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 (NPM1) as a major estrogen-regulated protein. Subsequently, we identified autoantibodies to nucleophosmin in the sera of breast cancer patients and showed that 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 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, RNAi, and small molecule inhibitors of implicated genes. Thus, we have directly implicated NPM1, NFκB (nuclear factor kappa B), IRF1 (interferon regulatory factor-1), XBP1 (X-box binding protein 1) and other genes in estrogen-independent and antiestrogen resistance gene network signaling (Gu et al. Cancer Res, 2002).


Over 10 years ago our research directly implicated changes in the unfolded protein response (UPR) in endocrine resistance, with the underling prosurvival signaling being driven at least partly by the UPR-driven splicing of XBP1 (Gu et al. 2002). The UPR is initiated following induction of endoplasmic reticulum (EnR) stress. Several stressors can induce EnR stress including oxidative stress, low oxygen tension, or low intracellular energy/glucose levels (Cook et al., Expert Rev Anticancer Ther, 2011). Subsequent work from our laboratory provided definitive evidence implicating ER-alpha (Wang et al. Cancer Cell, 2006; Kuske et al. Endocr Rel Cancer, 2006), IRF1 (Bouker et al. Cancer Res 2004; Ning et al., Mol Cancer Ther, 2010), XBP1 and the UPR (Gomez et al. FASEB J, 2007). The regulation of cell survival as driven by the UPR and XBP1 splicing incorporates the actions of NFκB (primarily RELA), BECN1 (Crawford et al., PLoS ONE, 2010), and several BCL2 family members (Riggins et al. Mol Cancer Ther, 2005; Nehra et al., FASEB J, 2010; Crawford et al., PLoS ONE, 2010). These affect the balance between apoptosis and a prosurvival autophagy (Clarke et al., J Steroid Biochem Mol Biol, 2009; Crawford et al., PLoS ONE, 2010). Our most recent data also implicate significant alterations in the metabolome, particularly in specific areas of carbohydrate, amino acid, and lipid metabolism, largely consistent with the activation of prosurvival in autophagy and the reuse of its products to feed intermediate cellular metabolism (Clarke et al. Cancer Res, 2012).


The integration of this molecular signaling is most clearly presented in our report of the first road-map for modeling ER signaling in the acquisition of endocrine resistance (Tyson et al., Nature Rev Cancer, 2011) and in a review of how endoplasmic reticulum stress, the UPR, autophagy, apoptosis, and changes in metabolism are coregulated to maintain the resistant phenotype in breast cancer(Clarke et al. Cancer Res, 2012). The challenge to modeling high dimentional data for systems biology are described in (Clarke et al, Nature Rev Cancer, 2008). Much of this most recent work represents research central to our NCI ICBP-funded Center for Cancer Systems Biology

Selected Publications
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Year 10s
  1. Chen C, Baumann WT, Xing J, Xu L, Clarke R, Tyson JJ., Mathematical models of the transitions between endocrine therapy responsive and resistant states in breast cancer. Interface Focus. 2014 May 7;11(96):20140206. doi: 10.1098/rsif.2014.0206. Print 2014 Jul 6. PubMed
