The O-glycosylating enzyme GALNT2 suppresses the malignancy of gastric adenocarcinoma by reducing EGFR activities.
ABSTRACT: Aberrant glycosylation affects the malignant progression of cancers. Here, we report that N-acetyl-galactosaminyltransferase 2 (GALNT2), an enzyme that initiates the mucin type-O glycosylation, suppresses malignant phenotypes in gastric adenocarcinoma (GCA) cells by modifying epidermal growth factor receptor (EGFR) activity. GALNT2 was knocked down using siRNA in AGS and MKN28 cells. The expression of phosphorylated EGFR (pEGFR), phosphorylated Akt (pAkt) and Tn antigen were detected by western blotting. Proliferation, migration and invasion of cells with/without GLANT2-knockdown were assessed. Expression of pEGFR in the resected gastric cancer tissue was analyzed by Immunohistochemical staining, and was correlated with clinicopathological factors. The results showed that GALNT2 knockdown enhanced phosphorylation of EGFR and decreased expression of the Tn antigen on EGFR. Inhibiting EGFR activity with Gefitinib decreased the migration/invasion abilities and reversed the increase pAkt caused by GALNT2 knockdown in GCA cells. The addition of MK2206 (Akt inhibitor) mitigated the migration and invasion abilities of the GALNT2-knockdown cells. Patients with increased expressions of pEGFR in their cancer tissues were associated more metastasis, advanced stage and recurrence after surgical resection. Our results indicate that GALNT2 suppresses the malignant potential of GCA cells through the EGFR-Akt signaling pathway. The significance of O-glycosylation in receptor tyrosine kinases activities and GCA progression deserve further studies.
Project description:Glycosylation affects malignancy in cancer. Here, we report that N- acetylgalactosaminyltransferase 2 (GALNT2), an enzyme that mediates the initial step of mucin type-O glycosylation, suppresses malignant phenotypes in gastric adenocarcinoma (GCA) by modifying MET (Hepatocyte growth factor receptor) activity. GALNT2 mRNA and protein were downregulated in GCAs, and this reduction was associated with more advanced disease stage and shorter recurrence-free survival. Suppressing GALNT2 expression in GCA cells increased cell growth, migration, and invasion in vitro, and tumor metastasis in vivo. GALNT2 knockdown enhanced phosphorylation of MET and decreased expression of the Tn antigen on MET. Inhibiting MET activity with PHA665752 decreased the malignant phenotypes caused by GALNT2 knockdown in GCA cells. Our results indicate that GALNT2 suppresses the malignant potential of GCA cells and provide novel insights into the significance of O-glycosylation in MET activity and GCA progression.
Project description:Aberrant expression of the simple mucin-type carbohydrate antigens such as Tn antigen is associated with malignant transformation and cancer progression. N-acetylgalactosaminyltransferase 2 (GALNT2), one of the enzymes that mediate the initial step of mucin-type O-glycosylation, is responsible for forming Tn antigen. GALNT2 is expressed differentially in nervous tissues during mouse embryogenesis; however, the role of GALNT2 in neuroblastoma (NB) remains unclear. Here we showed that increased GALNT2 expression evaluated using immunohistochemistry in NB tumor tissues correlated well with the histological grade of differentiation as well as younger age at diagnosis, early clinical stage, primary tumor originated from the extra-adrenal site, favorable INPC histology, and MYCN non-amplification. Multivariate analysis showed that GALNT2 expression is an independent prognostic factor for better survival for NB patients. GALNT2 overexpression suppressed IGF-1-induced cell growth, migration, and invasion of NB cells, whereas GALNT2 knockdown enhanced these NB phenotypes. Mechanistic investigations demonstrated that GALNT2 overexpression modified O-glycans on IGF-1R, which suppressed IGF-1-triggered IGF-1R dimerization and subsequent downstream signaling events. Conversely, these properties were reversed by GALNT2 knockdown in NB cells. Our findings suggest that GALNT2 regulates malignant phenotypes of NB cells through the IGF-1R signaling pathway, suggesting a critical role for GALNT2 in the pathogenesis of NB.
