Clarke Labs | Breast Cancer Research

Identification of Resistance Mechanisms to Hormone/Antihormone Therapies in Breast Cancer

Human breast cancer variants with different hormone dependent and antiestrogen responsive phenotypes. These models are based on the MCF-7 human breast cancer cell line.

Overview

A major focus of our studies is directed towards understanding, at the cellular and molecular levels, the factors conferring estrogen-independence and antiestrogen resistance and those responsible for affecting estrogen responsiveness. In collaboration with Dr. Leena Hilakivi-Clarke, our studies include those phytochemicals, environmental estrogens and nutrients that may influence or mimic endocrine effects in breast cancer. We apply a translational approach, where observations in experimental systems are further explored in human breast cancer specimens and vice versa.

We began by developing a novel series of variants of human breast cancer cell lines and xenografts that allow us to investigate these processes in vitro and in vivo. Initially, we applied 2-dimensional gel electrophoresis analyses to explore the proteomes of estrogen-dependent and estrogen-independent variants. We identified several interesting coordinates on these gels, definitively identifying nucleophosmin (NPM) as a major estrogen-regulated protein. Subsequently, we began studies to assess the clinical relevance of this observation, identifying autoantibodies to nucleophosmin in the sera of breast cancer patients. The levels of these autoantibodies increase in patients 6-months prior to recurrence. Consistent with the estrogenic regulation of the antigen, nucleophosmin autoantibodies are lower in patients that have received Tamoxifen.

Additional studies of our cell line variants have applied Serial Analysis of Gene Expression (SAGE) and gene expression microarray analysis to explore the transcriptomes of responsive and resistant human breast cancer cells and breast biopsies. The altered activity of candidate genes identified in these studies was confirmed in functional studies, including the use of promoter-reporter assays, gene overexpression analyses and gene repression studies using dominant negative, siRNA, and small molecule inhibitors of implicated transcription factors. Thus, we have indicated NFkB (nuclear factor keppa B), IRF-1 (interferon regulatory factor 1), hXBP-1 (human X-box binding protein 1) and other genes in estrogen-independent and antiestrogen resistance gene network signaling. Additional studies are evaluating other candidates. Our studies also implicate changes in oxidative stress responses in acquired antiestrogen resistance.

Selected Publications

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Clarke, R., Shajahan, A.N., Riggins, R.B., Cho, Y., Crawford, A., Xuan, J., Wang, Y., Zwart, A., Nehra, R. & Liu, M.C. “Gene network signaling in hormone responsiveness modifies apoptosis and autophagy in breast cancer cells.” J Steroid Biochem Mol Biol, 114: 8-20, 2009. PubMed

Zhang, B., Li, H., Riggins, R.B., Zhan, M., Xuan, J., Zhang, Z., Hoffman, E.P., Clarke, R., Wang, Y., “Differential dependency network analysis to identify condition-specific topological changes in biological networks.” Bioinformatics, 25: 526-532, 2009. PubMed

Olivo, S., Zhu, Y., Lee, R.Y., Cabanes, A., Khan, G., Zwart, A., Wang, Y, Clarke, R., Hilakivi-Clarke, L.A., “Identification of gene signaling pathways mediating the opposite effects of prepubertal low and high fat n-3 PUFA diets on breast cancer risk.” Cancer Prev Res, 1: 522-531, 2008. PubMed

Deb, T.B., Ramlijak, D., Dickson, R.B., Johnson, M.D. & Clarke, R. “Signaling pathways in the normal and neoplastic breast.” In: “Handbook of Cell Signaling.” Eds: Thompson, B.E.; Elsevier, Inc. New York, NY; in press.

Shajahan, A.N., Riggins, R.B. & Clarke, R. “Apoptosis, cell death and breast cancer” Chapter 8. In: “Breast cancer: prognosis, treatment, and prevention.” Eds: Pasqualini, J.R., Informa Healthcare; New York, NY; pp137-156, 2008.

