MEX3A regulates Lgr5+ stem cell maintenance in the developing intestinal epithelium.

Intestinal stem cells (ISCs) fuel the lifelong self-renewal of the intestinal tract and are paramount for epithelial repair. In this context, the Wnt pathway component LGR5 is the most consensual ISC marker to date. Still, the effort to better understand ISC identity and regulation remains a challenge. We have generated a Mex3a knockout mouse model and show that this RNA-binding protein is crucial for the maintenance of the Lgr5+ ISC pool, as its absence disrupts epithelial turnover during postnatal development and stereotypical organoid maturation ex vivo. Transcriptomic profiling of intestinal crypts reveals that Mex3a deletion induces the peroxisome proliferator-activated receptor (PPAR) pathway, along with a decrease in Wnt signalling and loss of the Lgr5+ stem cell signature. Furthermore, we identify PPARγ activity as a molecular intermediate of MEX3A-mediated regulation. We also show that high PPARγ signalling impairs Lgr5+ ISC function, thus uncovering a new layer of post-transcriptional regulation that critically contributes to intestinal homeostasis.

Mesothelin Expression Is Not Associated with the Presence of Cancer Stem Cell Markers SOX2 and ALDH1 in Ovarian Cancer.

Mesothelin (MSLN) overexpression (OE) is a frequent finding in ovarian carcinomas and increases cell survival and tumor aggressiveness. Since cancer stem cells (CSCs) contribute to pathogenesis, chemoresistance and malignant behavior in ovarian cancer (OC), we hypothesized that MSLN expression could be creating a favorable environment that nurtures CSCs. In this study, we analyzed the expression of MSLN and CSC markers SOX2 and ALDH1 by immunohistochemistry (IHC) in different model systems: primary high-grade serous carcinomas (HGSCs) and OC cell lines, including cell lines that were genetically engineered for MSLN expression by either CRISPR-Cas9-mediated knockout (Δ) or lentivirus-mediated OE. Cell lines, wild type and genetically engineered, were evaluated in 2D and 3D culture conditions and xenografted in nude mice. We observed that MSLN was widely expressed in HGSC, and restricted expression was observed in OC cell lines. In contrast, SOX2 and ALDH1 expression was limited in all tissue and cell models. Most importantly, the expression of CSC markers was independent of MSLN expression, and manipulation of MSLN expression did not affect CSC markers. In conclusion, MSLN expression is not involved in driving the CSC phenotype.

Digital image analysis of multiplex fluorescence IHC in colorectal cancer recognizes the prognostic value of CDX2 and its negative correlation with SOX2.

Flourescence-based multiplex immunohistochemistry (mIHC) combined with multispectral imaging and digital image analysis (DIA) is a quantitative high-resolution method for determination of protein expression in tissue. We applied this method for five biomarkers (CDX2, SOX2, SOX9, E-cadherin, and ß-catenin) using tissue microarrays of a Norwegian unselected series of primary colorectal cancer. The data were compared with previously obtained chromogenic IHC data of the same tissue cores, visually assessed by the Allred method. We found comparable results between the methods, although confirmed that DIA offered improved resolution to differentiate cases with high and low protein expression. However, we experienced inherent challenges with digital image analysis of membrane staining, which was better assessed visually. DIA and mIHC enabled quantitative analysis of biomarker coexpression on the same tissue section at the single-cell level, revealing a strong negative correlation between the differentiation markers CDX2 and SOX2. Both methods confirmed known prognostic associations for CDX2, but DIA improved data visualization and detection of clinicopathological and biological associations. In summary, mIHC combined with DIA is an efficient and reliable method to evaluate protein expression in tissue, here shown to recapitulate and improve detection of known clinicopathological and survival associations for the emerging biomarker CDX2, and is therefore a candidate approach to standardize CDX2 detection in pathology laboratories.

A panel of intestinal differentiation markers (CDX2, GPA33, and LI-cadherin) identifies gastric cancer patients with favourable prognosis.