Project description:BACKGROUND: In Non-small cell lung cancer (NSCLC), an overactive epidermal growth factor receptor (EGFR) pathway is a component of the malignant phenotype. Two tyrosine kinase inhibitors (TKIs) of EGFR, gefinitib and erlotinib, have been used with variable benefit. METHODS: We have analyzed outcome data of a population of NSCLC patients that received these TKIs to determine the benefit derived and to define the clinical and molecular parameters that correlate with response. Tumor tissue from a subgroup of these patients was analyzed by immunohistochemistry to measure the expression level of EGFR and four activated (phosphorylated) members of the pathway, pEGFR, pERK, pAKT, and pSTAT3. RESULTS: Erlotinib was slightly superior to gefitinib in all measures of response, although the differences were not statistically significant. The most robust clinical predictors of time to progression (TTP) were best response and rash (p < 0.0001). A higher level of pEGFR was associated with longer TTP, while the total EGFR level was not associated with response. Higher levels of pAKT and pSTAT3 were also associated with longer TTP. In contrast, a higher level of pERK1/2 was associated with shorter TTP. CONCLUSION: These observations suggest the hypothesis that tumor cells that have activated EGFR pathways, presumably being utilized for survival, are clinically relevant targets for pathway inhibition. An accurate molecular predictive model of TKI response should include activated members of the EGFR pathway. TKIs may be best reserved for tumors expressing pEGFR and pAKT or pSTAT, and little pERK. In the absence of molecular predictors of response, the appearance of a rash and a positive first scan are good clinical indicators of response.
Project description:Expression of CBL, an ubiquitin ligase, is decreased in 60% of human pancreatic ductal adenocarcinomas (PDAC) and is associated with shorter overall survival. We sought to determine how low CBL directly contributes to clinically more aggressive PDAC.Human PDACs were stained for CBL, pEGFR, and EGFR. CBL-low was modeled in PDAC cells (Panc-1, L3.6pl, and AsPC-1) via transient transfection (siRNA) or stable knockdown (shRNA). Cell viability and apoptosis were measured by MTT assays and FACS. Immunoblot and a phospho-receptor tyrosine kinase (pRTK) array were used to probe signal transduction. NOD-scid-IL2R?(null) mice were subcutaneously implanted with PDAC or PDAC(CBL-low) cells on opposite flanks and treated with gemcitabine ± erlotinib for ?4 weeks.There was an inverse correlation between CBL and pEGFR protein expression in 12 of 15 tumors. CBL knockdown increased PDAC resistance to gemcitabine and 5-fluorouracil (5-FU) by upregulating pEGFR (Y1068), pERK, and pAKT. A pRTK array of PDAC(CBL-low) cells revealed additional activated tyrosine kinases but all to a much lower magnitude than EGFR. Increased chemoresistance from low CBL was abrogated by the EGFR inhibitor erlotinib both in vitro and in vivo. Erlotinib+gemcitabine-treated PDAC(CBL-low) cells exhibited greater apoptosis by cleaved PARP, caspase-3, and Annexin V/PI.Low CBL causes chemoresistance in PDAC via stress-induced EGFR activation that can be effectively abrogated by EGFR inhibition. These results suggest that dysregulation of ubiquitination is a key mechanism of EGFR hyperactivation in PDAC and that low CBL may define PDAC tumors likely to respond to erlotinib treatment.
Project description:Only a minority of cancer patients benefits from the combination of EGFR-inhibition and radiotherapy in head and neck squamous cell carcinoma (HNSCC). A potential resistance mechanism is activation of EGFR and/or downstream pathways by stimuli in the microenvironment. The aim of this study was to find molecular targets induced by the microenvironment by determining the in vitro and in vivo expression of proteins of the EGFR-signaling network in 6 HNSCC lines. As hypoxia is an important microenvironmental parameter associated with poor outcome in solid tumors after radiotherapy, we investigated the relationship with hypoxia in vitro and in vivo.Six human HNSCC cell lines were both cultured as cell lines (in vitro) and grown as xenograft tumors (in vivo). Expression levels were determined via western blot analysis and localization of markers was assessed via immunofluorescent staining. To determine the effect of hypoxia and pAKT-inhibition on cell survival, cells were incubated at 0.5% O(2) and treated with MK-2206.We observed strong in vitro-in vivo correlations for EGFR, pEGFR and HER2 (rs = 0.77, p = 0.10, rs = 0.89, p = 0.03) and rs = 0.93, p = 0.02, respectively), but not for pAKT, pERK1/2 or pSTAT3 (all r(s)<0.55 and p>0.30). In vivo, pAKT expression was present in hypoxic cells and pAKT and hypoxia were significantly correlated (rs = 0.51, p = 0.04). We confirmed in vitro that hypoxia induces activation of AKT. Further, pAKT-inhibition via MK-2206 caused a significant decrease in survival in hypoxic cells (p<0.01), but not in normoxic cells.These data suggest that (p)EGFR and HER2 expression is mostly determined by intrinsic features of the tumor cell, while the activation of downstream kinases is highly influenced by the tumor microenvironment. We show that hypoxia induces activation of AKT both in vitro and in vivo, and that hypoxic cells can be specifically targeted by pAKT-inhibition. Targeting pAKT is thus a potential way to overcome therapy resistance induced by hypoxia and improve patient outcome.
