on 241 patients naïve to biologics before initial ileocolectomy to assess the effect of biologic therapy on time to recurrent surgery. RESULTS: There were 286 patients who underwent a single ileocolectomy, whereas 123 required multiple ileocolectomies. Ileocolonic involvement [hazard ratio (HR) 1.90, 95% confidence interval (CI) 1.21-3.00, P = 0.006] and rs2066844 in NOD2 (HR 1.8, 95% CI 1.17-2.77, P = 0.007) were associated with decreased time to surgical recurrence by multivariate analysis. In patients naïve to preoperative biologics, the initiation of postoperative biologics was associated with a 40% decreased incidence of surgical recurrence (HR 0.60, CI 0.39-0.93, P = 0.02) over time. CONCLUSIONS: Ileocolonic distribution of disease and the rs2066844 SNP in NOD2 are associated with shorter time to recurrent ileocolectomy. The initiation of postoperative biologics in naïve patients was associated with a reduced incidence of recurrence over time.

Transcriptomic analysis of ileal tissue from Crohn's disease patients identifies extracellular matrix genes that distinguish individuals by age at diagnosis.

Crohn's disease (CD) is a debilitating gastrointestinal (GI) disorder that can impact the entirety of the GI tract. While substantial progress has been made in the medical management of CD, it remains incurable, frequently relapses, and is a significant financial and medical burden. The pathophysiology of CD is not well understood, but it is thought to arise in genetically susceptible individuals upon an environmental insult. Further elucidation of the disease etiology promises to expose additional therapeutic avenues, with the hope of reducing the burden of CD. One approach to understanding disease pathophysiology is to identify clinically relevant molecular disease subsets by using transcriptomics. In this report, we use hierarchical clustering of the ileal transcriptomes of 34 patients and identify two CD subsets. Clinically, these clusters differed in the age of the patients at CD diagnosis, suggesting that age of onset affects disease pathophysiology. The clusters were segregated by three major gene ontology categories: developmental processes, ion homeostasis, and the immune response. Of the genes constituting the immune system category, expression of extracellular matrix-associated genes, COL4A1, S100A9, ADAMTS2, SERPINE1, and FCN1, exhibits the strongest correlation with an individual's age at CD diagnosis. Together these findings demonstrate that transcriptional profiling is a powerful approach to subclassify CD patients.

Dichotomous role of the human mitochondrial Na+/Ca2+/Li+ exchanger NCLX in colorectal cancer growth and metastasis.

Despite the established role of mitochondria in cancer, the mechanisms by which mitochondrial Ca2+ (mtCa2+) regulates tumorigenesis remain incompletely understood. The crucial role of mtCa2+ in tumorigenesis is highlighted by altered expression of proteins mediating mtCa2+ uptake and extrusion in cancer. Here, we demonstrate decreased expression of the mitochondrial Na+/Ca2+/Li+ exchanger NCLX (SLC8B1) in human colorectal tumors and its association with advanced-stage disease in patients. Downregulation of NCLX causes mtCa2+ overload, mitochondrial depolarization, decreased expression of cell-cycle genes and reduced tumor size in xenograft and spontaneous colorectal cancer mouse models. Concomitantly, NCLX downregulation drives metastatic spread, chemoresistance, and expression of epithelial-to-mesenchymal, hypoxia, and stem cell pathways. Mechanistically, mtCa2+ overload leads to increased mitochondrial reactive oxygen species, which activate HIF1α signaling supporting metastasis of NCLX-null tumor cells. Thus, loss of NCLX is a novel driver of metastasis, indicating that regulation of mtCa2+ is a novel therapeutic approach in metastatic colorectal cancer.

Colorectal cancer is the second largest cause of cancer deaths worldwide. Even in cases where the cancer is diagnosed and treated early, cells can sometimes survive treatment and spread to other organs. Once the cancer has spread, the survival rate is less than 15%. Mitochondria are compartments in the cell that produce energy, and they play an important role in supporting the rapid growth of cancer cells. The levels of calcium ions in mitochondria control how they produce energy, a process that is altered in cancer cells. To better understand how calcium ions influence colorectal cancer growth, Pathak, Gueguinou et al. studied a protein called NCLX, which controls calcium levels by pumping them out of the mitochondria. Two mouse strains that were used to study what happens if NCLX is missing. The first strain was genetically modified to disable the gene for NCLX and then exposed to carcinogens. The second strain was injected with colorectal cancer cells from a human tumor that were lacking NCLX. In both strains, the tumors that formed were smaller than in mice with NCLX. However, the human cancer cells in the second model were more likely to spread to other organs. This is likely because the build-up of calcium ions in the mitochondria of mice lacking NCLX led to an increase in the production of hypoxia-inducible factor-1a, a protein that is a common driver of cancer spread. Pathak, Gueguinou et al. demonstrated how NCLX can affect colorectal cancer progression. It suggests that it may have opposing effects during early and late-stage colorectal cancer, encouraging tumor growth but also decreasing the spread to other organs. Further research could help refine treatments at different stages of the disease.

The mRNA-binding protein IGF2BP1 maintains intestinal barrier function by up-regulating occludin expression.

Insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) is an mRNA-binding protein that has an oncofetal pattern of expression. It is also expressed in intestinal tissue, suggesting that it has a possible role in intestinal homeostasis. To investigate this possibility, here we generated Villin CreERT2:Igf2bp1flox/flox mice, which enabled induction of an IGF2BP1 knockout specifically in intestinal epithelial cells (IECs) of adult mice. Using gut barrier and epithelial permeability assays and several biochemical approaches, we found that IGF2BP1 ablation in the adult intestinal epithelium causes mild active colitis and mild-to-moderate active enteritis. Moreover, the IGF2BP1 deletion aggravated dextran sodium sulfate-induced colitis. We also found that IGF2BP1 removal compromises barrier function of the intestinal epithelium, resulting from altered protein expression at tight junctions. Mechanistically, IGF2BP1 interacted with the mRNA of the tight-junction protein occludin (Ocln), stabilizing Ocln mRNA and inducing expression of occludin in IECs. Furthermore, ectopic occludin expression in IGF2BP1-knockdown cells restored barrier function. We conclude that IGF2BP1-dependent regulation of occludin expression is an important mechanism in intestinal barrier function maintenance and in the prevention of colitis.

Clinical and Genetic Factors Associated With Complications After Crohn's Ileocolectomy.

BACKGROUND: Ileocolectomy is the most common surgery performed for Crohn's disease, and postoperative complications occur frequently. There has been minimal evaluation of complications after ileocolectomy as a function of both clinical and genetic factors. OBJECTIVE: The purpose of this study was to evaluate both genetic and clinical factors associated with complications after Crohn's ileocolectomy. DESIGN: This was a retrospective clinical and genetic cohort study. SETTINGS: This study was conducted at a high-volume tertiary care center. PATIENTS: We identified 269 patients with Crohn's disease who had undergone 287 ileocolectomies at our institution between July 2008 and October 2018. MAIN OUTCOME MEASURES: We measured the association of complications with a combination of clinical factors and 6 Crohn's-associated single nucleotide polymorphisms in NOD2 (rs2076756, rs2066844, and rs2066845), IRGM (rs4958847 and rs13361189), and ATG16L1 (rs2241880). RESULTS: There were 86 ileocolectomies of 287 (30%) with complications requiring intervention. The single nucleotide polymorphism rs13361189 in the gene IRGM was significantly associated with complications on univariate and multivariate analysis. There were 61 patients with a variant at the rs13361189 single nucleotide polymorphism and 26 of them had complications, although only 55 of the 208 wild-type patients had complications (43% vs 26%; OR = 2.1; p = 0.02). Other significant factors associated with complication after ileocolectomy were open surgery, placement of a proximal ileostomy, and a greater perioperative decrease in hematocrit. LIMITATIONS: This study was limited by its retrospective design and inherent selection bias. CONCLUSIONS: In addition to clinical risk factors, the rs13361189 single nucleotide polymorphism in the IRGM gene was independently associated with complications after ileocolectomy for Crohn's disease. The use of such genetic determinants may identify patients at increased risk for surgical complications after ileocolectomy. See Video Abstract at http://links.lww.com/DCR/B124. FACTORES CLÍNICOS Y GENÉTICOS ASOCIADOS CON COMPLICACIONES DESPUÉS DE LA ILEOCOLECTOMÍA DE CROHN: La ileocolectomía es la cirugía más común realizada para la enfermedad de Crohn y con frecuencia ocurren complicaciones postoperatorias. Ha habido una evaluación mínima de complicaciones después de la ileocolectomía, en función de factores clínicos y genéticos.Evaluar factores genéticos y clínicos asociados con complicaciones, después de la ileocolectomía por Crohn.Estudio retrospectivo de cohorte clínico y genético.Este estudio se realizó en un centro de atención terciaria de alto volumen.Identificamos a 269 pacientes con enfermedad de Crohn, sometidos a 287 ileocolectomías en nuestra institución, entre julio de 2008 y octubre de 2018.La asociación de complicaciones con una combinación de factores clínicos y seis polimorfismos de un solo nucleótido asociados a Crohn en NOD2 (rs2076756, rs2066844 y rs2066845), IRGM (rs4958847 y rs13361189) y ATG16L1 (rs2241880).Hubieron 86 ileocolectomías en 287 (30%) pacientes con complicaciones que requirieron intervención. El polimorfismo de un solo nucleótido rs13361189 en el gen IRGM se asoció significativamente con complicaciones en el análisis univariado y multivariado. Hubieron 61 pacientes con una variante en el polimorfismo de un solo nucleótido rs13361189 y 26 de ellos tuvieron complicaciones, mientras que solo 55 de los 208 pacientes de tipo salvaje (WT) tuvieron complicaciones (43% vs 26%, OR 2.1, p = 0.02). Otros factores significativos asociados con las complicaciones después de la ileocolectomía fueron, la cirugía abierta, la colocación de una ileostomía proximal y una mayor disminución perioperatoria del hematocrito.Este estudio estuvo limitado por su diseño retrospectivo y sesgo de selección inherente.Además de los factores de riesgo clínicos, el polimorfismo de un solo nucleótido rs13361189 en el gen IRGM se asoció independientemente con complicaciones después de la ileocolectomía, para la enfermedad de Crohn. El uso de tales determinantes genéticos puede identificar a los pacientes con mayor riesgo de complicaciones quirúrgicas, después de la ileocolectomía. Consulte Video Resumen en http://links.lww.com/DCR/B124.