  2. Cook KL, Clarke PA, Parmar J, Hu R, Schwartz-Roberts JL, Abu-Asab M, Wärri A, Baumann WT, Clarke R. Knockdown of estrogen receptor-α induces autophagy and inhibits antiestrogen-mediated unfolded protein response activation, promoting ROS-induced breast cancer cell death. FASEB J. 2014 May 23. pii: fj.13-247353. PubMed
  3. Cook KL, Wärri A, Soto-Pantoja DR, Clarke PA, Cruz MI, Zwart A, Clarke R. Hydroxychloroquine inhibits autophagy to potentiate antiestrogen responsiveness in ER+ breast cancer. Clin Cancer Res. 2014 Jun 15;20(12):3222-32. doi: 10.1158/1078-0432.CCR-13-3227. PubMed
  4. Cook KL, Soto-Pantoja DR, Abu-Asab M, Clarke PA, Roberts DD, Clarke R. Mitochondria directly donate their membrane to form autophagosomes during a novel mechanism of parkin-associated mitophagy. Cell Biosci. 2014 Mar 27;4(1):16. doi: 10.1186/2045-3701-4-16. PubMed
  5. Parmar JH, Cook KL, Shajahan-Haq AN, Clarke PA, Tavassoly I, Clarke R, Tyson JJ, Baumann WT. Modelling the effect of GRP78 on anti-oestrogen sensitivity and resistance in breast cancer. Interface Focus. 2013 Aug 6;3(4):20130012. doi: 10.1098/rsfs.2013.0012. PubMed
  6. Aguilar H, Urruticoechea A, Halonen P, Kiyotani K, Mushiroda T, Barril X, Serra-Musach J, Islam A, Caizzi L, Di Croce L, Nevedomskaya E, Zwart W, Bostner J, Karlsson E, Pérez Tenorio G, Fornander T, Sgroi DC, Garcia-Mata R, Jansen MP, García N, Bonifaci N, Climent F, Soler MT, Rodríguez-Vida A, Gil M, Brunet J, Martrat G, Gómez-Baldó L, Extremera AI, Figueras A, Balart J, Clarke R, Burnstein KL, Carlson KE, Katzenellenbogen JA, Vizoso M, Esteller M, Villanueva A, Rodríguez-Peña AB, Bustelo XR, Nakamura Y, Zembutsu H, Stål O, Beijersbergen RL, Pujana MA. VAV3 mediates resistance to breast cancer endocrine therapy. Breast Cancer Res. 2014 May 28;16(3):R53. doi: 10.1186/bcr3664. PubMed
  7. Chen C, Baumann WT, Clarke R, Tyson JJ. Modeling the estrogen receptor to growth factor receptor signaling switch in human breast cancer cells. FEBS Lett. 2013 Oct 11;587(20):3327-34. doi: 10.1016/j.febslet.2013.08.022. Epub 2013 Aug 28. PubMed
  8. Parmar JH, Cook KL, Shajahan-Haq AN, Clarke PA, Tavassoly I, Clarke R, Tyson JJ, Baumann WT. Modelling the effect of GRP78 on anti-oestrogen sensitivity and resistance in breast cancer. Interface Focus. 2013 Aug 6;3(4):20130012. doi: 10.1098/rsfs.2013.0012. PubMed
  9. Chen, L., Xuan, J., Riggins, R.B., Wang, Y. & Clarke, R. Identifying protein interaction subnetworks by a bagging Markov random field-based method. Nucleic Acid Res, 2013 Jan;41(2):e42. doi: 10.1093/nar/gks951. Epub 2012 Nov 17. PubMed
  10. Clarke R, Cook KL, Hu R, Facey CO, Tavassoly I, Schwartz JL, Baumann WT, Tyson JJ, Xuan J, Wang Y, Wärri A, Shajahan AN. “Endoplasmic reticulum stress, the unfolded protein response, autophagy, and the integrated regulation of breast cancer cell fate.” Cancer Res, 2012 Mar 15;72(6):1321-31. doi: 10.1158/0008-5472.CAN-11-3213. Review.
  11. Wang, C., Xuan, J., Shih, I.-M., Clarke, R. & Wang, Y. “Regulatory component analysis: a semi-blind extraction approach to infer gene regulatory networks with imperfect biological knowledge.” Signal Process, doi:/10.1016/j.sigpro.20112. 11.028, 2012.
  12. Tyson, J.J., Baumann, W.T., Chen, C., Verdugo, A., Tavassoly, I., Wang, Y., Weiner, L.M. & Clarke, R. “Dynamic models of estrogen signaling and cell fate in breast cancer cells.” Nature Rev Cancer, 11: 523-532, 2011.
