Clinic implication of MUC1 <Emphasis Type="Italic">O</Emphasis>-glycosylation and C1GALT1 in esophagus squamous cell carcinoma

Esophagus squamous cell carcinoma (ESCC) is one of the most aggressive malignant tumors in the world. Our previous data demonstrates that oncoprotein MUC1 is related with metastasis and poor outcome of ESCC. However, alteration of MUC1 in ESCC remains unclear. Using ONCOMINE and COSMIC databases, we analyzed MUC1 gene copy numbers and gene mutations and found that MUC1 had high expression level but few gene mutations in ESCC. Further study of ESCC samples indicated that MUC1 O-glycosylation levels were higher in tumor tissues than that in para-carcinoma tissues in 10 of 14 pairs of ESCC samples. Moreover, we verified a potential link between MUC1 O-glycosylation and C1GALT1, which was further supported by IHC analysis on 38 ESCC and 19 para-carcinoma samples. More importantly, co-expression of MUC1 Oglycosylation and C1GALT1 presented positive correlations with both lymph node metastasis and survival time of ESCC patients. Our work collectively indicates that C1GALT1 is associated with O-glycosylated MUC1 in ESCC, not only suggesting a diagnostic significance of C1GALT1 and MUC1 O-glycosylation in ESCC, but also opening novel insights into targeting C1GALT1 and MUC1 O-glycosylation to suppress ESCC cells metastasis in patients.     

MUC15 loss facilitates epithelial-mesenchymal transition and cancer stemness for prostate cancer metastasis through GSK3β/β-catenin signaling

Patients with prostate cancer (PCa) have a high incidence of relapse and metastasis. Unfortunately, the molecular mechanisms underlying these processes have not been fully elucidated. In our study, we demonstrate that MUC15, a member of the mucin family, is a novel tumor suppressor in PCa that modulates epithelial-mesenchymal transition (EMT) and cancer stemness, contributing to PCa metastasis. First, MUC15 expression was found to be decreased in PCa tissues compared with para-carcinoma tissues. Moreover, we observed that MUC15 suppressed cell migration and invasion, both in vitro and in vivo, but had no effect on cell proliferation. Mechanistically, knockdown of MUC15 increased GSK3β phosphorylation and promoted β-catenin nuclear translocation. Therefore, the β-catenin-specific inhibitors XAV939 and PRI-724 rescued EMT in MUC15-deficient cell lines. Taken together, these results indicate that MUC15 is downregulated in PCa tissues and serves as a potential target to prevent PCa metastasis, which can inhibit EMT and cancer stemness via the GSK3β/β-catenin signaling pathway.     

Inhibition of MUC1-C regulates metabolism by AKT pathway in esophageal squamous cell carcinoma

Esophageal squamous cell carcinoma (ESCC) is one of the most common digestive tumors worldwide. The Mucin 1 (MUC1) heterodimeric protein has been confirmed that is overexpressed in ESCC and induced adverse outcomes. However, the detailed mechanism(s) remained challenging. So, we investigated the relationship between MUC1-C and metabolism in ESCC cells. In the results, TP53-induced glycolysis and apoptosis regulator (TIGAR) was overexpressed and correlative with MUC1-C positively in ESCC tissue. Targeting MUC1-C inhibits AKT–mTORC–S6K1 signaling and blocks TIGAR translation. We found that the inhibitory effect of GO-203 on TIGAR was mediated by inhibition of AKT–mTOR–S6K1 pathway. The findings also demonstrated that the suppressive effect of GO-203 on TIGAR is related to the decrease of glutathione level, the increase of reactive oxygen species and the loss of mitochondrial transmembrane membrane potential. In xenograft tissues, GO-203 inhibited the growth of ESCC cells and lead to the low expression of transmembrane C-terminal subunit (MUC1-C) and TIGAR. This evidence supports the contention that MUC1-C is significant for metabolism in ESCC and indicated that MUC1-C is a potential target for the treatment of ESCC.     