Riggins, R.B., Lan, P.-J., Klimach, U., Zwart, A., Cavalli, L.R., Haddad, B.R, Chen, L., Xuan, J., Ethier, S.P. & Clarke, R. “ERRγ mediates Tamoxifen resistance in a novel model of invasive lobular breast carcinoma.” Cancer Res, 68: 8908-8917, 2008. PubMed

Gomez, B.P. Riggins, R.B., Shajahan A., Klimach, U., Zhu, Y., Zwart, A., Wang, M., Wang, A. & Clarke, R. “Human X-box binding protein-1 confers both estrogen-independence and antiestrogen resistance in breast cancer.” FASEB J, 21:4013-27, 2007. PubMed

Naughton, C., Kuske, B., MacLeod, K., Clarke, R. Cameron, D.A. & Langdon, S.P. “Progressive loss of estrogen receptor alpha (ERα) cofactor recruitment in endocrine resistance.” Mol Endocrinol, 21:2615-26, 2007. PubMed

Bouker, K.B., Skaar, T.C., Harburger, D.S., Riggins, R.B., Fernandez, D.R., Zwart, A. & Clarke, R. “The A4396G polymorphism in interferon regulatory factor-1 is frequently expressed in breast cancer.” Cancer Genet Cytogenet, 175: 61-4, 2007. PubMed

Wang, L.H., Yang, X.Y., Zhang, X., An, P., Kim, H.-J., Huang, J., Clarke, R., Osborne, C.K., Inman, J.K., Appella, E. & Farrar, W.L. “Disruption of estrogen receptor DNA-binding domain and related intramolecular communication restores tamoxifen sensitivity in resistant breast cancer.” Cancer Cell, 10: 487-499, 2006. PubMed

Kuske, B., Naughton, C., Moore, K., MacLeod, K.G., Miller, W.R., Clarke, R. Langdon, S.P. & Cameron, D.A. “Endocrine therapy resistance can be associated with high estrogen receptor alpha (ERα) expression and reduced ERα phosphorylation in breast cancer models.” Endocr Related Cancer, 13: 1121-1133, 2006. PubMed

Wong, L.-J.C., Dai, P., Lu, J.-F., Lou, M.A., Clarke, R. & Nazarov, N. “AIB1 gene amplification and the instability of polyQ encoding sequence in breast cancer cell lines.” BMC Cancer, 6: 111-132, 2006. PubMed

Trock, B.J., Hilakivi-Clarke, L.A. & Clarke, R. “Meta-analysis of soy intake and breast cancer risk.” J Natl Cancer Inst, 98: 459-471, 2006. PubMed

Zhu, Y., Wang, A., Liu, M.C., Zwart, A., Lee, R.Y., Gallagher, A., Wang, Y., Miller, W.R., Dixon, J.M. & Clarke, R. “Estrogen Receptor alpha (ER) positive breast tumors and breast cancer cell lines share similarities in their transcriptome data structures.” Int Oncol, in press.

Riggins, R.B., Thomas, K.S., Ta, H.Q., Donelan, S.S., Owen, K.A., Gibson, M.A., Shupnik, M.A., Silva, C.M., Parsons, S.J., Clarke, R. & Bouton, A.H. “Physical and functional interactions between Cas and c-Src induce tamoxifen resistance of breast cancer cells through pathways involving EGFR and STAT5b.” Cancer Res, 66: 7007-7015, 2006. PubMed

Zhu, Y., Singh, S., Hewitt, S., Liu, A., Gomez, B., Wang, A. & Clarke, R. “Expression Patterns Among Interferon Regulatory Factor-1 (IRF-1), Human X-Box Binding Protein-1 (hXBP-1), Nuclear Factor Kappa B (NFκB, Nucleophosmin (NPM), Estrogen Receptor-Alpha (ERα), and Progesterone Receptor (PgR) Proteins in Breast Cancer Tissue Microarrays.” Int J Oncol,28: 67-76, 2006.