BACKGROUND: Gastric cancer is the fifth most common cancer and the third cause of global cancer mortality. CDX2 is an intestinal differentiation marker with prognostic value in gastric cancer and transcriptionally regulates the expression of glycoprotein A33 (GPA33) and liver intestine cadherin (LI-cadherin). METHODS: This study evaluated the clinical significance of the combined expression of CDX2 and its targets GPA33 and LI-cadherin in gastric cancer by fluorescence-based multiplex immunohistochemistry together with digital image analysis and chromogenic immunohistochemistry in 329 gastric cancer samples arranged in tissue microarrays. Additionally, publicly available RNA-seq expression data from 354 gastric cancer samples from the TCGA database were used to validate the immunohistochemistry results. RESULTS: Expression of the three markers (CDX2, GPA33, and LI-cadherin) was strongly correlated, defining an intestinal differentiation panel. Low or negative protein expression of the intestinal differentiation panel identified patients with particularly poor overall survival, irrespective of the methodology used, and was validated in the independent series at the RNA-seq level. CONCLUSIONS: Expression of the intestinal differentiation panel (CDX2, GPA33, and LI-cadherin) defines a set of biomarkers with a strong biological rationale and favourable impact for prognostication of gastric cancer patients.

Precise integration of inducible transcriptional elements (PrIITE) enables absolute control of gene expression.

Tetracycline-based inducible systems provide powerful methods for functional studies where gene expression can be controlled. However, the lack of tight control of the inducible system, leading to leakiness and adverse effects caused by undesirable tetracycline dosage requirements, has proven to be a limitation. Here, we report that the combined use of genome editing tools and last generation Tet-On systems can resolve these issues. Our principle is based on precise integration of inducible transcriptional elements (coined PrIITE) targeted to: (i) exons of an endogenous gene of interest (GOI) and (ii) a safe harbor locus. Using PrIITE cells harboring a GFP reporter or CDX2 transcription factor, we demonstrate discrete inducibility of gene expression with complete abrogation of leakiness. CDX2 PrIITE cells generated by this approach uncovered novel CDX2 downstream effector genes. Our results provide a strategy for characterization of dose-dependent effector functions of essential genes that require absence of endogenous gene expression.

Mucins and Truncated O-Glycans Unveil Phenotypic Discrepancies between Serous Ovarian Cancer Cell Lines and Primary Tumours.

Optimal research results rely on the selection of cellular models capable of recapitulating the characteristics of primary tumours from which they originate. The expression of mucins (MUC16 and MUC1) and truncated O-glycans (Tn, STn and T) represents a characteristic footprint of serous ovarian carcinomas (SOCs). Therefore, selecting ovarian cancer (OVCA) cell lines that reflect this phenotype is crucial to explore the putative biological role of these biomarkers in the SOC setting. Here, we investigated a panel of OVCA cell lines commonly used as SOC models, and tested whether, when cultured in 2D and 3D conditions, these recapitulate the mucin and O-glycan expression profiles of SOCs. We further explored the role of truncating the O-glycosylation capacity in OVCAR3 cells through knockout of the COSMC chaperone, using in vitro and in vivo assays. We found that the majority of OVCA cell lines of serous origin do not share the mucin and truncated O-glycan footprint of SOCs, although 3D cultures showed a higher resemblance. We also found that genetic truncation of the O-glycosylation capacity of OVCAR3 cells did not enhance oncogenic features either in vitro or in vivo. This study underscores the importance of well-characterized cellular models to study specific features of ovarian cancer.

Helicobacter pylori chronic infection and mucosal inflammation switches the human gastric glycosylation pathways.

