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CA125/MUC16 is dispensable for mouse development and reproduction.
ABSTRACT: Cancer antigen 125 (CA125) is a blood biomarker that is routinely used to monitor the progression of human epithelial ovarian cancer (EOC) and is encoded by MUC16, a member of the mucin gene family. The biological function of CA125/MUC16 and its potential role in EOC are poorly understood. Here we report the targeted disruption of the of the Muc16 gene in the mouse. To generate Muc16 knockout mice, 6.0 kb was deleted that included the majority of exon 3 and a portion of intron 3 and replaced with a lacZ reporter cassette. Loss of Muc16 protein expression suggests that Muc16 homozygous mutant mice are null mutants. Muc16 homozygous mutant mice are viable, fertile, and develop normally. Histological analysis shows that Muc16 homozygous mutant tissues are normal. By the age of 1 year, Muc16 homozygous mutant mice appear normal. Downregulation of transcripts from another mucin gene (Muc1) was detected in the Muc16 homozygous mutant uterus. Lack of any prominent abnormal phenotype in these Muc16 knockout mice suggests that CA125/MUC16 is not required for normal development or reproduction. These knockout mice provide a unique platform for future studies to identify the role of CA125/MUC16 in organ homeostasis and ovarian cancer.
Project description:BACKGROUND: MUC16 (CA125) is a large transmembrane mucin protein (> 200 kDa) aberrantly expressed in approximately 80% of human epithelial ovarian cancers (EOC). MUC16 expression in EOC cells is associated with increased tumorigenesis and inhibiton of genotoxic drug-induced apoptosis. However, the mechanism by which MUC16 mediates these effects is unknown. In the present study, we investigated the mechanisms by which MUC16 attenuates TRAIL-induced apoptosis. METHODS: MUC16 expression was down-regulated by stably expressing an anti-MUC16 single-chain antibody (scFv) targeted to the endoplasmic reticulum (ER), which prevents cell surface localization of MUC16 in OVCAR3 cells. We also generated a MUC16 C-terminal domain (MUC16CTD) construct that was stably expressed in MUC16 negative SKOV3 cells. RESULTS: We show that MUC16 attenuates apoptosis, activation of caspase-8 and mitochondria activation in EOC cells in response to TRAIL. MUC16 decreases TRAIL receptor R2 (DR5) expression and inhibits pro-caspase-8 activation at the death-inducing signaling complex (DISC). MUC16CTD expression is sufficient to attenuate the TRAIL signaling cascade. MUC16 knockdown decreases caspase-8 inhibitor cFLIP mRNA levels, increases cFLIP degradation, and consequently increases TRAIL-induced apoptosis. Down-regulation of cFLIP following treatment of MUC16-expressing OVCAR3 cells with cFLIP siRNA also increases TRAIL-induced apoptosis. CONCLUSIONS: These findings indicate that MUC16 protects EOC cells against TRAIL-induced apoptosis through multiple mechanisms including the blockade of TRAIL R2 expression and the regulation of cFLIP expression at both the transcriptional and the protein level.
Project description:Over three decades have passed since the first report on the expression of CA125 by ovarian tumors. Since that time our understanding of ovarian cancer biology has changed significantly to the point that these tumors are now classified based on molecular phenotype and not purely on histological attributes. However, CA125 continues to be, with the recent exception of HE4, the only clinically reliable diagnostic marker for ovarian cancer. Many large-scale clinical trials have been conducted or are underway to determine potential use of serum CA125 levels as a screening modality or to distinguish between benign and malignant pelvic masses. CA125 is a peptide epitope of a 3-5 million Da mucin, MUC16. Here we provide an in-depth review of the literature to highlight the importance of CA125 as a prognostic and diagnostic marker for ovarian cancer. We focus on the increasing body of literature describing the biological role of MUC16 in the progression and metastasis of ovarian tumors. Finally, we consider previous and on-going efforts to develop therapeutic approaches to eradicate ovarian tumors by targeting MUC16. Even though CA125 is a crucial marker for ovarian cancer, the exact structural definition of this antigen continues to be elusive. The importance of MUC16/CA125 in the diagnosis, progression and therapy of ovarian cancer warrants the need for in-depth research on the biochemistry and biology of this mucin. A renewed focus on MUC16 is likely to culminate in novel and more efficient strategies for the detection and treatment of ovarian cancer.