Hilakivi-Clarke, L.A., Olivo, S.E., Shajahan, A., Khan, G., Zhu, Y., Zwart, A., Cho, E. & Clarke, R. “Mechanisms mediating the effects of prepubertal (n-3) polyunsaturated fatty acid diets on breast cancer risks in rats.” J Nutr, 135 (suppl 12): 2946-2952, 2005. PubMed

Kishimoto, H., Wang, Z., Bhat-Nakshatri, P., Chang, D., Clarke, R. & Nakshatri, H. “The p160 family of coactivators regulate breast cancer cell proliferation and invasion through autocrine/paracrine activity of SDF-1 α/CXCL12.” Carcinogenesis, 26: 1706-1715, 2005. PubMed

Bouker, K.B., Skaar, T.C., Riggins, R.B., Harburger, D.S., Fernandez, D.R., Zwart, A., Wang, A. & Clarke, R. “Interferon regulatory factor-1 (IRF-1) exhibits tumor suppressor activities in breast cancer associated with caspase activation and induction of apoptosis.” Carcinogenesis. 2005 Sep;26(9):1527-35. PubMed

Riggins, R.B., Bouton, A.H., Liu, M. C. and Clarke, R. “Antiestrogens, aromatase inhibitors, and apoptosis in breast cancer.” Vitam Horm, 71:202-237, 2005. PubMed

Riggins RB, Zwart A, Nehra R, Clarke R. “The nuclear factor kappa B inhibitor parthenolide restores ICI 182,780 (Faslodex; fulvestrant)-induced apoptosis in antiestrogen-resistant breast cancer cells.” Mol Cancer Ther. Jan;4(1):33-41, 2005. PubMed

Bouker, K.B., Skaar, T.C., Fernandez, D.R., O’Brien, K.A., Riggins, R.B., Honghua, C. & Clarke, R.“Interferon regulatory factor-1 mediates the proapoptotic but not cell cycle arrest effects of the steroidal antiestrogen ICI 182,780 (Faslodex, Fulvestrant).” Cancer Res, 64:4030-4039, 2004. PubMed

Han, F., Miksicek, R., Clarke, R. & Conrad, S.E. “Expression of an estrogen receptor variant lacking exon 3 in derivatives of MCF-7 cells with acquired estrogen independence or Tamoxifen resistance.” J Mol Endocrinol 32: 935-945, 2004. PubMed

Johnson, M., Kenney, N., Hilakivi-Clarke, L.A., Singh, B., Chepko, G., Newbold, R., Clarke, R., Sholler, P.F., Lirio, A.A., Foss, C., Trock, B., Paik, S., Stoica, A. & Martin, M.B. “Cadmium mimics the effects of estrogen in vivo in the uterus and mammary gland.” Nature Med, 9: 1081-1084, 2003. PubMed

Pratt, M.A.C., Bishop, T.E., White, D., Yasvinski, G., Ménard, M., Niu, M.Y. & Clarke, R. “Estrogen withdrawal-induced NFkB and Bcl-3 expression in breast cancer cells: roles in growth and hormone independence.” Mol Cell Biol, 23: 6887-6900, 2003.

Clarke, R., Liu, M.C., Bouker, K.B., Gu, Z., Lee, R.Y., Zhu, Y., Skaar, T.C., Gomez, B., O'Brien, K., Wang, Y., Hilakivi-Clarke, L.A. “Antiestrogen resistance in breast cancer and the role of estrogen receptor signaling.” Oncogene, 22: 7316-7339, 2003.

Gu, Z., Lee, R.Y., Skaar, T.C., Bouker, K.B., Welch, J.N., Lu, J., Liu, A., Davis, N., Leonessa, F., Brünner, N., Wang, Y. & Clarke, R. “Association of interferon regulatory factor-1, nucleophosmin, nuclear factor kappa-B and cAMP response element binding with acquired resistance to Faslodex (ICI 182,780).” Cancer Res, 62: 3428-3437, 2002.