C1GALT1 Promotes Invasive Phenotypes of Hepatocellular Carcinoma Cells by Modulating Integrin β1 Glycosylation and Activity

Cancer cell invasion and metastasis are the primary causes of treatment failure and death in hepatocellular carcinoma (HCC). We previously reported that core 1 β1,3-galactosyltransferase (C1GALT1) is frequently overexpressed in HCC tumors and its expression is associated with advanced tumor stage, metastasis, and poor survival. However, the underlying mechanisms of C1GALT1 in HCC malignancy remain unclear. In this study, we found that overexpression of C1GALT1 enhanced HCC cell adhesion to extracellular matrix (ECM) proteins, migration, and invasion, whereas RNAi-mediated knockdown of C1GALT1 suppressed these phenotypes. The promoting effect of C1GALT1 on the metastasis of HCC cells was demonstrated in a mouse xenograft model. Mechanistic investigations showed that the C1GALT1-enhanced phenotypic changes in HCC cells were significantly suppressed by anti-integrin β1 blocking antibody. Moreover, C1GALT1 was able to modify O-glycans on integrin β1 and regulate integrin β1 activity as well as its downstream signaling. These results suggest that C1GALT1 could enhance HCC invasiveness through integrin β1 and provide novel insights into the roles of O-glycosylation in HCC metastasis.     

An Old Story Retold: Loss of G1 Control Defines A Distinct Genomic Subtype of Esophageal Squamous Cell Carcinoma

Esophageal squamous cell carcinoma(ESCC) has a high mortality rate. To determine the molecular basis of ESCC development, this study sought to identify characteristic genome-wide alterations in ESCC, including exonic mutations and structural alterations. The clinical implications of these genetic alterations were also analyzed. Exome sequencing and verification were performed for nine pairs of ESCC and the matched blood samples, followed by validation with additional samples using Sanger sequencing. Whole-genome SNP arrays were employed to detect copy number alteration(CNA) and loss of heterozygosity(LOH) in 55 cases, including the nine ESCC samples subjected to exome sequencing. A total of 108 non-synonymous somatic mutations(NSSMs) in102 genes were verified in nine patients. The chromatin modification process was found to be enriched in our gene ontology(GO) analysis. Tumor genomes with TP53 mutations were significantly more unstable than those without TP53 mutations. In terms of the landscape of genomic alterations, deletion of 9p21.3 covering CDKN2A/2B(30.9%), amplification of 11q13.3 covering CCND1(30.9%), and TP53 point mutation(50.9%) occurred in two-thirds of the cases. These results suggest that the deregulation of the G1 phase during the cell cycle is a key event in ESCC.Furthermore, six minimal common regions were found to be significantly altered in ESCC samples and three of them, 9p21.3, 7p11.2, and 3p12.1, were associated with lymph node metastasis. With the high correlation of TP53 mutation and genomic instability in ESCC, the amplification of CCND1, the deletion of CDKN2A/2B, and the somatic mutation of TP53 appear to play pivotal roles via G1 deregulation and therefore helps to classify this cancer into different genomic subtypes.These findings provide clinical significance that could be useful in future molecular diagnoses and therapeutic targeting.     

Upregulation of long noncoding RNA SNHG20 promotes cell growth and metastasis in esophageal squamous cell carcinoma via modulating ATM-JAK-PD-L1 pathway

Increasing evidence have proved that long noncoding RNAs (lncRNAs) play significant roles in tumorigenesis and development of various cancers. However, the effect of small nucleolar RNA host gene 20 (SNHG20) on the progression of esophageal squamous cell carcinoma (ESCC) remains to be discovered. Herein, we aim to find out the function and the possible mechanism of SNHG20 in ESCC progression. In our study, we demonstrate that SNHG20 is markedly upregulated in ESCC tissues and cell lines. Besides, the level of SNHG20 is closely associated with tumor size, lymph node metastasis, TNM stage, and tumor grade. In addition, SNHG20 level is an independent predictor for clinical outcomes of ESCC patients. Then the gain- and loss-of-function assays reveal that SNHG20 overexpression promotes cell proliferation, migration, invasion, and epithelial-mesenchymal transition as well as represses apoptosis, whereas depletion of SNHG20 exhibits opposite effects. Moreover, we uncover that SNHG20 modulates the expression of ataxia telangiectasia–mutated kinase (p-ATM), p-JAK1/2, and programmed cell death 1 ligand 1 (PD-L1) in ESCC cells and ATM upregulation restores the suppressive effect of SNHG20 inhibition on ESCC progression. Therefore, we conclude that SNHG20 serves as a carcinogen in ESCC by promoting growth and metastasis via ATM-JAK-PD-L1 pathway, supplying a possibly effective therapeutic target for ESCC.     

