Search

Mucins play an essential role in protecting the respiratory tract against microbial infections while also acting as binding sites for bacterial and viral adhesins. The heavily O-glycosylated gel-forming mucins MUC5AC and MUC5B eliminate pathogens by mucociliary clearance. Transmembrane mucins MUC1, MUC4, and MUC16 can restrict microbial invasion at the apical surface of the epithelium. In this study, we determined the impact of host mucins and mucin glycans on epithelial entry of SARS-CoV-2. Human lung epithelial Calu-3 cells express the SARS-CoV-2 entry receptor ACE2 and high levels of glycosylated MUC1, but not MUC4 and MUC16, on their cell surface. The O-glycan-specific mucinase StcE specifically removed the glycosylated part of the MUC1 extracellular domain while leaving the underlying SEA domain and cytoplasmic tail intact. StcE treatment of Calu-3 cells significantly enhanced infection with SARS-CoV-2 pseudovirus and authentic virus, while removal of terminal mucin glycans sialic acid and fucose from the epithelial surface did not impact viral entry. In Calu-3 cells, the transmembrane mucin MUC1 and ACE2 are located to the apical surface in close proximity and StcE treatment results in enhanced binding of purified spike protein. Both MUC1 and MUC16 are expressed on the surface of human organoid-derived air-liquid interface (ALI) differentiated airway cultures and StcE treatment led to mucin removal and increased levels of SARS-CoV-2 replication. In these cultures, MUC1 was highly expressed in non-ciliated cells while MUC16 was enriched in goblet cells. In conclusion, the glycosylated extracellular domains of different transmembrane mucins might have similar protective functions in different respiratory cell types by restricting SARS-CoV-2 binding and entry., Competing Interests: The authors declare no competing interests., (Copyright: © 2023 Chatterjee et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Subsets of tumor-produced cell surface and secreted proteins can bind to IgG1 type antibodies and suppress their immune-effector activities. As they affect antibody and complement-mediated immunity, we call these proteins humoral immuno-oncology (HIO) factors. Antibody-drug conjugates (ADCs) use antibody targeting to bind cell surface antigens, internalize into the cell, then kill target cells upon liberation of the cytotoxic payload. Binding of the ADC antibody component by a HIO factor may potentially hamper ADC efficacy due to reduced internalization. To determine the potential effects of HIO factor ADC suppression, we evaluated the efficacy of a HIO-refractory, mesothelin-directed ADC (NAV-001) and a HIO-bound, mesothelin-directed ADC (SS1). The HIO factor MUC16/CA125 binding to SS1 ADC was shown to have a negative effect on internalization and tumor cell killing. The MUC16/CA125 refractory NAV-001 ADC was shown to have robust killing of MUC16/CA125 expressing and non-expressing tumor cells in vitro and in vivo at single, sub-mg/kg dosing. Moreover, NAV-001-PNU, which contains the PNU-159682 topoisomerase II inhibitor, demonstrated good stability in vitro and in vivo as well as robust bystander activity of resident cells while maintaining a tolerable safety profile in vivo. Single-dose NAV-001-PNU demonstrated robust tumor regression of a number of patient-derived xenografts from different tumor types regardless of MUC16/CA125 expression. These findings suggest that identification of HIO-refractory antibodies to be used in ADC format may improve therapeutic efficacy as observed by NAV-001 and warrants NAV-001-PNU's advancement to human clinical trials as a monotherapy to treat mesothelin-positive cancers., Competing Interests: All authors are employees of Navrogen Inc. The authors have declared that no competing interests exist. This affiliation does not alter our adherence to all PLOS ONE policies on sharing data and materials., (Copyright: © 2023 Nicolaides et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