Poola, I., Clarke, R., Dewitty, R. & Lafalle, L.D. “Constitutively active and dominant negative ER isoforms are elevated and ER is decreased during breast carcinogenesis in African American women. ”Cancer, 94: 615-623, 2002.

Hilakivi-Clarke, L.A., Cho, E., Cabanes, A., DeAssis, S., Olivo, S., Helferich, W., Lippman, M.E. & Clarke, R. “Dietary modulation of pregnancy estrogen levels and breast cancer risk among female rat offspring.” Clin Cancer Res, 8: 3601-3610, 2002.

Welch, J.N. & Clarke, R. “ErbB-2 expression and drug resistance in cancer.” Signal, 3: 4-9, 2002.

Hilakivi-Clarke, L.A., Cabannes, A., Olivo, S., Kerr, L., Bouker, K.B. & Clarke, R. “Do estrogens always increase breast cancer risk?” J Steroid Biochem Mol Biol, 80: 163-174, 2002.

James, M.R., Skaar, T.C., Lee, R.Y., MacPherson, A., Zwiebel, J.A., Ampy, F. & Clarke, R. “Constitutive expression of the steroid sulfatase gene supports MCF-7 breast cancer cell growth in vitro and in vivo.” Endocrinology, 142:1497-1505, 2001.

Clarke, R., Leonessa, F., Welch, J.N., & Skaar, T.C. “Cellular and molecular pharmacology of antiestrogen action and resistance.” Pharmacol Rev, 53: 1-41, 2001.

Hilakivi-Clarke, L.A., Cho, E., Onojafe, I. & Clarke, R. “Maternal exposure to tamoxifen during pregnancy increases mammary tumorigenesis among female offspring.” Clin Cancer Res, 6: 305-308, 2000.

Poola, I., Chatra, S., Koduri, S. & Clarke, R. “Analysis of single-, double-, and multiple exon deletion transcripts of estrogen receptor in breast cancer cell lines and tumors by a novel approach.” J Steroid Biochem Mol Biol, 72: 249-258, 2000.

Clarke, R., Skaar, T.C., Bouker, K.B, Davis, N., Lee, Y.R., Welch, J.N. & Clarke, R. “Molecular and pharmacological aspects of antiestrogen resistance.” J Steroid Biochem Mol Biol, 76: 71-84, 2001.

Clarke, R. Sex steroids in the mammary gland. J Mammary Gland Biol Neoplasia, 5: 245-250, 2000.

Arteaga, C.L., Koli, K.M., Dugger, T.C. & Clarke, R. “Reversal of tamoxifen resistance of human breast carcinomas in vivo with neutralizing anti-transforming growth factor (TGF)-b antibodies.” J Natl Cancer Inst, 91: 46-53, 1999.

Gopalakrishna, R., Gundimeda, U., Fontana, J.A. & Clarke, R. “Differential distribution and nuclear association of protein phosphatase 2A in human breast carcinoma cell lines and its relation to estrogen receptor status and tumor progression.” Cancer Lett, 136:134-151, 1999.

Lavigne, M.C., Ramwell, P.W. & Clarke, R. “The effects of estrogens and antiestrogens on the growth of porcine coronary artery smooth muscle cells.” Steroids, 64:472-480, 1999.

Lavigne, M.C., Ramwell, P.W. & Clarke, R. “Growth regulation and ultrastructural characterization of porcine coronary artery smooth muscle cells.” In Vitro Cell Dev Biol, 35: 136-143, 1999.

Clarke, R., Skaar, T., El-Ashry, D., Leonessa, F. & Hilakivi-Clarke, L.A. “Use of ERE and reporter gene constructs to assess putative estrogenic activity.” J Med Food, 2:127-133, 1999.