Helicobacter pylori exploits host glycoconjugates to colonize the gastric niche. Infection can persist for decades promoting chronic inflammation, and in a subset of individuals lesions can silently progress to cancer. This study shows that H. pylori chronic infection and gastric tissue inflammation result in a remodeling of the gastric glycophenotype with increased expression of sialyl-Lewis a/x antigens due to transcriptional up-regulation of the B3GNT5, B3GALT5, and FUT3 genes. We observed that H. pylori infected individuals present a marked gastric local pro-inflammatory signature with significantly higher TNF-α levels and demonstrated that TNF-induced activation of the NF-kappaB pathway results in B3GNT5 transcriptional up-regulation. Furthermore, we show that this gastric glycosylation shift, characterized by increased sialylation patterns, favors SabA-mediated H. pylori attachment to human inflamed gastric mucosa. This study provides novel clinically relevant insights into the regulatory mechanisms underlying H. pylori modulation of host glycosylation machinery, and phenotypic alterations crucial for life-long infection. Moreover, the biosynthetic pathways here identified as responsible for gastric mucosa increased sialylation, in response to H. pylori infection, can be exploited as drug targets for hindering bacteria adhesion and counteract the infection chronicity.

A novel monoclonal antibody to a defined peptide epitope in MUC16.

The MUC16 mucin is overexpressed and aberrantly glycosylated in ovarian carcinomas. Immunodetection of circulating MUC16 is one of the most used cancer biomarker assays, but existing antibodies to MUC16 fail to distinguish normal and aberrant cancer glycoforms. Although all antibodies react with the tandem-repeat region, their epitopes appear to be conformational dependent and not definable by a short peptide. Aberrant glycoforms of MUC16 may constitute promising targets for diagnostic and immunotherapeutic intervention, and it is important to develop well-defined immunogens for induction of potent MUC16 immunity. Here, we developed a MUC16 vaccine based on a 1.7TR (264 aa) expressed in Escherichia coli and in vitro enzymatically glycosylated to generate the aberrant cancer-associated glycoform Tn. This vaccine elicited a potent serum IgG response in mice and we identified two major immunodominant linear peptide epitopes within the tandem repeat. We developed one monoclonal antibody, 5E11, reactive with a minimum epitope with the sequence FNTTER. This sequence contains potential N- and O-glycosylation sites and, interestingly, glycosylation blocked binding of 5E11. In immunochemistry of ovarian benign and cancer lesions, 5E11 showed similar reactivity as traditional MUC16 antibodies, suggesting that the epitope is not efficiently glycosylated. The study provides a vaccine design and immunodominant MUC16 TR epitopes.

CDX2 homeoprotein is involved in the regulation of ST6GalNAc-I gene in intestinal metaplasia.

De novo expression of Sialyl-Tn (STn) antigen is one of the most common features of intestinal metaplasia (IM) and gastric carcinomas, and its biosynthesis has been mostly attributed to ST6GalNAc-I activity. However, the regulation of this glycosyltransferase expression is not elucidated. In IM lesions and in the intestine, CDX2 homeobox transcription factor is co-expressed with STn and ST6GalNAc-I. We therefore hypothesized that CDX2 might induce STn expression by positive regulation of ST6GalNAc-I. We showed that ST6GalNAc-I transcript levels and CDX2 have a coordinated expression upon Caco-2 in vitro differentiation, and overexpression of CDX2 in MKN45 gastric cells increases ST6GalNAc-I transcript levels. Nine putative CDX-binding sites in the ST6GalNAc-I-regulatory sequence were identified and analyzed by chromatin immunoprecipitation in Caco-2 cells and in IM. The results showed that CDX2 protein is recruited to all regions, being the most proximal sites preferentially occupied in vivo. Luciferase assays demonstrated that CDX2 is able to transactivate ST6GalNac-I-regulatory region. The induction was stronger for the regions mapped in the neighbourhood of ATG start codon and site-directed mutagenesis of these sites confirmed their importance. In conclusion, we show that CDX2 transcriptionally regulates ST6GalNAc-I gene expression, specifically in the preneoplastic IM lesion.

Differentiation reprogramming in gastric intestinal metaplasia and dysplasia: role of SOX2 and CDX2.

