GALNT6 promotes bladder cancer malignancy and immune escape by epithelial-mesenchymal transition and CD8+ T cells.

Bladder cancer (BC) ranks as the sixth cancer in males and the ninth most common cancer worldwide. Conventional treatment modalities, including surgery, radiation, chemotherapy, and immunotherapy, have limited efficacy in certain advanced instances. The involvement of GALNT6-mediated aberrant O-glycosylation modification in several malignancies and immune evasion is a subject of speculation. However, its significance in BC has not been investigated. Through the integration of bioinformatics analysis and laboratory experimentation, we have successfully clarified the role of GALNT6 in BC. Our investigation revealed that GALNT6 has significant expression in BC, and its high expression level correlates with advanced stage and high grade, leading to poor overall survival. Moreover, both in vitro and in vivo experiments demonstrate a strong correlation between elevated levels of GALNT6 and tumor growth, migration, and invasion. Furthermore, there is a negative correlation between elevated GALNT6 levels, the extent of CD8+ T cell infiltration in the tumor microenvironment, and the prognosis of patients. Functional experiments have shown that the increased expression of GALNT6 could enhance the malignant characteristics of cancer cells by activating the epithelial-mesenchymal transition (EMT) pathway. In brief, this study examined the impact of GALNT6-mediated abnormal O-glycosylation on the occurrence and progression of bladder cancer and its influence on immune evasion. It also explored the possible molecular mechanism underlying the interaction between tumor cells and immune cells, as well as the bidirectional signaling involved. These findings offer a novel theoretical foundation rooted in glycobiology for the clinical application of immunotherapy in BC.

CCDC88C, an O-GalNAc glycosylation substrate of GALNT6, drives breast cancer metastasis by promoting c-JUN-mediated CEMIP transcription.

Coiled-coil domain containing 88C (CCDC88C) is a component of non-canonical Wnt signaling, and its dysregulation causes colorectal cancer metastasis. Dysregulated expression of CCDC88C was observed in lymph node metastatic tumor tissues of breast cancer. However, the role of CCDC88C in breast cancer metastasis remains unclear. To address this, the stable BT549 and SKBR3 cell lines with CCDC88C overexpression or knockdown were developed. Loss/gain-of-function experiments suggested that CCDC88C drove breast cancer cell motility in vitro and lung and liver metastasis in vivo. We found that CCDC88C led to c-JUN-induced transcription activation. Overlapping genes were identified from the genes modulated by CCDC88C and c-JUN. CEMIP, one of these overlapping genes, has been confirmed to confer breast cancer metastasis. We found that CCDC88C regulated CEMIP mRNA levels via c-JUN and it exerted pro-metastatic capabilities in a CEMIP-dependent manner. Moreover, we identified the CCDC88C as a substrate of polypeptide N-acetylgalactosaminyltransferase 6 (GALNT6). GALNT6 was positively correlated with CCDC88C protein abundance in the normal breast and breast cancer tissues, indicating that GALNT6 might be associated with expression patterns of CCDC88C in breast cancer. Our data demonstrated that GALNT6 maintained CCDC88C stability by promoting its O-linked glycosylation, and the modification was critical for the pro-metastatic potential of CCDC88C. CCDC88C also could mediate the pro-metastatic potential of GALNT6 in breast cancer. Collectively, our findings uncover that CCDC88C may increase the risk of breast cancer metastasis and elucidate the underlying molecular mechanisms.

Pan-Cancer Analysis of GALNT6 with Potential Implications for Prognosis and Tumor Microenvironment in Human Cancer Based on Bioinformatics and qPCR Verification.