MUC16 is overexpressed in ovarian cancer and plays important roles in invasion and metastasis. Previously described monoclonal antibodies against cell surface expressed MUC16 recognize the N-terminal tandemly repeated epitopes present in cancer antigen 125 (CA125). MUC16 is cleaved at a specific location, thus, releasing CA125 into the extracellular space. Recent reports have indicated that the retained carboxy-terminal (CT) fragment of MUC16 might play an important role in tumorigenicity in diverse types of cancers. However, limited data is available on the fate and existence of CT fragment on the surface of the cancer cell. Herein, we characterize two monoclonal antibodies (mAbs) showing specificity to the retained juxtamembrane region of MUC16. For the first time, we demonstrate that MUC16 is cleaved in ovarian cancer cells (NIH:OVCAR-3 [OVCAR-3]) and that the cleaved MUC16 subunits remain associated with each other. Immunohistochemical analyses on different grades of ovarian tumor tissues indicated differential reactivity of CA125 and MUC16 CT mAbs. The CA125 (M11) mAb detected 32/40 (80%), while the CT mAb (5E6) detected 33/40 (82.5%) of total ovarian cancer cases. For serous and serous papillary cases, the CA125 (M11) mAb stained 27/31 cases (87%), while CT mAb (5E6) stained 29/31 cases (93.5%). The CT mAb(s) accurately predict expression of MUC16 since their epitopes are not tandemly repeated and their reactivity may not be dependent on O-linked glycosylation. These antibodies can serve as valuable reagents for understanding MUC16 cleavage and may also serve as potential therapeutic agents for treatment of ovarian cancer.

The use of bacteriophages to select novel ligands has been widely explored for cancer therapy. Their application is most warranted in cancer subtypes lacking knowledge on how to target the cancer cells in question, such as the triple negative breast cancer, eventually leading to the development of alternative nanomedicines for cancer therapeutics. Therefore, the following study aimed to select and characterize novel peptides for a triple negative breast cancer murine mammary carcinoma cell line- 4T1. Using phage display, 7 and 12 amino acid random peptide libraries were screened against the 4T1 cell line. A total of four rounds, plus a counter-selection round using the 3T3 murine fibroblast cell line, was performed. The enriched selective peptides were characterized and their binding capacity towards 4T1 tissue samples was confirmed by immunofluorescence and flow cytometry analysis. The selected peptides (4T1pep1 -CPTASNTSC and 4T1pep2-EVQSSKFPAHVS) were enriched over few rounds of selection and exhibited specific binding to the 4T1 cell line. Interestingly, affinity to the human MDA-MB-231 cell line was also observed for both peptides, promoting the translational application of these novel ligands between species. Additionally, bioinformatics analysis suggested that both peptides target human Mucin-16. This protein has been implicated in different types of cancer, as it is involved in many important cellular functions. This study strongly supports the need of finding alternative targeting systems for TNBC and the peptides herein selected exhibit promising future application as novel homing peptides for breast cancer therapy., Competing Interests: The authors declare they have no competing interests or other interests that might be perceived to influence the results and discussion herein reported.

Background: The aim of this study was to assess the diagnostic values of three breast tumor markers (i.e., CEA, CA153 and CA125) in milk and nipple discharge in the prediction of different breast diseases diagnoses., Methods: Three hundred thirty-six patients (96 breast cancer and 240 benign disease patients) with nipple discharge and a control group of 56 healthy parturient participants were enrolled in the present study. Nipple discharge samples were preoperatively collected from the patients, and milk was collected from the colostrum of the parturient participants. The samples were assayed for the CEA, CA153 and CA125 levels. Cutoff values were determined for the detection of breast diseases using ROC curves., Results: The levels of CEA, CA153 and CA125 were significantly different between the nipple discharge and the milk (all ps < 0.001). In the nipple discharge, the CEA and CA153 levels in the breast cancer group were significantly greater than those in the benign group (all ps < 0.001), and cutoff values of 263.3 ng/mL and 1235.3 U/mL, respectively, were established. However, the expression of CA125 did not differ significantly between the breast cancer and benign groups., Conclusion: Differences in the apparent expression levels of CEA, CA153 and CA125 in patients with nipple discharge and healthy persons were validated. The present data suggest that CEA and CA153 might potentially be useful in the differential diagnoses of benign tumors and breast cancer. CA125 did not seem to be useful for breast cancer detection.