AIMS: Intestinal metaplasia (IM), which results from de-novo expression of CDX2, and dysplasia are precursor lesions of gastric cancer that are associated with an increased risk for cancer development. There is much evidence suggesting a role for the transcription factor SOX2 in gastric differentiation. The aim of this study was to attempt to establish the relationship of SOX2 with CDX2 and with the differentiation reprogramming that characterizes gastric carcinogenesis, to assess their involvement in IM and dysplasia. METHODS AND RESULTS: Characterization of gastric (SOX2, MUC5AC, and MUC6) and intestinal (CDX2 and MUC2) markers in normal gastric mucosa, in 55 foci of IM and in 26 foci of dysplasia, was performed by immunohistochemistry. SOX2 was expressed in the normal gastric mucosa, in the presumptive stem cell compartment, and was maintained in 7% of the complete (MUC5AC-negative) and 85% of the incomplete (MUC5AC-positive) IM subtypes. Twelve per cent of the dysplastic lesions expressed SOX2, and the association with MUC5AC was lost. CDX2 was present in all IMs and dysplastic lesions. CONCLUSIONS: SOX2 is associated with gastric differentiation in incomplete IM and is lost in the progression to dysplasia, whereas CDX2 is acquired de novo in IM and maintained in dysplasia. This suggests that the balance between gastric and intestinal differentiation programmes impacts on the gastric carcinogenesis cascade progression.

Increase in genogroup II.4 norovirus host spectrum by CagA-positive Helicobacter pylori infection.

BACKGROUND: Noroviruses (NoVs) represent a considerable public health burden. Despite their enormous genetic diversity, most outbreaks are due to the single GII.4 genotype, but the reasons for this are poorly understood. NoVs use histo-blood group antigens (HBGAs) as attachment factors. Since HBGAs are present in saliva, binding of strains to saliva is commonly used as a surrogate for recognition of the gut surface by specific strains, although the relationship between saliva and gut tissue expression of HBGAs is not well defined. METHODS: The presence of fucosylated HBGAs in saliva and stomach biopsy specimens, as well as that of genogroup I.1 and genogroup II.4 virus-like particles, were compared in a series of 109 donors from Portugal. RESULTS: An overall good concordance between HBGA expression in saliva and stomach surface mucosa was observed. However, unexpected mucosal expression of α(1,2)fucosylated epitopes in nonsecretor individuals was frequently detected, allowing for GII.4 attachment. Although all individuals were infected with Helicobacter pylori, abnormal expression of α(1,2)fucosylated motifs and binding of GII.4 virus-like particles in nonsecretors' mucosa were associated with positivity for the H. pylori CagA virulence factor. CONCLUSIONS: Infection by CagA-positive H. pylori induces expression of GII.4 attachment factors in nonsecretors' mucosa, expanding the host range of these strains and thereby possibly contributing to their epidemiological dominance.

Characterization of binding epitopes of CA125 monoclonal antibodies.

The most used cancer serum biomarker is the CA125 immunoassay for ovarian cancer that detects the mucin glycoprotein MUC16. Several monoclonal antibodies (mAbs) including OC125 and M11 are used in CA125 assays. However, despite considerable efforts, our knowledge of the molecular characteristics of the recognized epitopes and the role played by glycosylation has remained elusive. Here a comprehensive set of recombinant MUC16 tandem repeats (TRs) expressed in glycoengineered mammalian cells and E. coli, together with overlapping peptides, was used to probe antigen-binding epitopes. We present a complete analysis of N- and O-glycosylation sites of a MUC16 TR expressed in CHO cells and demonstrate that neither N- nor O-glycosylation appear to substantially influence binding of OC125 and M11 mAbs. A series of successive N- and C-terminal truncations of a MUC16 TR construct expressed in E. coli narrowed down the epitopes for OC125 and M11 to a segment containing parts of two consecutive SEA domains with a linker. Thus, a complete SEA domain is not required. These findings suggest that binding epitopes of mAbs OC125 and M11 are dependent on conformation but not on glycosylation. The availability of recombinant TR constructs with and without aberrant glycosylation now opens the way for vaccine studies.

Immunohistochemical molecular phenotypes of gastric cancer based on SOX2 and CDX2 predict patient outcome.