Purpose: We explored the expression and prognostic value of GALNT6 and the tumor microenvironment of pan-cancer in humans. Methods: In this study, we explored the expression pattern of GALNT6 pan-cancer across multiple databases. The prognostic value of GALNT6 was evaluated using the Kaplan-Meier method. The types and numbers of GALNT6 gene alterations were exhibited using the cBio Cancer Genomics Portal. The correlations between GALNT6 expression and immune infiltration in cancers were analyzed using the database Tumor Immune Estimation Resource 2. We also used the Kyoto Encyclopedia of Genes and Genomes pathway and Gene Ontology analysis to investigate the molecular mechanisms of the GALNT6 gene in tumorigenesis. The expression of GALNT6 was also further verified by qPCR in lung adenocarcinoma tissues. Results: In general, compared with normal tissue, tumor tissue had a higher expression level of GALNT6. GALNT6 showed a protective effect in colon carcinoma and other cancers; however, a high expression level of GALNT6 was detrimental to survival in bladder cancer and in pheochromocytoma and paraganglioma. Mutation, amplification, and deep deletion were the three main types of GALNT6 mutations in tumors. There was a significant positive correlation between GALNT6 expression and immune infiltration of CD8+ T-cells in skin cutaneous melanoma metastasis, based on most of the algorithms used. Moreover, protein processing- and glycoprotein metabolic-associated functions were involved in the functional mechanisms of GALNT6. Conclusion: This first pan-cancer study offers a relatively comprehensive understanding of the oncogenic roles of GALNT6 across different cancer types.

Knocking down GALNT6 promotes pyroptosis of pancreatic ductal adenocarcinoma cells through NF-κB/NLRP3/GSDMD and GSDME signaling pathway.

Background: Pancreatic ductal adenocarcinoma (PDAC), the most prevalent type of pancreatic cancer, is a highly lethal malignancy with poor prognosis. Polypeptide N-acetylgalactosaminyltransferase-6 (GALNT6) is frequently overexpressed in PDAC. However, the role of GALNT6 in the PDAC remains unclear. Methods: The expression of GALNT6 in pancreatic cancer and normal tissues were analyzed by bioinformatic analyses and immunohistochemistry. CCK8 and colony formation were used to detect cell proliferation. Flow cytometry was applied to detect cell cycle.The pyroptosis was detected by scanning electron microscopy. The mRNA expression was detected by qRT-PCR. The protein expression and localization were detected by western blot and immunofluorescence assay. ELISA was used to detect the levels of inflammatory factors. Results: The expression of GALNT6 was associated with advanced tumor stage, and had an area under curve (AUC) value of 0.919 in pancreatic cancer based on the cancer genome atlas (TCGA) dataset. Knockdown of GALNT6 inhibited cell proliferation, migration, invasion and cell cycle arrest of PDAC cells. Meanwhile, knockdown of GALNT6 increased the expression levels of interleukin-1ß (IL-1ß), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and interleukin-18 (IL-18), the release of inflammasome and an increasing of Gasdermin D (GSDMD), N-terminal of GSDMD (GSDMD-N), Gasdermin E (GSDME) and N-terminal of GSDME (GSDME-N) in PDAC cells. GALNT6 suppressed the expression of NOD-like receptor thermal protein domain associated protein 3 (NLRP3) and GSDMD by glycosylation of NF-κB and inhibiting the nucleus localization of NF-κB. Additionally, GALNT6 promotes the degradation of GSDME by O-glycosylation. Conclusion: We found that GALNT6 is highly expressed in pancreatic cancer and plays a carcinogenic role. The results suggested that GALNT6 regulates the pyroptosis of PDAC cells through NF-κB/NLRP3/GSDMD and GSDME signaling. Our study might provides novel insights into the roles of GALNT6 in PDAC progression.

Early-Stage Loss of GALNT6 Predicts Poor Clinical Outcome in Colorectal Cancer.

Colorectal adenocarcinomas arise from luminal lining epithelium of the colorectal tract which is covered with highly glycosylated mucins. Mucin O-glycosylation is initiated by a family of polypeptide N-acteylgalactosaminyltransferases (GALNTs). This study examined GALNT6 protein expression in 679 colorectal tumors, including 574 early-stage and 105 late-stage cancers. GALNT6 expression in cancer tissue varied widely between patients ranging from high levels to complete loss. Loss of GALNT6 occurred in 9.9% of early-stage and 15.2% of late-stage cancers and was more prevalent in grade 3 or MSI subtype tumors. Survival analyses revealed that loss of GALNT6 expression is prognostic of reduced overall survival, and univariate and multivariate analyses demonstrated that loss of GALNT6 is an independent risk variable. We also analyzed 508-case TCGA and 63-case CPTAC colorectal cancer cohorts for all members of the GALNT enzyme family, the mucin family, as well as KRAS and BRAF mutations. GLANT6 mRNA expression showed no strong correlation with other GALNTs or mucins but was significantly higher in KRAS mutated or BRAF wild-type early-stage cancers. Using large cohorts of patients and different approaches, this study shows that loss of GALNT6 enzyme in early-stage colorectal cancer predicts poor clinical outcomes.