Epithelial ovarian cancer is one of the increasingly incident malignancies that is notorious because of its evasiveness for early diagnosis and high mortality rates. Epithelial ovarian cancers are highly dependent on pathologic vasculature and Vascular Endothelial Growth Factor is known to be one of the most efficient angiogenic factors. Polymorphisms of the VEGF gene, in this study, were assessed for association with the malignancy and other clinico-pathological factors. 300 case samples and 320 age and mensus status matched controls were inculcated into the study. rs699947, rs833061, rs1570360, rs2010963, rs1413711 and rs3025039 were the six single nucleotide polymorphisms that were scrutinized. Genotyping was carried out by polymerase chain reaction and restriction fragment length polymorphism. rs 3025039 showed immense promise as a marker for disease aggression and recurrence and a factor for poor prognosis. rs699947 showed least association with the disease and clinico-pathologic factors studied. rs833061, rs 1570360 showed significant association with some clinico-pathological factors such as bilateral affliction of ovaries and post operative CA-125 levels. rs2010963 associated with presence of ascites in higher volumes. The SNPs under consideration showed no formidable linkage in our study samples. A haplotype analysis (excluding rs699947 and rs1413711) revealed 5 frontrunners being present in >85% of the population with TGGC and CGCC associating significantly as protective and risk factors respectively. These haplotypes showed a dose dependent additive effect of their seeming functionality. This study is unique and a first of its kind carried out in the Indian population of South-east Asia.

The CA125 antigen is found in the serum of many patients with serous ovarian cancer and has been widely used as a disease marker. CA125 has been shown to be an independent factor for clinical outcome in this disease. In The Cancer Genome Atlas ovarian cancer project, MUC16 expression levels are frequently increased, and the highest levels of MUC16 expression are linked to a significantly worse survival. To examine the biologic effect of the proximal portion of MUC16/CA125, NIH/3T3 (3T3) fibroblast cell lines were stably transfected with the carboxy elements of MUC16. As few as 114 amino acids from the carboxy-terminal portion of MUC16 were sufficient to increase soft agar growth, promote matrigel invasion, and increase the rate of tumor growth in athymic nude mice. Transformation with carboxy elements of MUC16 was associated with activation of the AKT and ERK pathways. MUC16 transformation was associated with up-regulation of a number of metastases and invasion gene transcripts, including IL-1β, MMP2, and MMP9. All observed oncogenic changes were exclusively dependent on the extracellular "ectodomain" of MUC16. The biologic impact of MUC16 was also explored through the creation of a transgenic mouse model expressing 354 amino acids of the carboxy-terminal portion of MUC16 (MUC16c354). Under a CMV, early enhancer plus chicken β actin promoter (CAG) MUC16c354 was well expressed in many organs, including the brain, colon, heart, kidney, liver, lung, ovary, and spleen. MUC16c354 transgenic animals appear to be viable, fertile, and have a normal lifespan. However, when crossed with p53-deficient mice, the MUC16c354:p53+/- progeny displayed a higher frequency of spontaneous tumor development compared to p53+/- mice alone. We conclude that the carboxy-terminal portion of the MUC16/CA125 protein is oncogenic in NIH/3T3 cells, increases invasive tumor properties, activates the AKT and ERK pathways, and contributes to the biologic properties of ovarian cancer.

Membrane-anchored mucins are present in the apical surface glycocalyx of mucosal epithelial cells, each mucosal epithelium having at least two of the mucins. The mucins have been ascribed barrier functions, but direct comparisons of their functions within the same epithelium have not been done. In an epithelial cell line that expresses the membrane-anchored mucins, MUC1 and MUC16, the mucins were independently and stably knocked down using shRNA. Barrier functions tested included dye penetrance, bacterial adherence and invasion, transepithelial resistance, tight junction formation, and apical surface size. Knockdown of MUC16 decreased all barrier functions tested, causing increased dye penetrance and bacterial invasion, decreased transepithelial resistance, surprisingly, disruption of tight junctions, and greater apical surface cell area. Knockdown of MUC1 did not decrease barrier function, in fact, barrier to dye penetrance and bacterial invasion increased significantly. These data suggest that barrier functions of membrane-anchored mucins vary in the context of other membrane mucins, and MUC16 provides a major barrier when present.

