Overexpression of CD44 accompanies acquired tamoxifen resistance in MCF7 cells and augments their sensitivity to the stromal factors, heregulin and hyaluronan

BMC Cancer

Julia Gee³  Peter Barrett-Lee⁴  Robert I Nicholson³  Zaruhi Poghosyan¹  Nicola Jordan³  Lindy Goddard³  Rebecca Bellerby²  Chris Smith³  Bedanta Baruah⁴  Stephen Hiscox³ 

[1]School of Medicine, Cardiff University, Wales, UK

[2]School of Biosciences, Cardiff University, Wales, UK

[3]Welsh School of Pharmacy, Cardiff University, Wales, UK

[4]Academic Breast Unit, Velindre Cancer Centre, Wales, UK

关键词: Heregulin;    Hyaluronan;    erbB;    CD44;    Tamoxifen-resistance;   

Others  :  1128588
DOI  :  10.1186/1471-2407-12-458

 received in 2012-01-13, accepted in 2012-09-20,  发布年份 2012

【 摘 要 】

Background

Acquired resistance to endocrine therapy in breast cancer is a significant problem with relapse being associated with local and/or regional recurrence and frequent distant metastases. Breast cancer cell models reveal that endocrine resistance is accompanied by a gain in aggressive behaviour driven in part through altered growth factor receptor signalling, particularly involving erbB family receptors. Recently we identified that CD44, a transmembrane cell adhesion receptor known to interact with growth factor receptors, is upregulated in tamoxifen-resistant (TamR) MCF7 breast cancer cells. The purpose of this study was to explore the consequences of CD44 upregulation in an MCF7 cell model of acquired tamoxifen resistance, specifically with respect to the hypothesis that CD44 may influence erbB activity to promote an adverse phenotype.

Methods

CD44 expression in MCF7 and TamR cells was assessed by RT-PCR, Western blotting and immunocytochemistry. Immunofluorescence and immunoprecipitation studies revealed CD44-erbB associations. TamR cells (± siRNA-mediated CD44 suppression) or MCF7 cells (± transfection with the CD44 gene) were treated with the CD44 ligand, hyaluronon (HA), or heregulin and their in vitro growth (MTT), migration (Boyden chamber and wound healing) and invasion (Matrigel transwell migration) determined. erbB signalling was assessed using Western blotting. The effect of HA on erbB family dimerisation in TamR cells was determined by immunoprecipitation in the presence or absence of CD44 siRNA.

Results

TamR cells overexpressed CD44 where it was seen to associate with erbB2 at the cell surface. siRNA-mediated suppression of CD44 in TamR cells significantly attenuated their response to heregulin, inhibiting heregulin-induced cell migration and invasion. Furthermore, TamR cells exhibited enhanced sensitivity to HA, with HA treatment resulting in modulation of erbB dimerisation, ligand-independent activation of erbB2 and EGFR and induction of cell migration. Overexpression of CD44 in MCF7 cells, which lack endogenous CD44, generated an HA-sensitive phenotype, with HA-stimulation promoting erbB/EGFR activation and migration.

Conclusions

These data suggest an important role for CD44 in the context of tamoxifen-resistance where it may augment cellular response to erbB ligands and HA, factors that are reported to be present within the tumour microenvironment in vivo. Thus CD44 may present an important determinant of breast cancer progression in the setting of endocrine resistance.

