MUC16 Knockdown Research Articles

MUC1, a New Hypoxia Inducible Factor Target Gene, Is an Actor in Clear Renal Cell Carcinoma Tumor Progression

The hypoxia inducible factor (HIF) signaling pathway is known as the main renal carcinogenetic pathway. MUC1, an O-glycoprotein membrane-bound mucin, is overexpressed in clear renal cell carcinomas (cRCC) with correlation to two major prognostic factors: tumor-node-metastasis stage and nuclear Fürhman grade. We questioned whether there is a direct link between the HIF pathway and MUC1 overexpression in renal tumors. Interestingly, we observed concomitant increase of HIF-1alpha and MUC1 in metastatic cRCC group versus nonmetastatic cRCC group. Using different renal cell models and small interfering RNA assays targeting either HIF-1alpha or YC-1, a HIF-1 pharmacologic inhibitor, we showed induction of MUC1 expression under hypoxia by a HIF-dependent mechanism. Chromatin immunoprecipitation assay showed a direct binding of HIF-1alpha at the MUC1 promoter. In addition, combined site-directed mutagenesis and gel shift assay allowed the identification of two functional putative hypoxia responsive elements at -1488/-1485 and at -1510/-1507 in the promoter. Using a rat kidney model of ischemia/reperfusion, we confirmed in vivo that clamping renal pedicle for 1 hour followed by 2 hours of reperfusion induced increased MUC1 expression. Furthermore, MUC1 knockdown induced significant reduction of invasive and migration properties of renal cancer cells under hypoxia. Altogether, these results show that MUC1 is directly regulated by HIF-1alpha and affects the invasive and migration properties of renal cancer cells. Thus, MUC1 could serve as a potential therapeutic target in cRCC.

MUC1 is a downstream target of STAT3 and regulates lung cancer cell survival and invasion

Signal transducer and activator of transcription 3 (STAT3) is aberrantly activated in human cancer including lung cancer and has been implicated in transformation, tumorigenicity, and metastasis. One putative downstream gene regulated by Stat3 is MUC1 which also has important roles in tumorigenesis. We determined if Stat3 regulates MUC1 in lung cancer cell lines and what function MUC1 plays in lung cancer cell biology. We examined MUC1 expression in non-small cell lung cancer (NSCLC) cell lines and found high levels of MUC1 protein expression associated with higher levels of tyrosine phosphorylated STAT3. STAT3 knockdown downregulated MUC1 expression whereas constitutive STAT3 expression increased MUC1 expression at mRNA and protein levels. MUC1 knockdown induced cellular apoptosis concomitant with reduced Bcl-XL and sensitized cells to cisplatin treatment. MUC1 knockdown inhibited tumor growth and metastasis in an orthotopic mouse model of lung cancer by activating apoptosis and inhibiting cell proliferation in vivo. These results demonstrate that constitutively activated STAT3 regulates expression of MUC1, which mediates lung cancer cell survival and metastasis in vitro and in vivo. MUC1 appears to be a cooperating oncoprotein with multiple oncogenic tyrosine kinase pathways and could be an effective target for the treatment of lung cancer.

HSP70 STABILIZES THE LYSOSOMES BY TRANSPORTING MUC1 TO THE LYSOSOMES

We have previously shown that HSP70 is overexpressed in pancreatic cancer cells and human pancreatic cancer specimens and that inhibition of its expression leads to apoptotic cell death. Studies in our laboratory have also shown that inhibition of HSP70 expression leads lysosomal permeability with the activation of apoptotic cascade, thus suggesting that HSP70 stabilizes the lysosomes. However, the mechanism by which HSP70 stabilizes these two organelles and thus inhibits apoptosis is unclear. The aim ofthis study was to elucidate the role of MUC1, a mucin glycoprotein which is expressed in pancreatic cancer cells, in the stabilization of lysosomes and evaluating its interaction with HSP70. We hypothesize that HSP70 carries MUC1 to lysosomes and mitochondria which then stabilizes the lysosomes. Method: HSP70 and MUC1 were immunoprecipitated from the pancreatic cancer cell lysates. The immunoprecipitates where then immunoblotted for MUC1 and HSP70. To study the localization of MUC1 to lysosomes, co-localization of MUC1 and LAMP-2 (a lysosomal marker) was studied in pancreatic cancer cells by confocal microscopy. Effect of MUC1 knockdown by siRNA was studied by CCK-8 assay. Results: HSP70 and MUC1 co-immunoprecipitate suggesting their interaction. Confocal microscopy demonstrated co-localization of MUC1 and LAMP-1 suggesting that MUC1 localizes to lysosomes. Inhibition of MUC1 expression by siRNA leads to decreased viability in Panc-1 pancreatic cancer cells (54 ± 6 % of control treated with transfection reagent Lipofectamine 2000 only). Conclusion: the results suggest that MUC1 may play a role in the mechanism by which HSP70 stabilizes the lysosomes. This is the first report suggesting that MUC1 localizes to the lysosomes and may play a role in its stabilization. Implication: MUC1 may prove to be a potential target for the treatment of pancreatic cancer.