  13. Madhavan., S., Gusev, Y., Harris, M., Tanenbaum, D.M., Gauba, R., Bhuvaneshwar, K., Shinohara, A., Rosso, K., Carabet, L.A., Song, S., Riggins, R.B., Dakshanamurthyu, S., Wang, Y., Byers, S.W., Clarke, R. & Weiner, L.M. “G-DOC®: a systems medicine platform for personalized oncology.” Neoplasia 13: 771-783, 2011.
  14. Thangavel, C., Dean, J., Ertel, A., Knudsen, K., Aldaz, M., Clarke, R. & Knudsen, E. “Therapeutically activating RB: reestablishing cell cycle control in endocrine therapy resistant breast cancer.” Endocr-Relat Cancer, 18: 333-345, 2011.
  15. Clarke, R., Shajahan, A.N., Wang, Y., Tyson, J.J. Riggins, R.B. Weiner, L.M., Bauman, W.T., Xuan, J., Zhang, B., Facey, C., Aiyer, H., Cook, K., Hickman, E.F., Tavassoly, I., Verdugo, A., Chen, C., Zwart, A., Wärri, A., Hilakivi-Clarke, L.A. “Endoplasmic reticulum stress, the unfolded protein response, and gene network modeling in antiestrogen resistant breast cancer.” Hormone Mol Biol Clin Invest, 5: 35-44, 2011.
  16. Schwartz, J., Shajahan, A.N. & Clarke, R. “The role of interferon regulatory factor-1 (IRF1) in Overcoming antiestrogen resistance in the treatment of breast cancer.” Int J Breast Cancer, 2011:912102, 2011.
  17. Cook, K.L., Shajahan, A.N. & Clarke, R. “Autophagy and endocrine resistance in breast cancer.” Expert Rev Anticancer Ther, 11: 1283-1294, 2011, 2011.
  18. Clarke, R. “Cannibalism, cell survival, and endocrine resistance in breast cancer.” Breast Cancer Res, 13: 311 (3 pages as published on-line), 2011.
  19. McCarver, G., Bhatia, J., Chambers, C., Clarke, R., Etzel, R., Foster, W., Hoyer, P., Leeder, J.S., Peters, J., Rissman, E., Rybak, M., Sherman, C., Toppari, J. & Turner, K. “NTP-CERHR expert panel report on the developmental toxicity of soy infant formula.” Birth Defects Res B Dev Reprod Toxicol 92: 421-468, 2011.
  20. Riggins, R.B., Mazzotta, M.M., Maniya, O.Z. & Clarke, R. “Orphan nuclear receptors in breast cancer pathogenesis and therapeutic response.” Endocr-Relat Cancer, 17: R213-221, 2010.
  21. Crawford, A.C., Riggins, R.B., Shajahan, A.N., Zwart, A., & Clarke, R. “Co-Inhibition of BCL-W and BCL2 restores antiestrogen sensitivity through BECN1 and promotes an autophagy-associated necrosis.” PLoS ONE, 5 (1): e8604, 2010.
  22. Nehra, R., Riggins, R.B., Shajahan, A.N., Zwart, A., Crawford, A.C. & Clarke, R. “BCL2 and CASP8 regulation by NFκB differentially affect mitochondrial function and cell fate in antiestrogen-sensitive and -resistant breast cancer cells.” FASEB J, 24: 2039-2054, 2010.
  23. Ning, Y. Riggins, R.B., Mulla, J.E., Chung, H., Zwart, A. & Clarke, R. “Interferon-gamma restores breast cancer sensitivity to fulvestrant by regulating STAT1, IRF1, NFκB, BCL2 family members, and signaling to caspase-dependent apoptosis.” Mol Cancer Ther, 9: 1274-1285, 2010.
  24. Broustas, C.G., Ross, J.S., Yang, Q., Sheehan, C.E., Riggins, R.B., Cavalli, L.R., Haddad, B.R., Seillier-Moiseiwitsch, F., Kallakury, B.V.S., Haffty, B.G., Clarke, R. & Kasid, U.N. “The pro-apoptotic molecule BLID interacts with BCL-XL and its downregulation in breast cancer correlates with poor disease free and overall survival.” Clin Cancer Res, 16: 2939-48, 2010.