【 授权许可】

   
2012 Hiscox et al.; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Early Breast Cancer Trialists' Collaborative Group (EBCTCG): Relevance of breast cancer hormone receptors and other factors to the efficacy of adjuvant tamoxifen: patient-level meta-analysis of randomised trials. Lancet 2011, 378:771-784.
• [2](EBCTCG) EBCTCG: Effects of chemotherapy and hormonal therapy for early breast cancer on recurrence and 15-year survival: an overview of the randomised trials. Lancet 2005, 365:1687-1717.
• [3]Dowsett M, Smith IE, Ebbs SR, Dixon JM, Skene A, Griffith C, Boeddinghaus I, Salter J, Detre S, Hills M, et al.: Proliferation and apoptosis as markers of benefit in neoadjuvant endocrine therapy of breast cancer. Clin Cancer Res 2006, 12:1024s-1030s.
• [4]Goss PE, Ingle JN, Martino S, Robert NJ, Muss HB, Piccart MJ, Castiglione M, Tu D, Shepherd LE, Pritchard KI, et al.: A randomized trial of letrozole in postmenopausal women after five years of tamoxifen therapy for early-stage breast cancer. N Engl J Med 2003, 349:1793-1802.
• [5]Nicholson RI, Hutcheson IR, Jones HE, Hiscox SE, Giles M, Taylor KM, Gee JM: Growth factor signalling in endocrine and anti-growth factor resistant breast cancer. Rev Endocr Metab Disord 2007, 8:241-253.
• [6]Knowlden JM, Hutcheson IR, Jones HE, Madden T, Gee JM, Harper ME, Barrow D, Wakeling AE, Nicholson RI: Elevated levels of epidermal growth factor receptor/c-erbB2 heterodimers mediate an autocrine growth regulatory pathway in tamoxifen-resistant MCF-7 cells. Endocrinology 2003, 144:1032-1044.
• [7]Hiscox S, Barrett-Lee P, Nicholson RI: Therapeutic targeting of tumor-stroma interactions. Expert Opin Ther Targets 2011, 15:609-621.
• [8]Hiscox S, Jordan NJ, Jiang W, Harper M, McClelland R, Smith C, Nicholson RI: Chronic exposure to fulvestrant promotes overexpression of the c-Met receptor in breast cancer cells: implications for tumour-stroma interactions. Endocr Relat Cancer 2006, 13:1085-1099.
• [9]Hiscox S, Jiang WG, Obermeier K, Taylor K, Morgan L, Burmi R, Barrow D, Nicholson RI: Tamoxifen resistance in MCF7 cells promotes EMT-like behaviour and involves modulation of beta-catenin phosphorylation. Int J Cancer 2006, 118:290-301.
• [10]Ponta H, Sherman L, Herrlich PA: CD44: from adhesion molecules to signalling regulators. Nat Rev Mol Cell Biol 2003, 4:33-45.
• [11]Orian-Rousseau V, Chen L, Sleeman JP, Herrlich P, Ponta H: CD44 is required for two consecutive steps in HGF/c-Met signaling. Genes Dev 2002, 16:3074-3086.
• [12]Bourguignon LY, Singleton PA, Zhu H, Zhou B: Hyaluronan promotes signaling interaction between CD44 and the transforming growth factor beta receptor I in metastatic breast tumor cells. J Biol Chem 2002, 277:39703-39712.
• [13]Palyi-Krekk Z, Barok M, Kovacs T, Saya H, Nagano O, Szollosi J, Nagy P: EGFR and ErbB2 are functionally coupled to CD44 and regulate shedding, internalization and motogenic effect of CD44. Cancer Lett 2008, 263:231-242.
• [14]Meran S, Luo DD, Simpson R, Martin J, Wells A, Steadman R, Phillips AO: Hyaluronan facilitates transforming growth factor-{beta}1 dependent proliferation via CD44 and epidermal growth factor-receptor interaction. J Biol Chem 2011, 286(20):17618-17630.
• [15]Bourguignon LY, Zhu H, Chu A, Iida N, Zhang L, Hung MC: Interaction between the adhesion receptor, CD44, and the oncogene product, p185HER2, promotes human ovarian tumor cell activation. J Biol Chem 1997, 272:27913-27918.
• [16]Gotte M, Yip GW: Heparanase, hyaluronan, and CD44 in cancers: a breast carcinoma perspective. Cancer Res 2006, 66:10233-10237.
