NALCN-mediated sodium influx confers metastatic prostate cancer cell invasiveness.

There is growing evidence that ion channels are critically involved in cancer cell invasiveness and metastasis. However, the molecular mechanisms of ion signaling promoting cancer behavior are poorly understood and the complexity of the underlying remodeling during metastasis remains to be explored. Here, using a variety of in vitro and in vivo techniques, we show that metastatic prostate cancer cells acquire a specific Na+ /Ca2+ signature required for persistent invasion. We identify the Na+ leak channel, NALCN, which is overexpressed in metastatic prostate cancer, as a major initiator and regulator of Ca2+ oscillations required for invadopodia formation. Indeed, NALCN-mediated Na+ influx into cancer cells maintains intracellular Ca2+ oscillations via a specific chain of ion transport proteins including plasmalemmal and mitochondrial Na+ /Ca2+ exchangers, SERCA and store-operated channels. This signaling cascade promotes activity of the NACLN-colocalized proto-oncogene Src kinase, actin remodeling and secretion of proteolytic enzymes, thus increasing cancer cell invasive potential and metastatic lesions in vivo. Overall, our findings provide new insights into an ion signaling pathway specific for metastatic cells where NALCN acts as persistent invasion controller.

A Novel Anti-TRPV6 Antibody and Its Application in Cancer Diagnosis In Vitro.

Though the first discovery of TRPV6 channel expression in various tissues took place in the early 2000s, reliable tools for its protein detection in various cells and tissues are still missing. Here we show the generation and validation of rabbit polyclonal anti-TRPV6 channel antibodies (rb79-82) against four epitopes of 15 amino acids. Among them, only one antibody, rb79, was capable of detecting the full-length glycosylated form of the TRPV6 channel at around 100 kDa. The generated antibody was shown to be suitable for all in vitro applications, such as immunoblotting, immunoprecipitation, immunocytochemistry, immunofluorescence, etc. One of the most important applications is immunohistochemistry using the paraffin-embedded sections from cancer resection specimens. Using prostate cancer resection specimens, we have confirmed the absence of the TRPV6 protein in both healthy and benign hyperplasia, as well as its expression and correlation to the prostate cancer grades. Thus, the generated rabbit polyclonal anti-TRPV6 channel antibody rb79 is suitable for all in vitro diagnostic applications and particularly for the diagnosis in clinics using paraffin-embedded sections from patients suffering from various diseases and disorders involving the TRPV6 channel.

How do they stick together? Bacterial adhesins implicated in the binding of bacteria to the human gastrointestinal mucins.

The gastrointestinal mucosal surface is the primary interface between internal host tissues and the vast microbiota. Mucins, key components of mucus, are high-molecular-weight glycoproteins characterized by the presence of many O-linked oligosaccharides to the core polypeptide. They play many biological functions, helping to maintain cellular homeostasis and to establish symbiotic relationships with complex microbiota. Mucin O-glycans exhibit a huge variety of peripheral sequences implicated in the binding of bacteria to the mucosal tissues, thereby playing a key role in the selection of specific species and in the tissue tropism displayed by commensal and pathogenic bacteria. Bacteria have evolved numerous strategies to colonize host mucosae, and among these are modulation of expression of cell surface adhesins which allow bacteria to bind to mucins. However, despite well structurally characterized adhesins and lectins, information on the nature and structure of oligosaccharides recognized by bacteria is still disparate. This review summarizes the current knowledge on the structure of epithelial mucin O-glycans and the interaction between host and commensal or pathogenic bacteria mediated by mucins.

TRPV6 calcium channel translocates to the plasma membrane via Orai1-mediated mechanism and controls cancer cell survival.