Brankin, B., Skaar, T.C., Trock, B., Berris, M. & Clarke, R. “Autoantibodies to the nucleolar phosphoprotein nucleophosmin in breast cancer patients.” Cancer Epidemiol Biomark Prev, 7:1109-1115, 1998.

Skaar, T.C., Prasad, S.C., Sharareh, S., Brünner, N., Lippman, M.E. & Clarke, R. “Cellular protein patterns associated with acquired estrogen-independent growth in human breast cancer cells. Implication of nucleophosmin as a major estrogen-regulated protein associated with acquired estrogen-independence.” J Steroid Biochem Mol Biol, 67:391-402, 1998.

Brünner, N., Boysen, B., Jirus, S., Skaar, T.C., Holst-Hansen, C., Lippman, J., Frandsen, T., Spang-Thomsen, M., Fuqua, S.A.W. & Clarke, R. “MCF7/LCC9: an antiestrogen resistant MCF-7 variant where acquired resistance to the steroidal antiestrogen ICI 182,780 confers an early crossresistance to the non-steroidal antiestrogen tamoxifen.” Cancer Res, 57: 3486-3493, 1997.

Clarke, R. & Brünner, N. “Acquired estrogen independence and antiestrogen resistance in breast cancer: estrogen receptor driven phenotypes?” Trends Endocrinol Metab, 7: 25-35, 1996.

Bei, M., Lavigne, M.C., Foegh, M.L., Ramwell, P. & Clarke, R. “Specific binding of oestradiol to rat coronary artery heart smooth muscle cells.” J Steroid Biochem Mol Biol, 58: 83-88, 1996.

Ruiz-Cabello, J., Berghmans, K., Kaplan, O., Lippman, M.E., Clarke, R. & Cohen, J.S. “Hormone dependence of breast cancer cells and the effects of tamoxifen and estrogen: ³¹P NMR studies.” Breast Cancer Res Treat, 33: 209-217, 1995.

Leonessa, F., Jacobson, M., Boyle, B., Lippman, J., McGarvey, M. & Clarke, R. “The effect of tamoxifen on the multidrug resistant phenotype in human breast cancer cells: isobologram, drug accumulation, and gp-170 binding studies.” Cancer Res, 54: 441-447, 1994.

Coopman, P., Garcia, M., Brünner, N., Derocq, D., Clarke, R. & Rochefort, H. “Antiproliferative and antiestrogenic effects of ICI 164,384 in 4-OH-Tamoxifen-resistant human breast cancer cells.” Int J Cancer, 56: 295-300, 1994.

Brünner, N., Boulay, V., Fojo, A., Freter, C., Lippman, M.E. & Clarke, R. “Acquisition of hormone-independence in MCF-7 cells is accompanied by increased expression of estrogen regulated genes but without DNA amplifications.” Cancer Res, 53: 283-290, 1993.

Brünner, N., Frandsen, T.O., Holst-Hansen, C., Bei, M., Thompson, E.W., Wakeling, A.E., Lippman, M.E. & Clarke, R. “MCF7/LCC2: A 4-hydroxytamoxifen resistant human breast cancer variant which retains sensitivity to the steroidal antiestrogen ICI 182,780.” Cancer Res, 53: 3229-3232, 1993.

Thompson, E.W., Brünner, N., Torri, J., Boulay, V., Wright, A., Lippman, M.E., Steeg, P.S. & Clarke, R. “The invasive and metastatic properties of hormone-independent but hormone-responsive variants of MCF-7 human breast cancer cells.” Clin Exptl Metastasis, 11: 15-26, 1993.

Clarke R., Currier, S., Kaplan, O., Boulay, V., Gottesman, M. & Dickson, R.B. “Effect of P-glycoprotein expression on sensitivity to hormones in MCF-7 human breast cancer cells.” J Natl Cancer Inst, 84: 1506-1512, 1992.