• [17]Tamada M, Nagano O, Tateyama S, Ohmura M, Yae T, Ishimoto T, Sugihara E, Onishi N, Yamamoto T, Yanagawa H, et al.: Modulation of glucose metabolism by CD44 contributes to antioxidant status and drug resistance in cancer cells. Cancer Res 2012, 72:1438-1448.
• [18]Wang YC, Yo YT, Lee HY, Liao YP, Chao TK, Su PH, Lai HC: ALDH1-bright epithelial ovarian cancer cells are associated with CD44 expression, drug resistance, and poor clinical outcome. Am J Pathol 2012, 180:1159-1169.
• [19]Auvinen P, Tammi R, Parkkinen J, Tammi M, Agren U, Johansson R, Hirvikoski P, Eskelinen M, Kosma VM: Hyaluronan in peritumoral stroma and malignant cells associates with breast cancer spreading and predicts survival. Am J Pathol 2000, 156:529-536.
• [20]Gee JM, Robertson JF, Gutteridge E, Ellis IO, Pinder SE, Rubini M, Nicholson RI: Epidermal growth factor receptor/HER2/insulin-like growth factor receptor signalling and oestrogen receptor activity in clinical breast cancer. Endocr Relat Cancer 2005, 12(Suppl 1):S99-S111.
• [21]Koumakpayi IH, Le Page C, Delvoye N, Saad F, Mes-Masson AM: Macropinocytosis inhibitors and Arf6 regulate ErbB3 nuclear localization in prostate cancer cells. Mol Carcinog 2011, 50:901-912.
• [22]Andrique L, Fauvin D, El Maassarani M, Colasson H, Vannier B, Seite P: ErbB3(80 kDa), a nuclear variant of the ErbB3 receptor, binds to the Cyclin D1 promoter to activate cell proliferation but is negatively controlled by p14ARF. Cell Signal 2012, 24:1074-1085.
• [23]Hutcheson IR, Knowlden JM, Hiscox SE, Barrow D, Gee JM, Robertson JF, Ellis IO, Nicholson RI: Heregulin beta1 drives gefitinib-resistant growth and invasion in tamoxifen-resistant MCF-7 breast cancer cells. Breast Cancer Res 2007, 9:R50. BioMed Central Full Text
• [24]Jones JT, Akita RW, Sliwkowski MX: Binding specificities and affinities of egf domains for ErbB receptors. FEBS Lett 1999, 447:227-231.
• [25]Franklin MC, Carey KD, Vajdos FF, Leahy DJ, de Vos AM, Sliwkowski MX: Insights into ErbB signaling from the structure of the ErbB2-pertuzumab complex. Cancer Cell 2004, 5:317-328.
• [26]Toole BP: Hyaluronan: from extracellular glue to pericellular cue. Nat Rev Cancer 2004, 4:528-539.
• [27]Ahmed MA, Aleskandarany MA, Rakha EA, Moustafa RZ, Benhasouna A, Nolan C, Green AR, Ilyas M, Ellis IO: A CD44(-)/CD24 (+) phenotype is a poor prognostic marker in early invasive breast cancer. Breast Cancer Res Treat 2011, 133(3):979-995.
• [28]Gee JM, Howell A, Gullick WJ, Benz CC, Sutherland RL, Santen RJ, Martin LA, Ciardiello F, Miller WR, Dowsett M, et al.: Consensus statement. Workshop on therapeutic resistance in breast cancer: impact of growth factor signalling pathways and implications for future treatment. Endocr Relat Cancer 2005, 12(Suppl 1):S1-7.
• [29]Hiscox S, Morgan L, Green T, Nicholson RI: Src as a therapeutic target in anti-hormone/anti-growth factor-resistant breast cancer. Endocr Relat Cancer 2006, 13(Suppl 1):S53-59.
• [30]Ricardo S, Vieira AF, Gerhard R, Leitao D, Pinto R, Cameselle-Teijeiro JF, Milanezi F, Schmitt F, Paredes J: Breast cancer stem cell markers CD44, CD24 and ALDH1: expression distribution within intrinsic molecular subtype. J Clin Pathol 2011, 64(11):937-946.
• [31]Gordin M, Tesio M, Cohen S, Gore Y, Lantner F, Leng L, Bucala R, Shachar I: c-Met and its ligand hepatocyte growth factor/scatter factor regulate mature B cell survival in a pathway induced by CD74. J Immunol 2010, 185:2020-2031.