GALNT6 Knockdown Inhibits the Proliferation and Migration of Colorectal Cancer Cells and Increases the Sensitivity of Cancer Cells to 5-FU.

The incidence of colorectal cancer (CRC) is increasing annually worldwide, highlighting the need for novel therapeutics to be developed. GALNT6 is a member of the N-acetylgalactosyltransferase enzyme family, which exhibits oncogenic functions in several types of cancers, such as breast cancer and ovarian cancer. However, the function of GALNT6 in CRC has not received much attention in recent years and is therefore poorly understood. In this study, the GALNT6 gene was screened using RNA-seq data obtained from The Cancer Genome Atlas (TCGA). RNA-seq data from 50 pairs of matched CRC patients in TCGA were obtained and analyzed, and the protein expression levels of GALNT6 were verified in 10 pairs of clinical samples. These samples showed that GALNT6 was highly expressed in CRC tissues. Functional analysis also showed that GALNT6 knockdown inhibited the proliferation and migration of CRC cells and increased the number of apoptotic cells. Furthermore, GALNT6 knockdown suppressed tumor growth in vivo. GALNT6 also regulated the AKT pathway based on ingenuity pathway analysis and western blotting assay. Finally, GALNT6 knockdown was observed to increase the sensitivity of CRC cells to 5-Fluorouracil (5-FU). These results, taken together, show that GALNT6 knockdown inhibits proliferation and migration of CRC cells and increases cellular sensitivity to 5-FU. It is therefore possible that targeting GALNT6 might prove to be an effective avenue for exploration in any attempt to develop new therapies for the treatment of CRC.

GALNT6 promotes breast cancer metastasis by increasing mucin-type O-glycosylation of α2M.

Breast cancer is the most lethal malignancy in women. N-acetylgalactosaminyltransferase 6 (GALNT6) is an enzyme which mediates the initial step of mucin-type O-glycosylation, and has been reported to be involved in mammary carcinogenesis. However, the molecular mechanism of GALNT6 in breast cancer metastasis has not been fully explored. In this study, based on online database analyses and tissue microarrays, the overall survival (OS) of breast cancer patients with high expression of GALNT6 was found to be shorter than those with low expression of GALNT6. Also, high GALNT6 expression was positively correlated with advanced pN stage and pTNM stage. GALNT6 was shown to be able to promote the migration and invasion of breast cancer cells, and enhance the level of mucin-type O-glycosylation of substrates in the supernatants of breast cancer cells. Qualitative mucin-type glycosylomics analysis identified α2M as a novel substrate of GALNT6. Further investigation showed that GALNT6 increased O-glycosylation of α2M, and the following activation of the downstream PI3K/Akt signaling pathway was involved in the promotion of migration and invasion of breast cancer cells. This study identified a new substrate of GALNT6 and provides novel understanding of the role of GALNT6 in promoting metastasis and poor prognosis in breast cancer.

GALNT6 Promotes Tumorigenicity and Metastasis of Breast Cancer Cell via ß-catenin/MUC1-C Signaling Pathway.

Polypeptide N-acetylgalactosaminyl transferase-6 (GALNT6), a member of the N-acetyl-D-galactosamine transferase family, was shown to be over-expression in mammary cancer and could be used as a biomarker. However, its roles and underlying mechanisms in the pathogenesis of breast cancer are still unclear. In this study, we reported that GALNT6 was up-expression in breast cancer, and it was not associated with tumor stage. The expression level of GALNT6 and menopause status was associated with patient survival. Biological function results illustrated that knockdown of GALNT6 inhibited proliferation, migration and invasion of MDA-MB-231 cells, and increased cell apoptosis. Knockdown of GALNT6 in breast cancer cell attenuated the protein expression of PCNA, cyclin D1, C-myc and ß-catenin, and increased the expression of E-cadherin, caspase 3 and cleaved PARP1. Cell fractionation assay showed that knockdown of GALNT6 reduced the levels of ß-catenin and MUC1-C in nucleus. Simultaneously knockdown of GALNT6 and ß-catenin significantly reduced the level of C-myc. Co-IP experiments indicated that GALNT6 interacted with MUC1-N, ß-catenin interacting with MUC1-C in breast cancer cells. Together, our study reveals that GALNT6 promotes tumorigenicity and metastasis through ß-catenin/MUC1-C signaling pathway.