Prostatic Cell-Specific Regulation of the Synthesis of MUC1-Associated Sialyl Lewis a

Sialyl Lewis antigens are selectin ligands involved in leukocyte trafficking and cancer metastasis. Biosynthesis of these selectin ligands occurs by the sequential actions of several glycosyltransferases in the Golgi apparatus following synthesis of the protein backbone in the endoplasmic reticulum. In this study, we examine how the synthesis of sialyl Lewis a (sLe^a) is regulated in prostatic cells and identify a mucin that carries this glycotope. We treat human prostatic cells including one normal and three cancerous cells with histone deacetylase inhibitors, valproic acid, tricostatin A (TSA), and suberoylanilide hydroxamic acid (SAHA), and then monitor the expression of sLe^a. We have found that SAHA enhances the production of sLe^a in normal prostatic RWPE-1 cells but not prostatic cancer cells. Employing siRNA technology and co-immunoprecipitation, we show that the sLe^a is associated with MUC1, which is confirmed by confocal immunofluorescence microscopy and proximity ligation assay. The SAHA-induced production of sLe^a in RWPE-1 cells is resulted from upregulation of B3GALT1 gene via enhancement of acetylated histone-3 and histone-4. Interestingly, PC3 and LNCaP C-81 cells do not produce detectable amounts of sLe^a despite expressing high levels of B3GALT1. However, the MUC1-associated sLe^a is generated in these cells after introduction of MUC1 cDNA. We conclude that the synthesis of sLe^a is controlled by not only peptide backbone of the glycoprotein but also glycoprotein-specific glycosyltransferases involved in the synthesis of sLe^a. Further, the SAHA induction of this selectin ligand in normal prostatic cells may pose a potentially serious side effect of this drug recently approved by the US Food and Drug Administration.     

Glycosylated modification of MUC1 maybe a new target to promote drug sensitivity and efficacy for breast cancer chemotherapy

Breast cancer, the most common cancer in women, usually exhibits intrinsic insensitivity to drugs, even without drug resistance. MUC1 is a highly glycosylated transmembrane protein, overexpressed in breast cancer, contributing to tumorigenesis and worse prognosis. However, the molecular mechanism between MUC1 and drug sensitivity still remains unclear. Here, natural flavonoid apigenin was used as objective due to the antitumor activity and wide availability. MUC1 knockout (KO) markedly sensitized breast cancer cells to apigenin cytotoxicity in vitro and in vivo. Both genetical and pharmacological inhibition significantly enhanced the chemosensitivity to apigenin and clinical drugs whereas MUC1 overexpression conversely aggravated such drug resistance. Constitutively re-expressing wild type MUC1 in KO cells restored the drug resistance; however, the transmembrane domain deletant could not rescue the phenotype. Notably, further investigation discovered that membrane-dependent drug resistance relied on the extracellular glycosylated modification since removing O-glycosylation via inhibitor, enzyme digestion, or GCNT3 (MUC1 related O-glycosyltransferase) knockout markedly reinvigorated the chemosensitivity in WT cells, but had no effect on KO cells. Conversely, inserting O-glycosylated sites to MUC1-N increased the drug tolerance whereas the O-glycosylated deletant (Ser/Thr to Ala) maintained high susceptibility to drugs. Importantly, the intracellular concentration of apigenin measured by UPLC and fluorescence distribution firmly revealed the increased drug permeation in MUC1 KO and BAG-pretreated cells. Multiple clinical chemotherapeutics with small molecular were tested and obtained the similar conclusion. Our findings uncover a critical role of the extracellular O-glycosylation of MUC1-N in weakening drug sensitivity through acting as a barrier, highlighting a new perspective that targeting MUC1 O-glycosylation has great potential to promote drug sensitivity and efficacy.Subject terms: Breast cancer     