MUC16 Is Lost from the Uterodome (Pinopode) Surface of the Receptive Human Endometrium: In Vitro Evidence That MUC16 Is a Barrier to Trophoblast Adherence1

In order for the preimplantation embryo to implant into the uterus, the trophoblast cells must initially adhere to the uterine epithelial surface. In preparation, the luminal secretory cells of the epithelium lose their nonadhesive character and their surface microvilli and bulge into the lumen, forming uterodomes (pinopodes; uterodome is used instead of pinopode, since in humans the surface membrane exocytoses rather than endocytoses (Murphy, Hum Reprod 2000; 15:2451-2454). Previous research has led to the hypothesis that loss of the nonadhesive membrane-spanning mucin MUC1 from the uterodome surface allows trophoblast adherence. Immunofluorescence microscopic assay of luminal epithelia on human uterine biopsies taken from LH+0 to LH+13 show that another membrane-spanning mucin, MUC16, was lost from uterodome surfaces in all samples taken during the receptive phase, LH+6 to LH+8 (n = 12), and that MUC1 was present on uterodomes in 4 of 12 samples and on all ciliated cells of the epithelium in the receptive phase. Short interfering RNA (siRNA) knockdown of MUC16 in a uterine epithelial cell line ECC-1 that, like uterine epithelium, expresses MUC16 and MUC1 allowed increased adherence of cells of a trophoblast cell line. In parallel experiments, siRNA knockdown of MUC1 did not affect trophoblast cell adherence. These data indicate that MUC16 is a membrane component of the nonreceptive luminal uterine surface, which prevents cell adhesion, and that its removal during uterodome formation facilitates adhesion of the trophoblast.

Functions of MUC16 in Corneal Epithelial Cells

The membrane-associated mucin MUC16, a heavily O-glycosylated transmembrane protein, is expressed by the ocular surface epithelia and localized on the tips of the surface microplicae. Although its functions in the ocular surface glycocalyx are unknown, it is thought that MUC16 provides a disadhesive barrier to the epithelial membrane. Two other membrane-associated mucins expressed by ocular surface epithelia, MUC1 and MUC4, are multifunctional and have signaling capabilities through their cytoplasmic tails and EGF-like domains, respectively. The MUC16 cytoplasmic tail has not been characterized, but, because it contains a polybasic amino acid sequence, it potentially interacts with the actin cytoskeleton through ezrin/radixin/moesin (ERM) actin-binding proteins. The interaction of MUC16 with the actin cytoskeleton through ERMs was investigated using cytoplasmic tail peptides and ERM pull-down experiments. MUC16 functions were determined using RNA interference in immortalized human corneal-limbal epithelial (HCLE) cells. The effect of MUC16 knockdown on microplicae structure in HCLE cells was determined using scanning and immunoelectron microscopy. HCLE cells were incubated with rose bengal dye to measure the role of MUC16 in ocular surface barrier function. Binding of fluorescently labeled Staphylococcus aureus to HCLE cells was measured to determine the role of MUC16 in the protection of pathogen adherence on the ocular surface epithelium. MUC16 cytoplasmic tail peptides bound the N-terminus of ERMs, with no detectable binding of MUC1 and MUC4 peptides. No effect on surface membrane projections could be detected in HCLE cells after MUC16 suppression; however, HCLE cells incubated with rose bengal showed that exclusion of the dye was significantly reduced in cells with MUC16 suppression. In addition, S. aureus binding to HCLE cells was significantly increased with MUC16 suppression. These results suggest that MUC16 is a multifunctional molecule linked to the actin cytoskeleton. The expression of MUC16 in the ocular surface glycocalyx helps provide a disadhesive protective barrier for the epithelial surface.