Mucins have been associated with survival in various cancer patients, but there have been no studies of mucins in small bowel carcinoma (SBC). In this study, we investigated the relationships between mucin expression and clinicopathologic factors in 60 SBC cases, in which expression profiles of MUC1, MUC2, MUC3, MUC4, MUC5AC, MUC6 and MUC16 in cancer and normal tissues were examined by immunohistochemistry. MUC1, MUC5AC and MUC16 expression was increased in SBC lesions compared to the normal epithelium, and expression of these mucins was related to clinicopathologic factors, as follows: MUC1 [tumor location (p = 0.019), depth (p = 0.017) and curability (p = 0.007)], MUC5AC [tumor location (p = 0.063) and lymph node metastasis (p = 0.059)], and MUC16 [venous invasion (p = 0.016) and curability (p = 0.016)]. Analysis of 58 cases with survival data revealed five factors associated with a poor prognosis: poorly-differentiated or neuroendocrine histological type (p<0.001), lymph node metastasis (p<0.001), lymphatic invasion (p = 0.026), venous invasion (p<0.001) and curative resection (p<0.001), in addition to expression of MUC1 (p = 0.042), MUC5AC (p = 0.007) and MUC16 (p<0.001). In subsequent multivariate analysis with curability as the covariate, lymph node metastasis, venous invasion, and MUC5AC and/or MUC16 expression were significantly related to the prognosis. Multivariate analysis in curative cases (n = 45) showed that SBC with MUC5AC and/or MUC16 expression had a significantly independent high hazard risk after adjusting for the effects of venous invasion (hazard ratio: 5.6, 95% confidence interval: 1.8-17). In conclusion, the study shows that a MUC5AC-positive and/or MUC16-positive status is useful as a predictor of a poor outcome in patients with SBC.

Background: Bone morphogenetic protein receptor I B (BMPR1B) is a transmembrane receptor mediating TGF-β signal transduction. Recent studies indicate a tumor suppressor role for BMPR1B in ovarian cancer. Polymorphism at BMPR1B 3'UTR within the miR-125b binding site alters its binding affinity toward the miRNA, which may result in insufficient post-transcriptional repression., Methods: Single-nucleotide polymorphisms rs1970801, rs1434536, and rs11097457 near the miR-125b binding site in BMPR1B were genotyped by Taqman assay on 193 endometriosis patients and 202 healthy controls. BMPR1B and CA125 levels in ectopic endometrial tissues were evaluated by quantitative PCR and immunohistochemistry. Luciferase reporter assay was utilized to verify regulatory roles of BMPR1B 3'UTR with allelic variants of rs1434536 in a cell line model. Cell proliferation and migration were recorded, while expression of BMPR1B, CA125, glucocorticoid receptor (GCCR) and IL-1β were measured by quantitative PCR in endometrial cells transfected with wild-type or mutated miR-125b., Results: This study found two endometriosis-associated SNPs, rs1434536 (P = 0.010) and rs1970801 (P = 0.0087), located within and next to a miR-125b binding site on BMPR1B. Interestingly, patients with homozygous variant alleles at rs1434536 showed significantly lower serum CA125 levels. Immunohistochemistry staining further confirmed inverse correlation between BMPR1B and CA125 levels in three rs1434536 genotypes. Cell assays demonstrated the variant allele of rs1434536 up-regulating BMPR1B at both mRNA and protein levels, which negatively correlated with CA125 and IL-1β levels. Disruption of the binding between miR-125b and BMPR1B hampered abnormal cell proliferation., Conclusions: SNPs of BMPR1B within and next to the miR-125b binding site manifested strong correlation with endometriosis development in a Taiwanese cohort. Disrupting the binding of miR-125b toward BMPR1B would increase protein expression, diminishing abnormal cell proliferation as well as serum and cellular CA125 levels. Genetic variation at the miR-125b binding site may play functional roles to protect against endometriosis progression.

Transmembrane mucins, MUC4 and MUC16 are associated with tumor progression and metastatic potential in human pancreatic adenocarcinoma. We discovered that miR-200c interacts with specific sequences within the coding sequence of MUC4 and MUC16 mRNAs, and evaluated the regulatory nature of this association. Pancreatic cancer cell lines S2.028 and T3M-4 transfected with miR-200c showed a 4.18 and 8.50 fold down regulation of MUC4 mRNA, and 4.68 and 4.82 fold down regulation of MUC16 mRNA compared to mock-transfected cells, respectively. A significant reduction of glycoprotein expression was also observed. These results indicate that miR-200c overexpression regulates MUC4 and MUC16 mucins in pancreatic cancer cells by directly targeting the mRNA coding sequence of each, resulting in reduced levels of MUC4 and MUC16 mRNA and protein. These data suggest that, in addition to regulating proteins that modulate EMT, miR-200c influences expression of cell surface mucins in pancreatic cancer.