TWIST1, MMP-21, and HLAG-1 co-overexpression is associated with ESCC aggressiveness

Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive types of cancer, requiring reliable biomarkers for prognosis and therapeutic responsiveness. TWIST1, as an important factor responsible for metastasis of several cancers, is involved in tumor invasion and metastasis through indirectly regulation of MMP-21 expression. On the other hand, NF-ĸβ which is a regulator of HLAG-1 has direct interaction with TWIST1 protein. In this retrospective study we investigated the clinical significance of TWIST1, MMP-21, and HLAG-1 expression in ESCC, and the possible correlation between these genes and progression of the disease. The gene expression analyses of TWIST1, MMP-21, and HLA-G1 were performed by relative comparative real-time polymerase chain reaction in 58 ESCCs compared with corresponding margin-normal esophageal tissues. Significant overexpression of HLAG-1, TWIST1, and MMP-21 messenger RNA was observed in 22.4%, 41.4%, and 60.3% of tumor samples, respectively. Concomitant overexpression of TWIST1/MMP-21 and TWIST1/HLAG-1 were significantly correlated to each other in various clinicopathological features, including depth of tumor invasion, stage of tumor progression, lymphatic invasion, and grade of tumor cell differentiation ( P < 0.05). The current study is the first report of coexpression of TWIST1, MMP-21, and HLAG-1 in ESCC. Such findings suggest an oncogenic role for concomitant expression of these genes in ESCC invasion and metastasis.     

N-Glycosylation of the MUC1 mucin in epithelial cells and secretions

The MUC1 mucin is an important tumor-associated antigen that shows extensive glycosylation in vivo. The O-glycosylation of this molecule, which has been well characterized in many cell types and tissues, is important in conferring the unusual biochemical and biophysical properties on a mucin. N-Glycosylation is crucial to the folding, sorting, membrane trafficking, and secretion of many proteins. Here, we evaluated the N-glycosylation of MUC1 derived from two sources: endogenous MUC1 isolated from human milk and a recombinant epitope-tagged MUC1F overexpressed in Caco2 colon carcinoma cells. N-Glycans on purified MUC1F/MUC1 were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), gas chromatography-mass spectrometry (GC-MS), and CAD-ESI-MS/MS. The spectra indicate that MUC1F N-glycans have compositions consistent with high-mannose structures (Hex(5-9)HexNAc(2)) and complex/hybrid-type glycans (NeuAc(0-3)Fuc(0-3)Hex(3-8)HexNAc(3-7)). Many of the N-glycan structures are identical on MUC1F and native MUC1; however, a marked difference is seen between the N-glycans on membrane-bound and secreted forms of the native molecule.     

Clinical implication of elevated human cervical cancer oncogene-1 expression in esophageal squamous cell carcinoma

The human cervical cancer oncogene 1 (HCCR-1), a novel human oncoprotein, has been shown to be upregulated in various human tumors and plays a critical role in tumorigenesis and tumor progression. Here, the authors investigated HCCR-1 level in esophageal squamous cell carcinoma (ESCC) tissues and assessed the correlation between HCCR-1 level and prognosis of the patients with ESCC. HCCR-1 levels were investigated by immunohistochemistry, in situ hybridization, real-time quantitative RT-PCR and Western blotting methods; Kaplan-Meier curve was used to evaluate the prognostic value of HCCR-1 level in patients with ESCC using log-rank test. HCCR-1 displayed high levels in ESCC tissues compared to squamous dysplasia tissues and normal esophageal epithelial tissues. No significant correlation was observed between the levels of HCCR-1 mRNA and protein and gender and age (all p>0.05) but obviously related to histological grade, clinical stage, and lymph node metastasis (all p<0.001). Moreover, the survival rate of the patients with low HCCR-1 levels was higher than that of the patients with high HCCR-1 levels (both p<0.05). These data demonstrate that HCCR-1 may be used as a novel predictor for the prognosis of the patients with ESCC.     

