Title: Hyaluronan-CD44 Interaction Activates Stem Cell Marker Nanog, Stat-3-mediated MDR1 Gene Expression, and Ankyrin-regulated Multidrug Efflux in Breast and Ovarian Tumor Cells
Abstract: Hyaluronan (HA) is a major glycosaminoglycan in the extracellular matrix whose expression is tightly linked to multidrug resistance and tumor progression. In this study we investigated HA-induced interaction between CD44 (a HA receptor) and Nanog (an embryonic stem cell transcription factor) in both human breast tumor cells (MCF-7 cells) and human ovarian tumor cells (SK-OV-3.ipl cells). Using a specific primer pair to amplify Nanog by reverse transcriptase-PCR, we detected the expression of Nanog transcript in both tumor cell lines. In addition, our results reveal that HA binding to these tumor cells promotes Nanog protein association with CD44 followed by Nanog activation and the expression of pluripotent stem cell regulators (e.g. Rex1 and Sox2). Nanog also forms a complex with the “signal transducer and activator of transcription protein 3” (Stat-3) in the nucleus leading to Stat-3-specific transcriptional activation and multidrug transporter, MDR1 (P-glycoprotein) gene expression. Furthermore, we observed that HA-CD44 interaction induces ankyrin (a cytoskeletal protein) binding to MDR1 resulting in the efflux of chemotherapeutic drugs (e.g. doxorubicin and paclitaxel (Taxol)) and chemoresistance in these tumor cells. Overexpression of Nanog by transfecting tumor cells with Nanog cDNA stimulates Stat-3 transcriptional activation, MDR1 overexpression, and multidrug resistance. Down regulation of Nanog signaling or ankyrin function (by transfecting tumor cells with Nanog small interfering RNA or ankyrin repeat domain cDNA) not only blocks HA/CD44-mediated tumor cell behaviors but also enhances chemosensitivity. Taken together, these findings suggest that targeting HA/CD44-mediated Nanog-Stat-3 signaling pathways and ankyrin/cytoskeleton function may represent a novel approach to overcome chemotherapy resistance in some breast and ovarian tumor cells displaying stem cell marker properties during tumor progression. Hyaluronan (HA) is a major glycosaminoglycan in the extracellular matrix whose expression is tightly linked to multidrug resistance and tumor progression. In this study we investigated HA-induced interaction between CD44 (a HA receptor) and Nanog (an embryonic stem cell transcription factor) in both human breast tumor cells (MCF-7 cells) and human ovarian tumor cells (SK-OV-3.ipl cells). Using a specific primer pair to amplify Nanog by reverse transcriptase-PCR, we detected the expression of Nanog transcript in both tumor cell lines. In addition, our results reveal that HA binding to these tumor cells promotes Nanog protein association with CD44 followed by Nanog activation and the expression of pluripotent stem cell regulators (e.g. Rex1 and Sox2). Nanog also forms a complex with the “signal transducer and activator of transcription protein 3” (Stat-3) in the nucleus leading to Stat-3-specific transcriptional activation and multidrug transporter, MDR1 (P-glycoprotein) gene expression. Furthermore, we observed that HA-CD44 interaction induces ankyrin (a cytoskeletal protein) binding to MDR1 resulting in the efflux of chemotherapeutic drugs (e.g. doxorubicin and paclitaxel (Taxol)) and chemoresistance in these tumor cells. Overexpression of Nanog by transfecting tumor cells with Nanog cDNA stimulates Stat-3 transcriptional activation, MDR1 overexpression, and multidrug resistance. Down regulation of Nanog signaling or ankyrin function (by transfecting tumor cells with Nanog small interfering RNA or ankyrin repeat domain cDNA) not only blocks HA/CD44-mediated tumor cell behaviors but also enhances chemosensitivity. Taken together, these findings suggest that targeting HA/CD44-mediated Nanog-Stat-3 signaling pathways and ankyrin/cytoskeleton function may represent a novel approach to overcome chemotherapy resistance in some breast and ovarian tumor cells displaying stem cell marker properties during tumor progression. Multidrug resistance frequently contributes to the failure of chemotherapeutic drug treatments in patients diagnosed with solid tumors such as breast and ovarian cancers (1Harnett P.R. Kirsten F. Tattersall M.H. J. Clin. Oncol. 