Project description:Abstract: Previous studies have shown that total epidermal growth factor receptor (EGFR) protein is highly expressed in soft tissue sarcoma (STS). We aimed to investigate the significance of phosphorylated-EGFR (pEGFR) and its activated-downstream signal transducers in STS tissue samples. A tissue microarray comprising 87 STS samples was assessed for total EGFR, pEGFR and its phosphorylated signal transducers and expression was correlated with clinicopathlogical parameters including patient outcome. Although the expression of total EGFR was significantly associated with adverse STS histologic grade (p = 0.004) and clinical stage (p = 0.012) similar to pEGFR, phosphorylated protein kinase B (pAkt) and phosphorylated extracellular signal regulated kinase (pERK), it is not a prognostic factor for survival. By contrast, the expression of pEGFR is an independent factor for cancer specific survival, while pERK is an independent prognostic factor for both overall and cancer specific survival in STS (p < 0.05, Cox proportional hazard model and log-rank test) in addition to the recognised factors of tumour grade and clinical stage. pERK and pEGFR are new independent prognostic factors for overall and/or cancer specific survival in STS. The expression of EGFR/pEGFR, and their associated downstream signal transducers, was associated with STS progression, suggesting that EGFR downstream signalling pathways may jointly support STS cell survival.
Project description:BACKGROUND: In the present study, we have investigated the prognostic usefulness of phosphorylated Akt immunoreactivity (pAkt-IR) in prostate cancer using a well-characterised tissue microarray from men who had undergone transurethral resection due to lower urinary tract symptoms. METHODOLOGY/PRINCIPAL FINDINGS: pAkt-IR in prostate epithelial and tumour cells was assessed using a monoclonal anti-pAkt (Ser(473)) antibody. Immunoreactive intensity was determined for 282 (tumour) and 240 (non-malignant tissue) cases. Tumour pAkt-IR scores correlated with Gleason score, tumour Ki67-IR (a marker of cell proliferation) and tumour phosphorylated epidermal growth factor receptor (pEGFR)-IR. For cases followed with expectancy, a high tumour pAkt-IR was associated with a poor disease-specific survival, and the prognostic information provided by this biomarker was additive to that provided by either (but not both) tumour pEFGR-IR or Ki67-IR. Upon division of the cases with respect to their Gleason scores, the prognostic value of pAkt-IR was seen for patients with Gleason score 8-10, but not for patients with Gleason score 6-7. CONCLUSIONS/SIGNIFICANCE: Tumour pAkt-IR is associated with both disease severity and disease-specific survival. However, its clinical use as a biomarker is limited, since it does not provide prognostic information in patients with Gleason scores 6-7.