Sequence-variant repeats of MUC1 show higher conformational flexibility, are less densely O-glycosylated and induce differential B lymphocyte responses

The human epithelial cancer mucin MUC1 is able to break tolerance and to induce humoral immune responses in healthy subjects and in cancer patients. We recently showed that clusters of sequence-variant repeats are interspersed in the repeat domain of MUC1 at high frequency, which should contribute to the structural and immunological features of the mucin. Here we elucidated the potential effects exerted by sequence-variant repeats on their O-glycosylation. Evidence from in vitro glycosylation with polypeptide N-acetylgalactosaminyltransferases GalNAc-T1 and GalNAc-T2 in concert with mass spectrometric analyses of in vivo glycosylated MUC1 probes from transiently transfected HEK293 cells indicated reduced glycosylation densities of repeats with three concerted replacements: AHGVTSAPESRPAPGSTAPA. The Pro to Ala replacement in STAPA exerts not only proximal effects on the ppGalNAc-T2 preferred site at -3 and -4, but also more distant effects on the ppGalNAc-T1 preferred site at -15 (TSAPESRPAPGSTAPA). We also examined the conformational changes of MUC1 glycopeptides induced by the concerted DT to ES replacements and revealed a higher conformational flexibility of ES/P peptides compared to DT/P peptides. Differences in conformational flexibilities and in O-glycosylation densities could underlie the observed differential humoral responses in humans. We were able to show that the natural immunoglobulin G (IgG) responses to the repeat domain of MUC1 in sera from nonmalignant control subjects are preferentially directed to variant repeat clusters. In contrast, the IgG response in patients with adenocarcinoma shifted to higher frequencies of preferential DTR peptide binding.     

Targeted Knockdown of IQGAP1 Inhibits the Progression of Esophageal Squamous Cell Carcinoma In Vitro and In Vivo

IQGAP1 is a scaffolding protein that can regulate several distinct signaling pathways. The accumulating evidence has demonstrated that IQGAP1 plays an important role in tumorigenesis and tumor progression. However, the function of IQGAP1 in esophageal squamous cell carcinoma (ESCC) has not been thoroughly investigated. In the present study, we showed that IQGAP1 was overexpressed in ESCC tumor tissues, and its overexpression was correlated with the invasion depth of ESCC. Importantly, by using RNA interference (RNAi) technology we successfully silenced IQGAP1 gene in two ESCC cell lines, EC9706 and KYSE150, and for the first time found that suppressing IQGAP1 expression not only obviously reduced the tumor cell growth, migration and invasion in vitro but also markedly inhibited the tumor growth, invasion, lymph node and lung metastasis in xenograft mice. Furthermore, Knockdown of IQGAP1 expression in ESCC cell lines led to a reversion of epithelial to mesenchymal transition (EMT) progress. These results suggest that IQGAP1 plays crucial roles in regulating ESCC occurrence and progression. IQGAP1 silencing may therefore develop into a promising novel anticancer therapy.     

AKR1C2 acts as a targetable oncogene in esophageal squamous cell carcinoma via activating PI3K/AKT signaling pathway

The aldo‐keto reductases family 1 member C2 (AKR1C2) has critical roles in the tumorigenesis and progression of malignant tumours. However, it was also discovered to have ambiguous functions in multiple cancers and till present, its clinical significance and molecular mechanism in oesophageal squamous cell carcinoma (ESCC) has been unclear. The aim of this study was to explore the role of AKR1C2 in the tumorigenesis of ESCC. Here, we showed that AKR1C2 expression was found to be up‐regulated in ESCC tissues and was significantly associated with pathological stage, lymph node metastasis and worse outcomes. Functional assays demonstrated that an ectopic expression of AKR1C2 in ESCC cells resulted in increased proliferation, migration and cisplatin resistance, while knockdown led to inversing effects. Bioinformation analyses and mechanistic studies demonstrated that AKR1C2 activated the PI3K/AKT signalling pathway, furthermore, the inhibitor of PI3K or the selective inhibitor of AKR1C2 enzyme activity could reverse the aggressiveness and showed synergistic antitumour effect when combined with cisplatin, both in vitro and in vivo. In conclusion, Our findings revealed that AKR1C2 could function as an oncogene by activating the PI3K/AKT pathway, as a novel prognostic biomarker and/or as a potential therapeutic target to ESCC.     