Membrane bound mucins are up-regulated and aberrantly glycosylated during malignant transformation in many cancer cells. This results in a negatively charged glycoprotein coat which may protect cancer cells from immune surveillance. However, only limited data have so far demonstrated the critical steps in glycan elongation that make aberrantly glycosylated mucins affect the interaction between cancer cells and cytotoxic effector cells of the immune system. Tn (GalNAc-Ser/Thr), STn (NeuAcα2-6GalNAc-Ser/Thr), T (Galβ1-3GalNAc-Ser/Thr), and ST (NeuAcα2-6Galβ1-3GalNAc-Ser/Thr) antigens are recognized as cancer associated truncated glycans, and are expressed in many adenocarcinomas, e.g. breast- and pancreatic cancer cells. To investigate the role of the cancer associated glycan truncations in immune-mediated killing we created glyco-engineered breast- and pancreatic cancer cells expressing only the shortest possible mucin-like glycans (Tn and STn). Glyco-engineering was performed by zinc finger nuclease (ZFN) knockout (KO) of the Core 1 enzyme chaperone COSMC, thereby preventing glycan elongation beyond the initial GalNAc residue in O-linked glycans. We find that COSMC KO in the breast and pancreatic cancer cell lines T47D and Capan-1 increases sensitivity to both NK cell mediated antibody-dependent cellular-cytotoxicity (ADCC) and cytotoxic T lymphocyte (CTL)-mediated killing. In addition, we investigated the association between total cell surface expression of MUC1/MUC16 and NK or CTL mediated killing, and observed an inverse correlation between MUC16/MUC1 expression and the sensitivity to ADCC and CTL-mediated killing. Together, these data suggest that up-regulation of membrane bound mucins protects cells from immune mediated killing, and that particular glycosylation steps, as demonstrated for glycan elongation beyond Tn and STn, can be important for fine tuning of the immune escape mechanisms in cancer cells.

The majority of bacterial infections occur across wet-surfaced mucosal epithelia, including those that cover the eye, respiratory tract, gastrointestinal tract and genitourinary tract. The apical surface of all these mucosal epithelia is covered by a heavily glycosylated glycocalyx, a major component of which are membrane-associated mucins (MAMs). MAMs form a barrier that serves as one of the first lines of defense against invading bacteria. While opportunistic bacteria rely on pre-existing defects or wounds to gain entry to epithelia, non opportunistic bacteria, especially the epidemic disease-causing ones, gain access to epithelial cells without evidence of predisposing injury. The molecular mechanisms employed by these non opportunistic pathogens to breach the MAM barrier remain unknown. To test the hypothesis that disease-causing non opportunistic bacteria gain access to the epithelium by removal of MAMs, corneal, conjunctival, and tracheobronchial epithelial cells, cultured to differentiate to express the MAMs, MUCs 1, 4, and 16, were exposed to a non encapsulated, non typeable strain of Streptococcus pneumoniae (SP168), which causes epidemic conjunctivitis. The ability of strain SP168 to induce MAM ectodomain release from epithelia was compared to that of other strains of S. pneumoniae, as well as the opportunistic pathogen Staphylococcus aureus. The experiments reported herein demonstrate that the epidemic disease-causing S. pneumoniae species secretes a metalloproteinase, ZmpC, which selectively induces ectodomain shedding of the MAM MUC16. Furthermore, ZmpC-induced removal of MUC16 from the epithelium leads to loss of the glycocalyx barrier function and enhanced internalization of the bacterium. These data suggest that removal of MAMs by bacterial enzymes may be an important virulence mechanism employed by disease-causing non opportunistic bacteria to gain access to epithelial cells to cause infection.

