): MUC1 is a well characterized marker for many cancers of epithelial origin that also directly contributes to the aggressive metastatic phenotype of tumors. Cumulative data from studies of these cancer patients indicates that high levels of particularly intracellularMUC1 in the tumor and in serum correlates with a strong potential for metastasis and a poor prognosis for the patient. The anti-adhesive property of this normally apical, transmembrane mucin (with 40-90 heavily 0-glycosylated tandem repeats of 20 aa) disrupts both cell-cell and cell-matrix interactions in non-polarized tumor cells, thus enhancing metastasis. The soluble MUC1 in patient serum and ascites also contributes to the aggressive phenotype of the cancer by acting as a tumor decoy by forming immunocomplexes with antibodies, by binding T-cells and blocking their proliferation, and by rebinding to the tumor and initiating signal transduction pathways. Since MUC1 has fewer and smaller 0-glycans in many tumors, preliminary studies were designed to understand how this contributes to the intracellular accumulation and shedding of MUC1 since the mechanisms for either event is unknown. Using normal and glycosylation-defective CHO cells, metabolic labeling and biotinylation, we find that cell surface expression of recombinant human MUC1 is dependent on 0-glycosylation. Surprisingly, we find that this enormous molecule is internalized by clathrin-mediated endocytosis indicating that the 40-90 tandem-repeat immunodominanat epitopes of MUCI are an ideal target for immunotoxin therapy. Thus, it is essential to understand what regulates MUC1 endocytosis and shedding to better target treatments to MUC1-positive tumors and avoid soluble MUCI in serum/ascites. We find that MUC1 endocytosis is blocked by mutation of a potential site for dual palmitoylation and enhanced by smaller 0-glycans, indicating that its trafficking may be modulated by homotypic clustering and association with lipid microdomains involved in signal transduction. Since we also find that MUCI shedding is endocytosis-dependent, our specific aims are designed to understand how MUCI trafficking and shedding is modulated by its 0-glycan structure, palmitoylation, and phosphorylation. Initial experiments will utilize non-polarized CHO cells while later experiments in new glycosylation-defective MDCK cells will define apical-specific modulation of MUCI trafficking.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
1R01DK054787-01A2
Application #
6327141
Study Section
Pathobiochemistry Study Section (PBC)
Program Officer
Scherbenske, M James
Project Start
2001-04-01
Project End
2005-03-31
Budget Start
2001-04-01
Budget End
2002-03-31
Support Year
1
Fiscal Year
2001
Total Cost
$293,296
Indirect Cost
Name
University of Pittsburgh
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
053785812
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
Poland, Paul A; Kinlough, Carol L; Hughey, Rebecca P (2015) Cloning, expression, and purification of galectins for in vitro studies. Methods Mol Biol 1207:37-49
Razawi, Hanieh; Kinlough, Carol L; Staubach, Simon et al. (2013) Evidence for core 2 to core 1 O-glycan remodeling during the recycling of MUC1. Glycobiology 23:935-45
Cascio, Sandra; Farkas, Adam M; Hughey, Rebecca P et al. (2013) Altered glycosylation of MUC1 influences its association with CIN85: the role of this novel complex in cancer cell invasion and migration. Oncotarget 4:1686-97
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Mo, Di; Costa, Simone A; Ihrke, Gudrun et al. (2012) Sialylation of N-linked glycans mediates apical delivery of endolyn in MDCK cells via a galectin-9-dependent mechanism. Mol Biol Cell 23:3636-46
Poland, Paul A; Rondanino, Christine; Kinlough, Carol L et al. (2011) Identification and characterization of endogenous galectins expressed in Madin Darby canine kidney cells. J Biol Chem 286:6780-90
Rondanino, Christine; Poland, Paul A; Kinlough, Carol L et al. (2011) Galectin-7 modulates the length of the primary cilia and wound repair in polarized kidney epithelial cells. Am J Physiol Renal Physiol 301:F622-33
Kinlough, Carol L; Poland, Paul A; Gendler, Sandra J et al. (2011) Core-glycosylated mucin-like repeats from MUC1 are an apical targeting signal. J Biol Chem 286:39072-81
Cui, Shanshan; Guerriero, Christopher J; Szalinski, Christina M et al. (2010) OCRL1 function in renal epithelial membrane traffic. Am J Physiol Renal Physiol 298:F335-45

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