Project description:To examine single nucleotide polymorphism (SNPs) in MUC16 (CA125) and MUC1 (CA15.3) in relation to ovarian cancer risk and survival.We genotyped germline variants of MUC16 (rs2547065, rs1559168, rs12984471, rs2121133) and MUC1 (rs2070803, rs4072037, rs1045253) using samples collected from 758 ovarian cancer cases and 788 controls enrolled in the New England Case-Control Study between 2003 and 2008. We calculated age-adjusted odds ratios (OR) and 95% confidence intervals (CIs) for disease risk using unconditional and polytomous logistic regression and hazard ratios (HR) for survival using Cox proportional hazard ratios. In a subset of cases, we compared log-normalized CA125 values by genotype using generalized linear models.Cases homozygous for the variant allele of MUC16 SNP, rs12984471, had poorer overall survival (log-rank p = 0.03) and higher CA125 levels, especially cases over age 65 (p = 0.01). For MUC1 SNP, rs4072037, women homozygous for the G variant had a non-significantly decreased risk for serous invasive types but elevated risk for serous borderline tumors, mucinous borderline and invasive tumors, and endometrioid tumors. Women with the variant allele of MUC16 SNP, rs2547065, especially those who were homozygous had an elevated risk for ovarian cancer; but this association was not confirmed in an independent dataset.This targeted screen of seven polymorphisms of MUC16 and MUC1 genes failed to identify and confirm effects on ovarian cancer risk overall. However, there may be effects of MUC16 rs12984471 on survival and MUC1 rs4072037 on risk for histologic types of ovarian cancer other than invasive serous. Further study is warranted.
Project description:Mucin 16 (MUC16) is a type I transmembrane protein, the extracellular portion of which is shed after proteolytic degradation and is denoted as CA125 antigen, a well known tumor marker for ovarian cancer. Regarding its polypeptide and glycan structures, as yet there is no detailed insight into their heterogeneity and ligand properties, which may greatly influence its function and biomarker potential. This study was aimed at obtaining further insight into the biological capacity of MUC16/CA125, using in silico analysis of corresponding mucin sequences, including similarity searches as well as GO (gene ontology)-based function prediction. The results obtained pointed to the similarities within extracellular serine/threonine rich regions of MUC16 to sequences of proteins expressed in evolutionary distant taxa, all having in common an annotated role in adhesion-related processes. Specifically, a homology to conserved domains from the family of herpesvirus major outer envelope protein (BLLF1) was found. In addition, the possible involvement of MUC16/CA125 in carbohydrate-binding interactions or cellular transport of protein/ion was suggested.
Project description: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-1?, 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 ? 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.
Project description:CA125, the most widely used ovarian cancer biomarker, was first identified approximately 35 years ago in an antibody screen against ovarian cancer antigen. Two decades later, it was cloned and characterized to be a transmembrane mucin, MUC16. Since then, several studies have investigated its expression, functional, and mechanistic involvement in multiple cancer types. Antibody-based therapeutic approaches primarily using antibodies against the tandem repeat domains of MUC16 (e.g., oregovomab and abagovomab) have been the modus operandi for MUC16-targeted therapy, but have met with very limited success. In addition, efforts have been also made to disrupt the functional cooperation of MUC16 and its interacting partners; for example, use of a novel immunoadhesin HN125 to interfere MUC16 binding to mesothelin. Since the identification of CA125 to be MUC16, it is hypothesized to undergo proteolytic cleavage, a process that is considered to be critical in determining the kinetics of MUC16 shedding as well as generation of a cell-associated carboxyl-terminal fragment with potential oncogenic functions. In addition to our experimental demonstration of MUC16 cleavage, recent studies have demonstrated the functional importance of carboxyl terminal fragments of MUC16 in multiple tumor types. Here, we provide how our understanding of the basic biologic processes involving MUC16 influences our approach toward MUC16-targeted therapy.
Project description:High-grade epithelial ovarian cancer kills more women than any other gynecologic cancer and is rarely diagnosed at an early stage. We sought to identify tumor-associated antigens (TAA) as candidate diagnostic and/or immunotherapeutic targets by taking advantage of tumor autoantibody responses in individuals with ovarian cancer. Plasma-derived IgG from a pool of five patients with advanced ovarian cancer was subjected to iterative biopanning using a library of bacteriophage MS2 virus-like particles (MS2-VLPs) displaying diverse short random peptides. After two rounds of biopanning, we analyzed the selectant population of MS2-VLPs by Ion Torrent deep sequencing. One of the top 25 most abundant peptides identified (DISGTNTSRA) had sequence similarity to cancer antigen 125 (CA125/MUC16), a well-known ovarian cancer-associated antigen. Mice immunized with MS2-DISGTNTSRA generated antibodies that cross-reacted with purified soluble CA125 from ovarian cancer cells but not membrane-bound CA125, indicating that the DISGTNTSRA peptide was a CA125/MUC16 peptide mimic of soluble CA125. Preoperative ovarian cancer patient plasma (n = 100) was assessed for anti-DISGTNTSRA, anti-CA125, and CA125. Patients with normal CA125 (<35 IU/mL) at the time of diagnosis had significantly more antibodies to DISGTNTSRA and to CA125 than those patients who had high CA125 (>35 IU/mL). A statistically significant survival advantage was observed for patients who had either normal CA125 and/or higher concentrations of antibodies to CA125 at the time of diagnosis. These data show the feasibility of using deep sequence-coupled biopanning to identify TAA autoantibody responses from cancer patient plasma and suggest a possible antibody-mediated mechanism for low CA125 plasma concentrations in some ovarian cancer patients.