1986; 4: 952-957Crossref PubMed Scopus (11) Google Scholar). It is now certain that oncogenic signaling and cytoskeleton function are directly involved in chemotherapeutic drug resistance and tumor progression (2Mollinedo F. 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It is well known that HA is enriched in many types of tumors (9Knudson W. Biswa C. Li X. Nemec R.E. Toole B.P. CIBA Found. Symp. 1989; 143: 150-159PubMed Google Scholar, 10Toole B.P. Wight T. Tammi M. J. Biol. Chem. 2002; 277: 4593-4596Abstract Full Text Full Text PDF PubMed Scopus (431) Google Scholar). In cancer patients, HA concentrations are usually higher in malignant tumors than in corresponding benign or normal tissues. Actually, in some tumor types the level of HA is predictive of malignancy (10Toole B.P. Wight T. Tammi M. J. Biol. Chem. 2002; 277: 4593-4596Abstract Full Text Full Text PDF PubMed Scopus (431) Google Scholar). For example, HA levels have been shown to be significantly elevated in the serum of breast cancer patients (11Delpech B. Cheyallier B. Reinhardt N. Julien J.P. Duval C. Maingonnat C. Bastit P. Asselain B. Int. J. Cancer. 1990; 46: 388-390Crossref PubMed Scopus (83) Google Scholar). Also it is believed that tumor cell adhesion to the HA-containing pericellular coat of mesothelial cells is one of the important mechanisms for the peritoneal spread of ovarian cancer (12Jones L.M.H. Gardner J.B. Catterall J.B. Turner G.A. Clin. Exp. Metastasis. 1995; 13: 373-380Crossref PubMed Scopus (82) Google Scholar). HA interacts with a specific cell surface receptor, CD44, which belongs to a family of multifunctional transmembrane glycoproteins expressed in numerous cells and tissues, including breast and ovarian tumor cells and various carcinoma tissues (13Iida N. Bourguignon L.Y.W. J. Cell Physiol. 1995; 162: 127-133Crossref PubMed Scopus (128) Google Scholar, 14Zhu D. Bourguignon L.Y.W. Cell Motil. Cytoskeleton. 1998; 39: 209-222Crossref PubMed Scopus (66) Google Scholar, 15Iida N. Bourguignon L.Y.W. J. Cell Physiol. 1997; 171: 152-160Crossref PubMed Scopus (59) Google Scholar, 16Bourguignon L.Y.W. Zhu H.B. Chu A. Zhang L. Hung M.C. J. Biol. 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CD44 is often expressed in a variety of isoforms that are products of a single gene generated by alternative splicing of variant exons into an extracellular membrane-proximal site (29Screaton G.R. Bell M.V. Jackson D.G. Cornelis F.B. Gerth U. Bell J.I. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 12160-12164Crossref PubMed Scopus (989) Google Scholar, 30Screaton G.R. Bell M.V. Bell J.I. Jackson D.G. J. Biol. Chem. 1993; 268: 12235-12238Abstract Full Text PDF PubMed Google Scholar). In fact, the expression of certain CD44 variant (CD44v) isoforms has been shown to be closely associated with tumor progression (13Iida N. Bourguignon L.Y.W. J. Cell Physiol. 1995; 162: 127-133Crossref PubMed Scopus (128) Google Scholar, 14Zhu D. Bourguignon L.Y.W. Cell Motil. Cytoskeleton. 1998; 39: 209-222Crossref PubMed Scopus (66) Google Scholar, 15Iida N. Bourguignon L.Y.W. J. Cell Physiol. 1997; 171: 152-160Crossref PubMed Scopus (59) Google Scholar, 16Bourguignon L.Y.W. Zhu H.B. Chu A. Zhang L. 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All CD44 isoforms contain an HA-binding site in their extracellular domain that serves as the major cell surface receptor for HA (31Underhill C. J. Cell Sci. 1992; 103: 293-298Crossref PubMed Google Scholar). Most importantly, HA binding to CD44 stimulates the cytoplasmic domain of CD44 to interact with a number of downstream effectors, including the cytoskeletal protein, ankyrin (14Zhu D. Bourguignon L.Y.W. Cell Motil. Cytoskeleton. 1998; 39: 209-222Crossref PubMed Scopus (66) Google Scholar, 19Bourguignon L.Y.W. J. Mammary Gland Biol. Neoplasia. 2001; 6: 287-297Crossref PubMed Scopus (145) Google Scholar, 20Bourguignon L.Y.W. Zhu D. Zhu H. Front. Biosci. 1998; 3: 637-649Crossref PubMed Scopus (108) Google Scholar, 21Turley E.A. Nobel P.W. Bourguignon L.Y.W. J. Biol. Chem. 2002; 277: 4589-4592Abstract Full Text Full Text PDF PubMed Scopus (882) Google Scholar, 32Zhu D. Bourguignon L.Y.W. J. Cell. Physiol. 2000; 183: 182-195Crossref PubMed Scopus (59) Google Scholar). 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