• [32]Singleton PA, Salgia R, Moreno-Vinasco L, Moitra J, Sammani S, Mirzapoiazova T, Garcia JG: CD44 regulates hepatocyte growth factor-mediated vascular integrity. Role of c-Met, Tiam1/Rac1, dynamin 2, and cortactin. J Biol Chem 2007, 282:30643-30657.
• [33]Tremmel M, Matzke A, Albrecht I, Laib AM, Olaku V, Ballmer-Hofer K, Christofori G, Heroult M, Augustin HG, Ponta H, Orian-Rousseau V: A CD44v6 peptide reveals a role of CD44 in VEGFR-2 signaling and angiogenesis. Blood 2009, 114:5236-5244.
• [34]Bourguignon LY, Zhu H, Zhou B, Diedrich F, Singleton PA, Hung MC: Hyaluronan promotes CD44v3-Vav2 interaction with Grb2-p185(HER2) and induces Rac1 and Ras signaling during ovarian tumor cell migration and growth. J Biol Chem 2001, 276:48679-48692.
• [35]Hatano H, Shigeishi H, Kudo Y, Higashikawa K, Tobiume K, Takata T, Kamata N: RHAMM/ERK interaction induces proliferative activities of cementifying fibroma cells through a mechanism based on the CD44-EGFR. Lab Invest 2011, 91:379-391.
• [36]Hiscox S, Morgan L, Barrow D, Dutkowskil C, Wakeling A, Nicholson RI: Tamoxifen resistance in breast cancer cells is accompanied by an enhanced motile and invasive phenotype: inhibition by gefitinib ('Iressa', ZD1839). Clin Exp Metastasis 2004, 21:201-212.
• [37]Zhao Y, Planas-Silva MD: Mislocalization of cell-cell adhesion complexes in tamoxifen-resistant breast cancer cells with elevated c-Src tyrosine kinase activity. Cancer Lett 2009, 275:204-212.
• [38]Soni S, Lin BT, August A, Nicholson RI, Kirsch KH: Expression of a phosphorylated p130(Cas) substrate domain attenuates the phosphatidylinositol 3-kinase/Akt survival pathway in tamoxifen resistant breast cancer cells. J Cell Biochem 2009, 107:364-375.
• [39]Bourguignon LY, Gilad E, Peyrollier K: Heregulin-mediated ErbB2-ERK signaling activates hyaluronan synthases leading to CD44-dependent ovarian tumor cell growth and migration. J Biol Chem 2007, 282:19426-19441.
• [40]Sadlonova A, Bowe DB, Novak Z, Mukherjee S, Duncan VE, Page GP, Frost AR: Identification of molecular distinctions between normal breast-associated fibroblasts and breast cancer-associated fibroblasts. Cancer Microenviron 2009, 2:9-21.
• [41]Cho SJ, Chae MJ, Shin BK, Kim HK, Kim A: Akt- and MAPK-mediated activation and secretion of MMP-9 into stroma in breast cancer cells upon heregulin treatment. Mol Med Report 2008, 1:83-88.
• [42]Normanno N, Kim N, Wen D, Smith K, Harris AL, Plowman G, Colletta G, Ciardiello F, Salomon DS: Expression of messenger RNA for amphiregulin, heregulin, and cripto-1, three new members of the epidermal growth factor family, in human breast carcinomas. Breast Cancer Res Treat 1995, 35:293-297.
• [43]Hudelist G, Singer CF, Manavi M, Pischinger K, Kubista E, Czerwenka K: Co-expression of ErbB-family members in human breast cancer: Her-2/neu is the preferred dimerization candidate in nodal-positive tumors. Breast Cancer Res Treat 2003, 80:353-361.
• [44]Visscher DW, Sarkar FH, Kasunic TC, Reddy KB: Clinicopathologic analysis of amphiregulin and heregulin immunostaining in breast neoplasia. Breast Cancer Res Treat 1997, 45:75-80.
• [45]Van Phuc P, Nhan PL, Nhung TH, Tam NT, Hoang NM, Tue VG, Thuy DT, Ngoc PK: Downregulation of CD44 reduces doxorubicin resistance of CD44CD24 breast cancer cells. Onco Targets Ther 2011, 4:71-78.
• [46]Cain JW, Hauptschein RS, Stewart JK, Bagci T, Sahagian GG, Jay DG: Identification of CD44 as a Surface Biomarker for Drug Resistance by Surface Proteome Signature Technology. Mol Cancer Res 2011, 9(5):637-647.