Engineering mammalian mucin-type O-glycosylation in plants

Mucin-type O-glycosylation is an important post-translational modification that confers a variety of biological properties and functions to proteins. This post-translational modification has a particularly complex and differentially regulated biosynthesis rendering prediction and control of where O-glycans are attached to proteins, and which structures are formed, difficult. Because plants are devoid of GalNAc-type O-glycosylation, we have assessed requirements for establishing human GalNAc O-glycosylation de novo in plants with the aim of developing cell systems with custom-designed O-glycosylation capacity. Transient expression of a Pseudomonas aeruginosa Glc(NAc) C4-epimerase and a human polypeptide GalNAc-transferase in leaves of Nicotiana benthamiana resulted in GalNAc O-glycosylation of co-expressed human O-glycoprotein substrates. A chimeric YFP construct containing a 3.5 tandem repeat sequence of MUC1 was glycosylated with up to three and five GalNAc residues when co-expressed with GalNAc-T2 and a combination of GalNAc-T2 and GalNAc-T4, respectively, as determined by mass spectrometry. O-Glycosylation was furthermore demonstrated on a tandem repeat of MUC16 and interferon α2b. In plants, prolines in certain classes of proteins are hydroxylated and further substituted with plant-specific O-glycosylation; unsubstituted hydroxyprolines were identified in our MUC1 construct. In summary, this study demonstrates that mammalian type O-glycosylation can be established in plants and that plants may serve as a host cell for production of recombinant O-glycoproteins with custom-designed O-glycosylation. The observed hydroxyproline modifications, however, call for additional future engineering efforts.     

In vivo glycosylation of MUC1 in airway epithelial cells

The O-glycans that decorate mucin glycoproteins contribute to the biophysical and biochemical properties of these molecules and hence their function as a barrier and lubricant on epithelial surfaces. Alterations in mucin O-glycosylation in certain diseases may contribute to pathology. It is known that both the host cell type and the amino acid sequence of the mucin tandem repeat contribute to the O-glycosylation of a mucin molecule. We expressed an epitope-tagged MUC1 mucin cDNA construct in the airway cell line 16HBE14o- and the colon carcinoma cell line Caco2 and used Fast Atom Bombardment Mass Spectrometry to evaluate the contribution of the host cell to differences in O-glycosylation of a single mucin. Many of the glycans detected on the MUC1 mucin were common to both cell types, as would be predicted from biosynthetic constraints. However, MUC1 synthesized in the airway cell line showed comparatively low levels of sialylation but carried a range of oligo-N-acetyllactosamine structures that were not seen in the colon carcinoma cell line.     

Association between MUC1 gene polymorphism and expected progeny differences in Nelore cattle (Bos primigenius indicus)

MUC1 is a heavily glycosylated mammalian transmembrane protein expressed by mucosal secretory tissues for both protection against microbial infection and lubrication. An important characteristic of MUC1 is its variable number of tandem repeats (VNTR) containing several sites for O-glycosylation. VNTR length has been associated with many human diseases and with certain economically important traits in domestic ruminants. The aim of the present study was to correlate the length of MUC1 gene VNTR with expected progeny differences (EPDs) obtained for growth, fertility and carcass traits. Five alleles were identified, with alleles containing short VNTRs being more frequent than those with long, thereby demonstrating that Brazilian Nelore cattle are characterized by high frequencies in short MUC1 VNTRs. Statistical analyses revealed there to be no significant association between VNTR length and EPDs for weight at 120 days (W(120) ), scrotal circumference at 365 (SC (365) ) and 450 (SC (450) ) days, age at first calving (AFC), and rib eye area (REA).