  25. Chen, L., Xuan, J., Riggins, R.B., Wang, Y., Hoffman, E.P. & Clarke, R. “Multi-level support vector regression analysis to identify condition-specific regulatory networks.” Bioinformatics, 26: 1426-1422, 2010.
  26. Trauernicht, A.M., Kim, S.J., Kim, N.H., Clarke, R. & Boyer, T.G. “DBC-1 mediated endocrine resistance in breast cancer cell survival.” Cell Cycle, 9: 1218-1219, 2010.
  27. Cavalli, L.R., Riggins, R.B., Wang, A., Clarke, R., & Haddad, B.R. “Frequent loss of heterozygosity at the interferon regulatory factor-1 (IRF1) gene locus in breast cancer" Breast Cancer Res Treat, 121: 227-231, 2010.
  28. Shajahan, A.N., Goel, S., de Assis, S., Yu, B., Clarke, R. & Hilakivi-Clarke, L.A. “Changes in mammary caveolin-1 sigaling pathways are associated with breast cancer risk in rats exposed to estradiol in utero or during prepuberty.” Hormone Mol Biol Clin Invest, 2: 227-234, 2010.

Year 00s
  1. Clarke, R. “The role of preclinical animal models in breast cancer drug development.” Breast Cancer Research, 11(Suppl 3): S22, 2009.
  2. Shajahan, A.N., Riggins, R.B. & Clarke, R. “The role of X-box binding protein-1 in tumorigenicity” Drug News Perspect, 22: 241-246, 2009.
  3. Gong, T., Xuan, J., Riggins, R.B. & Clarke, R. “A systems biology approach to identify affected regulatory and signaling circuits in protein interaction networks.” Proc Int Conf Bioinf Sys Biol Intell Comp, 297-300, 2009.
  4. Chen, L. Xuan, J., Riggins, R.B., Wang, Y. Hoffman, E.P., Riggins, R.B., & Clarke, R. “Identification of condition-specific regulatory modules through multi-level motif and mRNA expression analysis," Int J Comp Biology Drug Design, 2: 1-20, 2009.
  5. 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
  6. 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
  7. 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
  8. 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.
  9. 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.
  10. 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
  11. 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
  12. 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
  13. 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
  14. 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
  15. 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
  16. 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.
  17. 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.
  18. 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.
  19. 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.
  20. 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.
  21. 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.
  22. 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.
  23. 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.
  24. Riggins, R.B., Bouton, A.H., Liu, M. C. and Clarke, R. “Antiestrogens, aromatase inhibitors, and apoptosis in breast cancer.” Vit Horm, 71:202-237, 2005.
  25. 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.
  26. 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.
  27. 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.
  28. 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.
  29. 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.
  30. 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.
  31. 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.
  32. 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.
  33. Welch, J.N. & Clarke, R. “ErbB-2 expression and drug resistance in cancer.” Signal, 3: 4-9, 2002.
  34. 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.
  35. 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.
  36. 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.
  37. 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.
  38. 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.
  39. 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.
  40. 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.
  41. Clarke, R. Sex steroids in the mammary gland. J Mammary Gland Biol Neoplasia, 5: 245-250, 2000.

Year 90s
  1. 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.
  2. 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.
  3. 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.
  4. 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.
  5. 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.
  6. 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.
  7. 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.
  8. 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.
  9. Clarke, R. & Brünner, N. “Acquired estrogen independence and antiestrogen resistance in breast cancer: estrogen receptor driven phenotypes?” Trends Endocrinol Metab, 7: 25-35, 1996. 
  10. 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.
  11. 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.
  12. 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.
  13. 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.
  14. 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.
  15. 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.
  16. 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.
  17. 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.
  18. 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.
  19. 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.
  20. 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.

Year 80s
  1. 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.
  2. 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.
  3. 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.
  4. 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.
  5. 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.
  6. 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.
  7. 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.
  8. 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.
  9. 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.
  10. 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