• [47]Bourguignon LY: Hyaluronan-mediated CD44 activation of RhoGTPase signaling and cytoskeleton function promotes tumor progression. Semin Cancer Biol 2008, 18:251-259.
• [48]Peng ST, Su CH, Kuo CC, Shaw CF, Wang HS: CD44 crosslinking-mediated matrix metalloproteinase-9 relocation in breast tumor cells leads to enhanced metastasis. Int J Oncol 2007, 31:1119-1126.
• [49]Urakawa H, Nishida Y, Wasa J, Arai E, Zhuo L, Kimata K, Kozawa E, Futamura N, Ishiguro N: Inhibition of hyaluronan synthesis in breast cancer cells by 4-methylumbelliferone suppresses tumorigenicity in vitro and metastatic lesions of bone in vivo. Int J Cancer 2011, 30(2):454-466.
• [50]Hosono K, Nishida Y, Knudson W, Knudson CB, Naruse T, Suzuki Y, Ishiguro N: Hyaluronan oligosaccharides inhibit tumorigenicity of osteosarcoma cell lines MG-63 and LM-8 in vitro and in vivo via perturbation of hyaluronan-rich pericellular matrix of the cells. Am J Pathol 2007, 171:274-286.
• [51]Udabage L, Brownlee GR, Waltham M, Blick T, Walker EC, Heldin P, Nilsson SK, Thompson EW, Brown TJ: Antisense-mediated suppression of hyaluronan synthase 2 inhibits the tumorigenesis and progression of breast cancer. Cancer Res 2005, 65:6139-6150.
• [52]Toole BP: Hyaluronan-CD44 Interactions in Cancer: Paradoxes and Possibilities. Clin Cancer Res 2009, 15:7462-7468.
• [53]Wallach-Dayan SB, Rubinstein AM, Hand C, Breuer R, Naor D: DNA vaccination with CD44 variant isoform reduces mammary tumor local growth and lung metastasis. Mol Cancer Ther 2008, 7:1615-1623.
• [54]Ma W, Deng Y, Zhou L: The prognostic value of adhesion molecule CD44v6 in women with primary breast carcinoma: a clinicopathologic study. Clin Oncol (R Coll Radiol) 2005, 17:258-263.
• [55]Diaz LK, Zhou X, Wright ET, Cristofanilli M, Smith T, Yang Y, Sneige N, Sahin A, Gilcrease MZ: CD44 expression is associated with increased survival in node-negative invasive breast carcinoma. Clin Cancer Res 2005, 11:3309-3314.
• [56]Afify A, Purnell P, Nguyen L: Role of CD44s and CD44v6 on human breast cancer cell adhesion, migration, and invasion. Exp Mol Pathol 2009, 86:95-100.
• [57]Ouhtit A: Abd Elmageed ZY, Abdraboh ME, Lioe TF, Raj MH: In vivo evidence for the role of CD44s in promoting breast cancer metastasis to the liver. Am J Pathol 2007, 171:2033-2039.
• [58]Brown RL, Reinke LM, Damerow MS, Perez D, Chodosh LA, Yang J, Cheng C: CD44 splice isoform switching in human and mouse epithelium is essential for epithelial-mesenchymal transition and breast cancer progression. J Clin Invest 2011, 121:1064-1074.
• [59]Afify AM, Tate S, Durbin-Johnson B, Rocke DM, Konia T: Expression of CD44s and CD44v6 in lung cancer and their correlation with prognostic factors. Int J Biol Markers 2011, 26:50-57.
• [60]Neame SJ, Isacke CM: The cytoplasmic tail of CD44 is required for basolateral localization in epithelial MDCK cells but does not mediate association with the detergent-insoluble cytoskeleton of fibroblasts. J Cell Biol 1993, 121:1299-1310.
• [61]Morgan L, Gee J, Pumford S, Farrow L, Finlay P, Robertson J, Ellis I, Kawakatsu H, Nicholson R, Hiscox S: Elevated Src kinase activity attenuates Tamoxifen response in vitro and is associated with poor prognosis clinically. Cancer Biol Ther 2009, 8:1550-1558.
• [62]Gee JM, Robertson JF, Ellis IO, Willsher P, McClelland RA, Hoyle HB, Kyme SR, Finlay P, Blamey RW, Nicholson RI: Immunocytochemical localization of BCL-2 protein in human breast cancers and its relationship to a series of prognostic markers and response to endocrine therapy. Int J Cancer 1994, 59:619-628.