BACKGROUND: Gastric cancer remains a serious health concern worldwide. Patients would greatly benefit from the discovery of new biomarkers that predict outcome more accurately and allow better treatment and follow-up decisions. Here, we used a retrospective, observational study to assess the expression and prognostic value of the transcription factors SOX2 and CDX2 in gastric cancer. METHODS: SOX2, CDX2, MUC5AC and MUC2 expression were assessed in 201 gastric tumors by immunohistochemistry. SOX2 and CDX2 expression were crossed with clinicopathological and follow-up data to determine their impact on tumor behavior and outcome. Moreover, SOX2 locus copy number status was assessed by FISH (N = 21) and Copy Number Variation Assay (N = 62). RESULTS: SOX2 was expressed in 52% of the gastric tumors and was significantly associated with male gender, T stage and N stage. Moreover, SOX2 expression predicted poorer patient survival, and the combination with CDX2 defined two molecular phenotypes, SOX2+CDX2- versus SOX2-CDX2+, that predict the worst and the best long-term patients' outcome. These profiles combined with clinicopathological parameters stratify the prognosis of patients with intestinal and expanding tumors and in those without signs of venous invasion. Finally, SOX2 locus copy number gains were found in 93% of the samples reaching the amplification threshold in 14% and significantly associating with protein expression. CONCLUSIONS: We showed, for the first time, that SOX2 combined with CDX2 expression profile in gastric cancer segregate patients into different prognostic groups, complementing the clinicopathological information. We further demonstrate a molecular mechanism for SOX2 expression in a subset of gastric cancer cases.

Glycoproteomic analysis of serum from patients with gastric precancerous lesions.

Gastric cancer is preceded by a carcinogenesis pathway that includes gastritis caused by Helicobacter pylori infection, chronic atrophic gastritis that may progress to intestinal metaplasia (IM), dysplasia, and ultimately gastric carcinoma of the more common intestinal subtype. The identification of glycosylation changes in circulating serum proteins in patients with precursor lesions of gastric cancer is of high interest and represents a source of putative new biomarkers for early diagnosis and intervention. This study applies a glycoproteomic approach to identify altered glycoproteins expressing the simple mucin-type carbohydrate antigens T and STn in the serum of patients with gastritis, IM (complete and incomplete subtypes), and control healthy individuals. The immunohistochemistry analysis of the gastric mucosa of these patients showed expression of T and STn antigens in gastric lesions, with STn being expressed only in IM. The serum glycoproteomic analysis using 2D-gel electrophoresis, Western blot, and MALDI-TOF/TOF mass spectrometry led to the identification of circulating proteins carrying these altered glycans. One of the glycoproteins identified was plasminogen, a protein that has been reported to play a role in H. pylori chronic infection of the gastric mucosa and is involved in extracellular matrix modeling and degradation. Plasminogen was further characterized and showed to carry STn antigens in patients with gastritis and IM. These results provide evidence of serum proteins displaying abnormal O-glycosylation in patients with precursor lesions of gastric carcinoma and include a panel of putative targets for the non-invasive clinical diagnosis of individuals with gastritis and IM.

Helicobacter pylori and the BMP pathway regulate CDX2 and SOX2 expression in gastric cells.

Helicobacter pylori infection is the main risk factor for intestinal metaplasia (IM) and gastric cancer development. IM is a pre-neoplastic lesion, induced by the transcription factor CDX2, where the gastric mucosa is converted to an intestinal phenotype. We previously demonstrated that key elements of the bone morphogenetic protein (BMP) pathway co-localize with CDX2 in IM and upregulate CDX2 expression in gastric cell lines. These observations, together with the hypothesis that CDX2 could be repressed by SOX2, led us to test whether H. pylori, through BMPs, SOX2 and CDX2 could participate in a molecular network critical for the development of IM. AGS cells with and without SMAD4 knock-down were co-cultured with H. pylori or BMP2 to assess the expression of BMP pathway members as well as CDX2 and SOX2 by qPCR and western blot. Proximity ligation assay (PLA) was also performed to evaluate SMAD proteins interaction. Immunohistochemistry and western blot were performed in gastric samples from mice infected with Helicobacter spp. to measure Smad4, pSmad1/5/8, Cdx2 and Sox2 expression in vivo. Increased expression and activity of the BMP pathway accompanied by CDX2 upregulation and SOX2 downregulation were observed in AGS cells co-cultured with H. pylori or BMP2. These effects were impaired by downregulation of the BMP pathway. Finally, infected mice present BMP pathway upregulation, focal Cdx2 expression and decreased Sox2. These results provide a novel link between H. pylori infection and the BMP pathway in the regulation of intestinal and gastric-specific genes that might be relevant for gastric IM.