Transient receptor potential vanilloid subfamily member 6 (TRPV6) is a highly selective calcium channel that has been considered as a part of store-operated calcium entry (SOCE). Despite its first discovery in the early 2000s, the role of this channel in prostate cancer (PCa) remained, until now, obscure. Here we show that TRPV6 mediates calcium entry, which is highly increased in PCa due to the remodeling mechanism involving the translocation of the TRPV6 channel to the plasma membrane via the Orai1/TRPC1-mediated Ca(2+)/Annexin I/S100A11 pathway, partially contributing to SOCE. The TRPV6 calcium channel is expressed de novo by the PCa cell to increase its survival by enhancing proliferation and conferring apoptosis resistance. Xenografts in nude mice and bone metastasis models confirmed the remarkable aggressiveness of TRPV6-overexpressing tumors. Immunohistochemical analysis of these demonstrated the increased expression of clinical markers such as Ki-67, prostate specific antigen, synaptophysin, CD31, and CD56, which are strongly associated with a poor prognosis. Thus, the TRPV6 channel acquires its oncogenic potential in PCa due to the remodeling mechanism via the Orai1-mediated Ca(2+)/Annexin I/S100A11 pathway.

Alteration or adaptation, the two roads for human gastric mucin glycosylation infected by Helicobacter pylori.

Helicobacter pylori is a Gram-negative bacterium that colonizes the mucus niche of the gastric mucosa and infects more than half of the world's human population. Chronic infection may cause gastritis, duodenal ulcer, intestinal metaplasia or gastric cancer. In the stomach, H. pylori interacts with O-glycans of gastric mucins but the mechanism by which the bacteria succeed in altering the mucosa remains mainly unknown. To better understand the physiopathology of the infection, inhibitory adhesion assays were performed with various O-glycans expressed by human gastric mucins, and topographic expression of gastric mucins MUC5AC and MUC6 was analyzed for healthy uninfected individuals, for infected asymptomatic individuals and for patients infected by H. pylori and having the incomplete type of intestinal metaplasia. The glycosylation of the gastric mucosa of asymptomatic individuals infected by H. pylori was determined and compared with the glycosylation pattern found for patients with the incomplete type of intestinal metaplasia. Results show that H. pylori manages to modulate host's glycosylation during the course of infection in order to create a favorable niche, whereas asymptomatic infected individuals seem to counteract further steps of infection development by adapting their mucus glycosylation.

Claudin 3, 4, and 15 expression in solid tumors of lung adenocarcinoma versus malignant pleural mesothelioma.

Epithelioid malignant pleural mesothelioma (MPM) can easily be confused with lung adenocarcinomas (ACAs). In serous effusion, claudin (cldn) 3 is shown to be useful in the diagnosis of mesothelioma vs ACAs. Cldn15 is reported to be overexpressed in epithelioid mesothelioma and absent in human airway epithelium. The aim was to assess the value of cldn3 and cldn4 compared to that of BerEp4 and thyroid transcription factor-1 (TTF1) in differentiating lung ACA from epithelioid MPM and to examine the expression of cldn15 in these tumors. The expression of cldn3, cldn4, cldn15, BerEp4, and TTF1 was examined by immunohistochemistry in a total of 62 human specimen including 28 epithelioid MPMs and 34 ACAs of the lung. In lung ACA, cldn4 was strongly expressed in all 34 (100%) specimens followed by cldn3 in 33 (97%) of 34. BerEp4 was expressed in 32 (94.1%) of 34. TTF1 reacted for only 20 (58.82%) of 34 cases of lung ACA. In MPM specimens, the expression of cldn3 and4 as well as that of TTF1 was completely absent. In contrast, BerEp4 was focally expressed in 5 (17.85%) of 28 cases of epithelioid MPM. Cldn15 was strongly expressed in 53% pf epithelioid MPMs but also in 50% of lung ACAs. Its expression was moderate in normal pleura and limited in normal lung. Cldn3 and cldn4 appear to be the best performing carcinoma markers in discriminating lung ACA from mesothelioma compared with BerEp4 and TTF1. There is no differential expression of cldn15 between the 2 pathologies. However, the limited cldn15 expression in normal tissues and high expression in tumors make it an attractive candidate for cancer therapy.

Trpv6 channel targeting using monoclonal antibody induces prostate cancer cell apoptosis and tumor regression.