Project description:We hypothesized that normal human mesothelial cells acquire resistance to asbestos-induced toxicity via induction of one or more epidermal growth factor receptor (EGFR)-linked survival pathways (phosphoinositol-3-kinase/AKT/mammalian target of rapamycin and extracellular signal-regulated kinase [ERK] 1/2) during simian virus 40 (SV40) transformation and carcinogenesis. Both isolated HKNM-2 mesothelial cells and a telomerase-immortalized mesothelial line (LP9/TERT-1) were more sensitive to crocidolite asbestos toxicity than an SV40 Tag-immortalized mesothelial line (MET5A) and malignant mesothelioma cell lines (HMESO and PPM Mill). Whereas increases in phosphorylation of AKT (pAKT) were observed in MET5A cells in response to asbestos, LP9/TERT-1 cells exhibited dose-related decreases in pAKT levels. Pretreatment with an EGFR phosphorylation or mitogen-activated protein kinase kinase 1/2 inhibitor abrogated asbestos-induced phosphorylated ERK (pERK) 1/2 levels in both LP9/TERT-1 and MET5A cells as well as increases in pAKT levels in MET5A cells. Transient transfection of small interfering RNAs targeting ERK1, ERK2, or AKT revealed that ERK1/2 pathways were involved in cell death by asbestos in both cell lines. Asbestos-resistant HMESO or PPM Mill cells with high endogenous levels of ERKs or AKT did not show dose-responsive increases in pERK1/ERK1, pERK2/ERK2, or pAKT/AKT levels by asbestos. However, small hairpin ERK2 stable cell lines created from both malignant mesothelioma lines were more sensitive to asbestos toxicity than shERK1 and shControl lines, and exhibited unique, tumor-specific changes in endogenous cell death-related gene expression. Our results suggest that EGFR phosphorylation is causally linked to pERK and pAKT activation by asbestos in normal and SV40 Tag-immortalized human mesothelial cells. They also indicate that ERK2 plays a role in modulating asbestos toxicity by regulating genes critical to cell injury and survival that are differentially expressed in human mesotheliomas.
Project description:Epidermal growth factor receptor (EGFR) activation events and the mammalian target of rampamycin (mTOR) are considered important therapeutic targets in alleviating cancer conditions. The current treatment paradigm has shifted to personalized treatment strategies with tyrosine kinase inhibitors (TKIs) or anaplastic lymphoma kinase (ALK) inhibitors, due to low survival rates in non-small cell lung cancer (NSCLC) in terms of the prevailing platinum-based therapy. In the present study, we examined the anticancer potential of Verrucarin J (VJ), a small molecule, in NSCLC cell lines (H460 and A549). The small molecule significantly inhibited cell growth, proliferation, colony forming ability, and induced apoptosis in both lung cancer cell lines. The inhibitory effects on EGFR (pEGFR -tyr1173) and AKT (pAKT Serine473) signaling, downregulates downstream pro-survival signaling (mTOR and NF-?B) in cancer cell lines. In addition, VJ abrogated invasive and migratory potential of A549 and H460 cells. We also observed a downregulation of mesenchymal markers such as N-cadherin, Slug, ?-catenin, and vimentin expression in both cell lines. Our results suggest that VJ inhibited cancer cell growth and could be a potent molecule to inhibit EGFR and AKT signaling in NSCLC.
Project description:Nuclear factor-erythroid 2-related factor 3 (NRF3), a nuclear transcription factor, has been implicated in various cellular processes including carcinogenesis. However, mechanisms underlying its regulation in carcinogenesis are unclear. Herein, we found that NRF3 is lowly expressed in colorectal cancer (CRC) tissues and cells, and NRF3 low-expressions in CRC tissue samples are associated with CRC carcinogenesis and poor patient outcomes. Nrf3-knockdown increased CRC cell growth, colony formation, and cell motility and invasion, and Nrf3-knockin dramatically decreased CRC cell growth and colony formation. Mechanistically, NRF3 increased CRC cell apoptosis and arrested cell G2/M stage. NRF3 was found to be reversely with epidermal growth factor receptor (EGFR) and p38. Strikingly, Nrf3-knockin dramatically decreased phosphorylated-EGFR at Tyrosine845 (pEGFR Tyr845) and phosphorylated-p38 at Threonine180/Tyrosine182 (p-p38 Thr180/Tyr182) expressions, and Nrf3-knockdown increased pEGFR Tyr845 and p-p38 Thr180/Tyr182. Moreover, NRF3 regulated EGFR and p38 down-stream molecules, protein kinase B (AKT), activating transcription factor (ATF) 2, and C/EBP homologous protein (CHOP) expressions. NRF3 loss-increased CRC growth through EGFR and p38 was confirmed in nude mice. Collectively, NRF3-loss in CRC cell increases EGFR and p38 phosphorylation activation, enhances cell proliferation and decreases cell apoptosis, and finally promotes CRC malignance.