Identification of new cancer biomarkers based on aberrant mucin glycoforms by in situ proximity ligation.

Mucin glycoproteins are major secreted or membrane-bound molecules that, in cancer, show modifications in both the mucin proteins expression and in the O-glycosylation profile, generating some of the most relevant tumour markers in clinical use for decades. Thus far, the identification of these biomarkers has been based on the detection of either the protein or the O-glycan modifications. We therefore aimed to identify the combined mucin and O-glycan features, that is, specific glycoforms, in an attempt to increase specificity of these cancer biomarkers. Using in situ proximity ligation assays (PLA) based on existing monoclonal antibodies directed to MUC1, MUC2, MUC5AC and MUC6 mucins and to cancer-associated carbohydrate antigens Tn, Sialyl-Tn (STn), T, Sialyl-Le(a) (SLe(a)) and Sialyl-Le(x) (SLe(x)) we screened a series of 28 mucinous adenocarcinomas from different locations (stomach, ampulla of Vater, colon, lung, breast and ovary) to detect specific mucin glycoforms. We detected Tn/STn/SLe(a)/SLe(x)-MUC1 and STn/SLe(a)/SLe(x)-MUC2 glycoforms in ≥50% of the cases, with a variable distribution among organs. Some new glycoforms-T/SLe(a)-MUC2, STn/T/SLe(a) SLe(x)-MUC5AC and STn/T/SLe(a)/SLe(x)-MUC6-were identified for the first time in the present study in a variable percentage of cases from different organs. In conclusion, application of the PLA technique allowed sensitive detection of specific aberrant mucin glycoforms in cancer, increasing specificity to the use of antibodies either to the mucin protein backbone or to the O-glycan haptens alone.

CDX2 autoregulation in human intestinal metaplasia of the stomach: impact on the stability of the phenotype.

BACKGROUND AND AIMS: Intestinal metaplasia (IM) is a gastric preneoplastic lesion that appears following Helicobacter pylori infection and confers an increased risk for development of cancer. It is induced by gastric expression of the intestine-specific transcription factor CDX2. The regulatory mechanisms involved in triggering and maintaining gastric CDX2 expression have not been fully elucidated. The Cdx2(+/-) mouse develops intestinal polyps with gastric differentiation and total loss of Cdx2 expression in the absence of structural loss of the second allele, suggesting a regulatory defect. This putative haplo-insufficiency, together with the apparent stability of IM, led to the hypothesis that CDX2 regulates its own expression through an autoregulatory loop in both contexts. METHODS: Gastrointestinal cell lines were co-transfected with wild-type or mutated Cdx2 promoter constructs and CDX2 expression vector for luciferase assays. Transfection experiments were also used to assess endogenous CDX2 autoregulation, evaluated by RT-PCR, qPCR and western blotting. Chromatin immunoprecipitation was performed in a cell line, mouse ileum and human IM. RESULTS: CDX2 binds to and transactivates its own promoter and positively regulates its expression in gastrointestinal human carcinoma cell lines. Furthermore, CDX2 is bound to its promoter in the mouse ileum and in human gastric IM, providing a major contribution to understanding the relevance of this autoregulatory pathway in vivo. CONCLUSION: The results of this study demonstrate another layer of complexity in CDX2 regulation by an effective autoregulatory loop which may have a major impact on the stability of human IM, possibly resulting in the inevitable progression of the gastric carcinogenesis pathway.