Yano, T., Pinski, J., Szepeshazi, K., Korkut, E., Groot, K., Clarke, R., Comaru-Schally, A.M. & Schally, A.V. “Growth inhibition of human breast carcinoma (MCF-7 MIII) in athymic nude mice with sustained delivery systems of luteinizing hormone-releasing hormone antagonist SB-75 and agonist D-Trp⁶-LH-RH.” Breast Cancer Res Treat, 21: 35-45, 1992.

Clarke, R., Lippman, M.E. & Dickson, R.B. “Hormonal aspects of breast cancer: growth factors, drugs and stromal interactions.” Crit Rev Oncol Hematol, 12: 1-23, 1992.

Clarke, R., van den Berg, H.W. & Murphy, R.F. “Tamoxifen and 17b-estradiol reduce the membrane fluidity of human breast cancer cells.” J Natl Cancer Inst, 82: 1702-1705, 1990.

Clarke, R., Brünner, N., Katzenellenbogen, B.S., Thompson E.W., Norman, M.J., Koppi, C., Paik, S., Lippman, M.E. & Dickson, R.B. “Progression from hormone dependent to hormone independent growth in MCF-7 human breast cancer cells.” Proc Natl Acad Sci USA, 86: 3649-3653, 1989.

Clarke, R., Brünner, N., Katz, D., Glanz, P., Dickson, R.B., Lippman, M.E. & Kern, F. “The effects of a constitutive production of TGF-alpha on the growth of MCF-7 human breast cancer cells in vitro and in vivo.” Mol Endocrinol, 3: 372-380, 1989.

Clarke, R. & van den Berg, H.W. “Adverse interactions between cytotoxic drugs and hormonal agents in human breast cancer cells.” J Clin Oncol, 7: 1580-1582, 1989.

Clarke, R., Brünner, N., Thompson, E.W., Glanz, P., Katz, D., Dickson, R.B. & Lippman, M.E. “The inter-relationships between ovarian-independent growth, antioestrogen resistance and invasiveness in the malignant progression of human breast cancer.” J Endocrinol, 122: 331-340, 1989.

van den Berg, H.W., Leahey, W.J., Lynch, M., Clarke, R. & Nelson, J. “Recombinant human interferon alpha increases oestrogen receptor expression in human breast cancer cells (ZR-75-1) and sensitises them to the anti-proliferative effects of Tamoxifen.” Br J Cancer, 55: 255-257, 1987.

Nelson, J., Clarke, R., McFerran, N.V. & Murphy, R.F. “Morpho-functional effects of phenol red on oestrogen sensitive human breast cancer cells.” Biochem Soc Trans, 15: 244-245, 1987.

Clarke, R., Morwood, J., van den Berg, H.W., Nelson, J. & Murphy, R.F. “The effect of cytotoxic drugs on estrogen receptor expression and response to Tamoxifen in MCF-7 cells.” Cancer Res, 46: 6116-6119, 1986.

Clarke, R., Morwood, J., van den Berg, H.W., Nelson, J. & Murphy, R.F. “Influence of vincristine on the oestrogen binding capacity of two human breast cancer cell lines in vitro.” Biochem Soc Trans, 14: 449-450, 1986.

Nelson, J., Clarke, R., Dickson, G.R., van den Berg, H.W. & Murphy, R.F. “The effects of Mg⁺⁺ ions on nuclear integrity and apparent subcellular distribution of unoccupied oestrogen receptor in breast cancer cells.” J Steroid Biochem, 25: 619-626, 1986.

Clarke, R., van den Berg, H.W., Kennedy, D.G. & Murphy, R.F. “Oestrogen receptor status and the response of human breast cancer cells to a combination of methotrexate and 17 beta-oestradiol.” Br J Cancer, 51: 365-369, 1985.

Other manuscripts submitted and in preparation