Stromal elements within a tumor interact with cancer cells to create a microenvironment that supports tumor growth and survival. Adrenomedullin (ADM) is an autocrine/paracrine factor produced by both stromal cells and cancer cells to create such a microenvironment. During differentiation of macrophages, ADM is produced in response to pro-inflammatory stimuli and hypoxia. In this study we investigated the role of ADM as a growth factor for ovarian cancer cells and as a modulator of macrophages. We also analyzed ADM expression levels in a retrospective clinical study using nanofluidic technology and assessment of ADM at the gene level in 220 ovarian cancer patients. To study the effects of ADM, ovarian cancer cell lines A2780, OVCAR-3, and HEY and their drug-resistant counterparts were used for proliferation assays, while monocytes from healthy donors were differentiated in vitro. ADM was a weak growth factor, as revealed by proliferation assays and cell cycle analysis. After culturing cancer cells under stressing conditions, such as serum starvation and/or hypoxia, ADM was found to be a survival factor in HEY but not in other cell lines. In macrophages, ADM showed activity on proliferation/differentiation, primarily in type 2 macrophages (M2). Unexpectedly, the clinical study revealed that high expression of ADM was linked to positive outcome and to cancer with low Ca125. In conclusion, although in vitro ADM was a potential factor in biological aggressiveness, this possibility was not confirmed in patients. Therefore, use of an ADM antagonist would be inappropriate in managing ovarian cancer patients.

Ovarian cancer is the most common cause of death from gynecological cancer. Understanding the biology of this disease, particularly how tumor-associated lymphocytes and fibroblasts contribute to the progression and metastasis of the tumor, has been impeded by the lack of a suitable tumor xenograft model. We report a simple and reproducible system in which the tumor and tumor stroma are successfully engrafted into NOD-scid IL2Rγ(null) (NSG) mice. This is achieved by injecting tumor cell aggregates derived from fresh ovarian tumor biopsy tissues (including tumor cells, and tumor-associated lymphocytes and fibroblasts) i.p. into NSG mice. Tumor progression in these mice closely parallels many of the events that are observed in ovarian cancer patients. Tumors establish in the omentum, ovaries, liver, spleen, uterus, and pancreas. Tumor growth is initially very slow and progressive within the peritoneal cavity with an ultimate development of tumor ascites, spontaneous metastasis to the lung, increasing serum and ascites levels of CA125, and the retention of tumor-associated human fibroblasts and lymphocytes that remain functional and responsive to cytokines for prolonged periods. With this model one will be able to determine how fibroblasts and lymphocytes within the tumor microenvironment may contribute to tumor growth and metastasis, and will make it possible to evaluate the efficacy of therapies that are designed to target these cells in the tumor stroma.

MUC16 (CA125) belongs to a family of high-molecular weight O-glycosylated proteins known as mucins. While MUC16 is well known as a biomarker in ovarian cancer, its expression pattern in pancreatic cancer (PC), the fourth leading cause of cancer related deaths in the United States, remains unknown. The aim of our study was to analyze the expression of MUC16 during the initiation, progression and metastasis of PC for possible implication in PC diagnosis, prognosis and therapy. In this study, a microarray containing tissues from healthy and PC patients was used to investigate the differential protein expression of MUC16 in PC. MUC16 mRNA levels were also measured by RT-PCR in the normal human pancreatic, pancreatitis, and PC tissues. To investigate its expression pattern during PC metastasis, tissue samples from the primary pancreatic tumor and metastases (from the same patient) in the lymph nodes, liver, lung and omentum from Stage IV PC patients were analyzed. To determine its association in the initiation of PC, tissues from PC patients containing pre-neoplastic lesions of varying grades were stained for MUC16. Finally, MUC16 expression was analyzed in 18 human PC cell lines. MUC16 is not expressed in the normal pancreatic ducts and is strongly upregulated in PC and detected in pancreatitis tissue. It is first detected in the high-grade pre-neoplastic lesions preceding invasive adenocarcinoma, suggesting that its upregulation is a late event during the initiation of this disease. MUC16 expression appears to be stronger in metastatic lesions when compared to the primary tumor, suggesting a role in PC metastasis. We have also identified PC cell lines that express MUC16, which can be used in future studies to elucidate its functional role in PC. Altogether, our results reveal that MUC16 expression is significantly increased in PC and could play a potential role in the progression of this disease.