A method for Boolean analysis of protein interactions at a molecular level.

Determining the levels of protein-protein interactions is essential for the analysis of signaling within the cell, characterization of mutation effects, protein function and activation in health and disease, among others. Herein, we describe MolBoolean - a method to detect interactions between endogenous proteins in various subcellular compartments, utilizing antibody-DNA conjugates for identification and signal amplification. In contrast to proximity ligation assays, MolBoolean simultaneously indicates the relative abundances of protein A and B not interacting with each other, as well as the pool of A and B proteins that are proximal enough to be considered an AB complex. MolBoolean is applicable both in fixed cells and tissue sections. The specific and quantifiable data that the method generates provide opportunities for both diagnostic use and medical research.

Searching for SARS-CoV-2 in Cancer Tissues: Results of an Extensive Methodologic Approach based on ACE2 and Furin Expression.

SARS-CoV-2 pandemics have been massively characterized on a global scale by the rapid generation of in-depth genomic information. The main entry gate of SARS-CoV-2 in human cells is the angiotensin-converting enzyme 2 (ACE2) receptor. The expression of this protein has been reported in several human tissues, suggesting a correlation between SARS-CoV-2 organotropism and ACE2 distribution. In this study, we selected (a series of) 90 patients who were submitted to surgery for tumor removal between the beginning of the SARS-CoV-2 pandemic and the closure of operating rooms (by the end of March 2020) in two different countries-Portugal and Brazil. We evaluated the expressions of ACE2 and furin (another important factor for virus internalization) in colon (n = 60), gastric (n = 19), and thyroid (n = 11) carcinomas. In a subseries of cases with PCR results for SARS-CoV-2 detection in the peri-operatory window (n = 18), we performed different methodological approaches for viral detections in patient tumor samples. Our results show that colon and gastric carcinomas display favorable microenvironments to SARS-CoV-2 tropism, presenting high expression levels of ACE2 and furin. From the subseries of 18 cases, 11 tested positive via PCR detection performed in tumor blocks; however, a direct association between the ACE2 expression and SARS-CoV-2 infection was not demonstrated in cancer cells using histology-based techniques, such as immunohistochemistry or in situ hybridization. This study raises the possibility of ACE2-mediated viral tropism in cancer tissues to be clarified in future studies.

Helicobacter pylori induces beta3GnT5 in human gastric cell lines, modulating expression of the SabA ligand sialyl-Lewis x.

Chronic Helicobacter pylori infection is recognized as a cause of gastric cancer. H. pylori adhesion to gastric cells is mediated by bacterial adhesins such as sialic acid-binding adhesin (SabA), which binds the carbohydrate structure sialyl-Lewis x. Sialyl-Lewis x expression in the gastric epithelium is induced during persistent H. pylori infection, suggesting that H. pylori modulates host cell glycosylation patterns for enhanced adhesion. Here, we evaluate changes in the glycosylation-related gene expression profile of a human gastric carcinoma cell line following H. pylori infection. We observed that H. pylori significantly altered expression of 168 of the 1,031 human genes tested by microarray, and the extent of these alterations was associated with the pathogenicity of the H. pylori strain. A highly pathogenic strain altered expression of several genes involved in glycan biosynthesis, in particular that encoding beta3 GlcNAc T5 (beta3GnT5), a GlcNAc transferase essential for the biosynthesis of Lewis antigens. beta3GnT5 induction was specific to infection with highly pathogenic strains of H. pylori carrying a cluster of genes known as the cag pathogenicity island, and was dependent on CagA and CagE. Further, beta3GnT5 overexpression in human gastric carcinoma cell lines led to increased sialyl-Lewis x expression and H. pylori adhesion. This study identifies what we believe to be a novel mechanism by which H. pylori modulates the biosynthesis of the SabA ligand in gastric cells, thereby strengthening the epithelial attachment necessary to achieve successful colonization.