GALNT6 suppresses progression of colorectal cancer.

Colorectal cancer (CRC) is one of the most common malignant tumors in the world. Invasion and metastasis are the main cause of mortality in most CRC patients. Polypeptide N-acetylgalactosaminyltransferase 6 (GALNT6) regulated glycosylation, which is frequently altered in cancers, and play an important role in cancer development. However, the role of GALNT6 in CRC remains unknown. To investigate the role of GALNT6 in CRC, first we studied correlation of GALNT6 expression levels with outcomes of CRC patients and found CRC patients with higher expression of GALNT6 had a better overall survival than those with lower expression. In addition, GALNT6 expression were significantly associated with tumor size, histological differentiation and lymph node metastasis. In vitro, GALNT6 overexpression dramatically inhibited cellular colony formation, migration, and invasion, and promoted the apoptosis of CRC cells. In vivo, CRC with GALNT6 overexpression showed reduced pulmonary metastasis in recipient mice compared with the controls. GALNT6 expression was significantly increased in SW480 and SW1116 cells cultured in hypoxic condition, and decreased in HT29 and LOVO cells with oxidative stress. Affimetrix microarray analysis showed that GALNT6 overexpression induced 279 genes up-regulated and 215 genes down-regulated in CRC. GALNT6 overexpression dramatically suppressed AKT and activated CD28 signaling pathway in CRC. AKT rescue experiment found that AKT was involved in GALNT6-induced CRC cell migration and invasion. In conclusion, our results first suggest that GALNT6 plays an important role in development and progression of CRC as a tumor suppressor gene.

Polypeptide N-acetylgalactosaminyltransferase-6 expression in gastric cancer.

Gastric cancer (GC) is one of the leading causes of cancer-related deaths, with limited improvement in its clinical outcome worldwide. Aberrant mucin-type O-glycosylation is a critical event widespread in the development of GC. Polypeptide N-acetylgalactosaminyltransferases (GALNTs) regulate the initial step and determine the sites of mucin-type O-glycoprotein bio-synthesis. GALNT6 has considerable potential as a biomarker in various cancers. The roles of GALNT6 in GC were analyzed, and the results showed that GALNT6 expression markedly increased in GC tissues compared with those in adjacent gastric tissues. High intratumoral GALNT6 density was associated with the clinicopathological parameters of TNM stage and distant metastasis. GALNT6 was identified as an independent prognosticator for the poor prognosis of GC patients. Moreover, the high expression level of GALNT6 was significantly associated with the low expression levels of E-cadherin and ß-catenin and the high expression levels of MMP9. These findings indicated that GALNT6 could provide new insights into the characterization of GC as well as contribute to the development of an efficient prognostic indicator and novel therapeutic modalities for GC.

GALNT6 expression enhances aggressive phenotypes of ovarian cancer cells by regulating EGFR activity.

Ovarian cancer is the most lethal of the gynecologic malignancies. N-acetylgalactosaminyltransferase 6 (GALNT6), an enzyme that mediates the initial step of mucin type-O glycosylation, has been reported to regulate mammary carcinogenesis. However, the expression and role of GALNT6 in ovarian cancer are still unclear. Here we showed that high GALNT6 expression correlates with increased recurrence, lymph node metastasis, and chemoresistance in ovarian endometrioid and clear cell carcinomas; and higher GALNT6 levels are significantly associated with poorer patient survivals. GALNT6 knockdown with two independent siRNAs significantly suppressed viability, migration, and invasion of ovarian cancer cells. Using phospho-RTK array and Western blot analyses, we identified EGFR as a critical target of GALNT6. GALNT6 knockdown decreased phosphorylation of EGFR, whereas GALNT6 overexpression increased the phosphorylation. Lectin pull-down assays with Vicia villosa agglutinin (VVA) indicated that GALNT6 was able to modify O-glycans on EGFR. Moreover, the GALNT6-enhanced invasive behavior was significantly reversed by erlotinib, an EGFR inhibitor. Our results suggest that GALNT6 expression is associated with poor prognosis of ovarian cancer and enhances the aggressive behavior of ovarian cancer cells by regulating EGFR activity.