MUC1 Mucin Expressed no Activated T Cells has a Regulatory Function (87.17)

Abstract MUC1 (CD227) is a large (>400kDa), heavily glycosylated, type-I transmembrane protein present most notably on the apical surface of ductal epithelia. In epithelial adenocarcinomas MUC1 is overexpressed as a hypoglycosylated protein in a non-polarized fashion. Because of these attributes, MUC1 has been studied foremostly as a tumor-associated antigen. The role of MUC1 in other cell types remained largely unexplored until MUC1 was found to be expressed by activated human T cells from healthy donors. Preliminary studies we have performed on the role of MUC1 in T cells point towards MUC1 as a negative regulator of effector T cell function. Crosslinking of MUC1 limits the proliferative capacity of primary human T cells activated with anti-CD3, as well as the ability of these cells to make IL-2 and upregulate IL-2Rα. Studies using the Jurkat T cell line have yielded intriguing results. When MUC1 is present and crosslinked, Jurkat T cells activated with anti-CD3 do not secrete IL-2. Paradoxically, knockdown of MUC1 in Jurkat T cells with siRNA also renders them unable to produce IL-2 when similarly activated. Because failure to secrete IL-2 and proliferate following TCR ligation are characteristics of T cell hyporesponsiveness, elucidating the exact role of MUC1 in T cell activation in the context of other co-inhibitory or co-activating receptors is important. To do so, we have been employing luciferase reporter assays to determine the effect of MUC1 expression and crosslinking on well-characterized signaling pathways activated during the engagement of TCR/CD3 and CD28. This will add to our current, limited, knowledge on MUC1 signaling in T cells.

Mesothelin-MUC16 binding is a high affinity, N-glycan dependent interaction that facilitates peritoneal metastasis of ovarian tumors

BackgroundThe mucin MUC16 and the glycosylphosphatidylinositol anchored glycoprotein mesothelin likely facilitate the peritoneal metastasis of ovarian tumors. The biochemical basis and the kinetics of the binding between these two glycoproteins are not clearly understood. Here we have addressed this deficit and provide further evidence supporting the role of the MUC16-mesothelin interaction in facilitating cell-cell binding under conditions that mimic the peritoneal environment.ResultsIn this study we utilize recombinant-Fc tagged human mesothelin to measure the binding kinetics of this glycoprotein to MUC16 expressed on the ovarian tumor cell line OVCAR-3. OVCAR-3 derived sublines that did not express MUC16 showed no affinity for mesothelin. In a flow cytometry-based assay mesothelin binds with very high affinity to the MUC16 on the OVCAR-3 cells with an apparent Kd of 5–10 nM. Maximum interaction occurs within 5 mins of incubation of the recombinant mesothelin with the OVCAR-3 cells and significant binding is observed even after 10 sec. A five-fold molar excess of soluble MUC16 was unable to completely inhibit the binding of mesothelin to the OVCAR-3 cells. Oxidation of the MUC16 glycans, removal of its N-linked oligosaccharides, and treatment of the mucin with wheat germ agglutinin and erythroagglutinating phytohemagglutinin abrogates its binding to mesothelin. These observations suggest that at least a subset of the MUC16-asscociated N-glycans is required for binding to mesothelin. We also demonstrate that MUC16 positive ovarian tumor cells exhibit increased adherence to A431 cells transfected with mesothelin (A431-Meso+). Only minimal adhesion is observed between MUC16 knockdown cells and A431-Meso+ cells. The binding between the MUC16 expressing ovarian tumor cells and the A431-Meso+ cells occurs even in the presence of ascites from patients with ovarian cancer.ConclusionThe strong binding kinetics of the mesothelin-MUC16 interaction and the cell adhesion between ovarian tumor cells and A431-Meso+ even in the presence of peritoneal fluid strongly support the importance of these two glycoproteins in the peritoneal metastasis of ovarian tumors. The demonstration that N-linked glycans are essential for mediating mesothlein-MUC16 binding may lead to novel therapeutic targets to control the spread of ovarian carcinoma.

Cutting Edge: Enhanced Pulmonary Clearance of Pseudomonas aeruginosa by Muc1 Knockout Mice

MUC1 (MUC1 in human and Muc1 in nonhumans) is a membrane-tethered mucin that interacts with Pseudomonas aeruginosa (PA) through flagellin. In this study, we compared PA pulmonary clearance and proinflammatory responses by Muc1(-/-) mice with Muc1(+/+) littermates following intranasal instillation of PA or flagellin. Compared with Muc1(+/+) mice, Muc1(-/-) mice showed increased PA clearance, greater airway recruitment of neutrophils, higher levels of TNF-alpha and KC in bronchoalveolar lavage fluid, higher levels of TNF-alpha in media of flagellin-stimulated alveolar macrophages, and higher levels of KC in media of tracheal epithelial cells. Knockdown of MUC1 enhanced flagellin-induced IL-8 production by primary human bronchial epithelial cells. Expression of MUC1 in HEK293T cells attenuated TLR5-dependent IL-8 release in response to flagellin, which was completely ablated when its cytoplasmic tail was deleted. We conclude that MUC1/Muc1 suppresses pulmonary innate immunity and speculate its anti-inflammatory activity may play an important modulatory role during microbial infection.