TRPV6 calcium channel is a prospective target in prostate cancer (PCa) since it is not expressed in healthy prostate while its expression increases during cancer progression. Despite the role of TRPV6 in PCa cell survival and apoptotic resistance has been already established, no reliable tool to target TRPV6 channel in vivo and thus to reduce tumor burden is known to date. Here we report the generation of mouse monoclonal antibody mAb82 raised against extracellular epitope of the pore region of the channel. mAb82 inhibited TRPV6 currents by 90% at 24 µg/ml in a dose-dependent manner while decreasing store-operated calcium entry to 56% at only 2.4 µg/ml. mAb82 decreased PCa survival rate in vitro by 71% at 12 µg/ml via inducing cell death through the apoptosis cascade via activation of the protease calpain, following bax activation, mitochondria enlargement, and loss of cristae, Cyt C release, pro-caspase 9 cleavage with the subsequent activation of caspases 3/7. In vivo, mice bearing either PC3Mtrpv6+/+ or PC3Mtrpv6-/-+pTRPV6 tumors were successfully treated with mAb82 at the dose as low as 100 µg/kg resulting in a significant reduction tumor growth by 31% and 90%, respectively. The survival rate was markedly improved by 3.5 times in mice treated with mAb82 in PC3Mtrpv6+/+ tumor group and completely restored in PC3Mtrpv6-/-+pTRPV6 tumor group. mAb82 showed a TRPV6-expression dependent organ distribution and virtually no toxicity in the same way as mAbAU1, a control antibody of the same Ig2a isotype. Overall, our data demonstrate for the first time the use of an anti-TRPV6 monoclonal antibody in vitro and in vivo in the treatment of the TRPV6-expressing PCa tumors.

TRPV6 Channel Is Involved in Pancreatic Ductal Adenocarcinoma Aggressiveness and Resistance to Chemotherapeutics.

Pancreatic ductal adenocarcinoma (PDAC) stands as a highly aggressive and lethal cancer, characterized by a grim prognosis and scarce treatment alternatives. Within this context, TRPV6, a calcium-permeable channel, emerges as a noteworthy candidate due to its overexpression in various cancers, capable of influencing the cell behavior in different cancer entities. Nonetheless, the exact expression pattern and functional significance of TRPV6 in the context of PDAC remains enigmatic. This study scrutinizes the expression of TRPV6 in tissue specimens obtained from 46 PDAC patients across distinct stages and grades. We manipulated TRPV6 expression (knockdown, overexpression) in the human PDAC cell lines Panc-1 and Capan-1. Subsequently, we analyzed its impact on multiple facets, encompassing Ca2+ influx, proliferation, apoptosis, migration, chemoresistance, and tumor growth, both in vitro and in vivo. Notably, the data indicate a direct correlation between TRPV6 expression levels, tumor stage, and grade, establishing a link between TRPV6 and PDAC proliferation in tissue samples. Decreasing TRPV6 expression via knockdown hampered Ca2+ influx, resulting in diminished proliferation and viability in both cell lines, and cell cycle progression in Panc-1. The knockdown simultaneously led to an increase in apoptotic rates and increased the susceptibility of cells to 5-FU and gemcitabine treatments. Moreover, it accelerated migration and promoted collective movement among Panc-1 cells. Conversely, TRPV6 overexpression yielded opposing outcomes in terms of proliferation in Panc-1 and Capan-1, and the migration of Panc-1 cells. Intriguingly, both TRPV6 knockdown and overexpression diminished the process of tumor formation in vivo. This intricate interplay suggests that PDAC aggressiveness relies on a fine-tuned TRPV6 expression, raising its profile as a putative therapeutic target.

Persistent Cryptosporidium parvum Infection Leads to the Development of the Tumor Microenvironment in an Experimental Mouse Model: Results of a Microarray Approach.