Project description:The ovarian tumor marker CA125 is expressed on human MUC16, a cell surface bound mucin that is also shed by proteolytic cleavage. Human MUC16 is overexpressed by ovarian cancer cells. MUC16 facilitates the binding of ovarian tumor cells to mesothelial cells lining the peritoneal cavity. Additionally, MUC16 also is a potent inhibitor of natural killer cell mediated anti-tumor cytotoxic responses. Extensive studies using human as well as murine ovarian tumor cell models are required to clearly define the function of MUC16 in the progression of ovarian tumors. The major objective of this study was to determine if the murine ovarian tumor cells, MOVCAR, express Muc16 and to characterize antibodies that recognize this mucin.RT-PCR analysis was used for detecting the Muc16 message and size exclusion column chromatography for isolating Muc16 produced by MOVCAR cells. Soluble and cell-associated murine Muc16 were analyzed, respectively, by Western blotting and flow cytometry assays using a new panel of antibodies. The presence of N-linked oligosaccharides on murine Muc16 was determined by ConA chromatography.We demonstrate that murine Muc16 is expressed by mouse ovarian cancer cells as an ~250 kDa glycoprotein that carries both O-linked and N-linked oligosaccharides. In contrast to human MUC16, the murine ortholog is primarily released from the cells and cannot be detected on the cell surface. Since the released murine Muc16 is not detected by conventional anti-CA125 assays, we have for the first time identified a panel of anti-human MUC16 antibodies that also recognizes the murine counterpart.The antibodies identified in this study can be used in future purification of murine Muc16 and exhaustive study of its properties. Furthermore, the initial identification and characterization of murine Muc16 is a vital preliminary step in the development of effective murine models of human ovarian cancer. These models will aid in the further elucidation of the role that human MUC16 plays in the etiology and progression of ovarian tumors.
Project description:The present study aimed to design and develop a nanocomposite drug delivery system employing an antineoplastic-loaded antibody functionalized nanomicelle encapsulated within a Chitosan?Poly(vinylpyrrolidone)?Poly(N-isopropylacrylamide) (C?P?N) hydrogel to form an in situ forming implant (ISFI), responsive to temperature and pH for cancer cell-targeting following intraperitoneal implantation. The optimum nanomicelle formulation was surface-functionalized with anti-MUC 16 (antibody) for the targeted delivery of methotrexate to human ovarian carcinoma (NIH:OVCAR-5) cells in Athymic nude mice that expressed MUC16, as a preferential form of intraperitoneal ovarian cancer (OC) chemotherapy. The cross-linked interpenetrating C?P?N hydrogel was synthesized for the preparation of an in situ-forming implant (ISFI). Subsequently, the ISFI was fabricated by encapsulating a nanocomposite comprising of anti-MUC16 (antibody) functionalized methotrexate (MTX)-loaded poly(N-isopropylacrylamide)-b-poly(aspartic acid) (PNIPAAm-b-PASP) nanomicelles (AF(MTX)NM's) within the cross-linked C?P?N hydrogel. This strategy enabled specificity and increased the residence time of the nanomicelles at tumor sites over a period exceeding one month, enhancing uptake of drugs and preventing recurrence and chemo-resistance. Chemotherapeutic efficacy was tested on the optimal ovarian tumor-bearing Athymic nude mouse model and the results demonstrated tumor regression including reduction in mouse weight and tumor size, as well as a significant (p < 0.05) reduction in mucin 16 levels in plasma and ascitic fluid, and improved survival of mice after treatment with the experimental anti-MUC16/CA125 antibody-bound nanotherapeutic implant drug delivery system (ISFI) (p < 0.05). The study also concluded that ISFI could potentially be considered an important immuno-chemotherapeutic agent that could be employed in human clinical trials of advanced, and/or recurring, metastatic epithelial ovarian cancer (EOC). The development of this ISFI may circumvent the treatment flaws experienced with conventional systemic therapies, effectively manage recurrent disease and ultimately prolong disease-free intervals in ovarian cancer patients.
Project description:The CA125 assay detects circulating MUC16 and is one of the most widely used cancer biomarkers for the follow-up of ovarian cancer. We previously demonstrated that detection of aberrant cancer-associated glycoforms of MUC16 as well as MUC1 in circulation could improve the yield of these serum assays. Our aim was to refine ovarian cancer biomarkers by detection of aberrant glycoforms (Tn, STn, and T) of MUC16 and MUC1 in ovarian cancer tissue using Proximity Ligation Assays (PLA). We studied two series of serous ovarian tumours, a pilot series of 66 ovarian tumours (27 cystadenomas, 16 borderline tumours and 23 adenocarcinomas) from Centro Hospitalar S. João, Porto and a validation series of 89 ovarian tumours (17 cystadenomas, 25 borderline tumours and 47 adenocarcinomas) from the Portuguese Institute of Oncology Francisco Gentil, Lisbon. PLA reactions for MUC16/Tn, MUC16/STn, MUC1/Tn and MUC1/STn were negative in benign lesions but often positive in borderline and malignant lesions, in both series. An even better yield was obtained based on positivity for any of the four glyco-mucin profiles, further increasing sensitivity to 72% and 83% in the two series, respectively, with 100% specificity. The strategy is designated glyco-mucin profiling and provides strong support for development of PLA-based serum assays for early diagnosis.