Cryptosporidium spp. are enteric protozoa parasites that infect a variety of vertebrate hosts. These parasites are capable of inducing life-threatening gastrointestinal disease in immunocompromised individuals. With the rising epidemiological evidence of the occurrence of Cryptosporidium infections in humans with digestive cancer, the tumorigenic potential of the parasite has been speculated. In this regard, Cryptosporidium parvum has been reported to induce digestive adenocarcinoma in a rodent model of chronic cryptosporidiosis. However, the processes by which the parasite could induce this carcinogenesis are still unknown. Therefore, the transcriptomes of C. parvum infected ileo-cecal regions of mice developing tumors were analyzed in the current study. For the first time, downregulation of the expression of α-defensin, an anti-microbial target of the parasite in response to C. parvum infection was observed in the transformed tissues. This phenomenon has been speculated to be the result of resistance of C. parvum to the host defense through the upregulated expression of interferon γ-stimulated genes. The inflammatory response generated as result of attenuated expression of anti-microbial peptides highlights the role of immune evasion in the C. parvum-induced tumorigenesis. The study has also succeeded in the characterization of the tumor microenvironment (TME) which is characterized by the presence of cancer associated fibroblasts, myeloid-derived suppressor cells, tumor-associated macrophages and extracellular matrix components. Identification of immune suppressor cells and accumulation of pro-inflammatory mediators speculates that chronic inflammation induced by persistent C. parvum infection assists in development of an immunosuppressive tumor microenvironment.

Structural Characterization of Mucin O-Glycosylation May Provide Important Information to Help Prevent Colorectal Tumor Recurrence.

Although colorectal cancer is a preventable and curable disease if early stage tumors are removed, it still represents the second cause of cancer-related death worldwide. Surgical resection is the only curative treatment but once operated the patient is either subjected to adjuvant chemotherapy or not, depending on the invasiveness of the cancer and risks of recurrence. In this context, we investigated, by mass spectrometry (MS), alterations in the repertoire of glycosylation of mucins from colorectal tumors of various stages, grades, and recurrence status. Tumors were also compared with their counterparts in resection margins from the same patients and with healthy controls. The obtained data showed an important decrease in the level of expression of sialylated core 3-based O-glycans in tumors correlated with an increase in sialylated core 1 structures. No correlation was established between stages of the tumor samples and mucin O-glycosylation. However, with the notable exception of sialyl Tn antigens, tumors with recurrence presented a milder alteration of glycosylation profile than tumors without recurrence. These results suggest that mucin O-glycans from tumors with recurrence might mimic a healthier physiological situation, hence deceiving the immune defense system.

B4GALNT2 gene expression controls the biosynthesis of Sda and sialyl Lewis X antigens in healthy and cancer human gastrointestinal tract.

The histo blood group carbohydrate Sd(a) antigen and its cognate biosynthetic enzyme B4GALNT2 show the highest level of expression in normal colon. Their dramatic down regulation previously observed in colon cancer tissues could play a role in the concomitant elevation of the selectin ligand sLe(x), involved in metastasis. However, down regulation of sLe(x) expression by B4GALNT2 has been so far demonstrated in vitro, but not in tissues. The human B4GALNT2 gene specifies at least two transcripts, diverging in the first exon, never studied in normal and cancer tissues. The long form contains a 253 nt exon 1L; the short form contains a 38 nt exon 1S. Using qPCR, we showed that cell lines and normal or cancerous colon, expressed almost exclusively the short form, while the long form was mainly expressed by the embryonic colon fibroblast cell line CCD112CoN. Immunochemistry approaches using colon cancer cells permanently expressing either B4GALNT2 cDNAs as controls, led to the observation of several protein isoforms in human normal and cancerous colon, and cell lines. We showed that tissues expressing B4GALNT2 protein isoforms were able to induce Sd(a) and to inhibit sLe(x) expression; both of which are expressed mainly on PNGase F-insensitive carbohydrate chains. Concomitant expression of B4GALNT2 and siRNA-mediated inhibition of FUT6, the major fucosyltransferase involved in sLe(x) synthesis in colon, resulted in a cumulative inhibition of sLe(x). In normal colon samples a significant relationship between sLe(x) expression and the ratio between FUT6/B4GALNT2 activities exists, demonstrating for the first time a role for B4GALNT2 in sLe(x) inhibition in vivo.