The dual ErbB1/ErbB2 inhibitor, lapatinib (GW572016), cooperates with tamoxifen to inhibit both cell proliferation- and estrogen-dependent gene expression in antiestrogen-resistant breast cancer.

Effective treatment of estrogen receptor (ER)-positive breast cancers with tamoxifen is often curtailed by the development of drug resistance. Antiestrogen-resistant breast cancers often show increased expression of the epidermal growth factor receptor family members, ErbB1 and ErbB2. Tamoxifen activates the cyclin-dependent kinase inhibitor, p27 to mediate G(1) arrest. ErbB2 or ErbB1 overexpression can abrogate tamoxifen sensitivity in breast cancer lines through both reduction in p27 levels and inhibition of its function. Here we show that the dual ErbB1/ErbB2 inhibitor, lapatinib (GW572016), can restore tamoxifen sensitivity in ER-positive, tamoxifen-resistant breast cancer models. Treatment of MCF-7(pr), T-47D, and ZR-75 cells with lapatinib or tamoxifen alone caused an incomplete cell cycle arrest. Treatment with both drugs led to a more rapid and profound cell cycle arrest in all three lines. Mitogen-activated protein kinase and protein kinase B were inhibited by lapatinib. The two drugs together caused a greater reduction of cyclin D1 and a greater p27 increase and cyclin E-cdk2 inhibition than observed with either drug alone. In addition to inhibiting mitogenic signaling and cell cycle progression, lapatinib inhibited estrogen-stimulated ER transcriptional activity and cooperated with tamoxifen to further reduce ER-dependent transcription. Lapatinib in combination with tamoxifen effectively inhibited the growth of tamoxifen-resistant ErbB2 overexpressing MCF-7 mammary tumor xenografts. These data provide strong preclinical data to support clinical trials of ErbB1/ErbB2 inhibitors in combination with tamoxifen in the treatment of human breast cancer.

[1]  D. Picard,et al.  Activation of the unliganded estrogen receptor by EGF involves the MAP kinase pathway and direct phosphorylation. , 1996, The EMBO journal.

[2]  B. Katzenellenbogen,et al.  Phosphorylation of the human estrogen receptor. Identification of hormone-regulated sites and examination of their influence on transcriptional activity. , 1994, The Journal of biological chemistry.

[3]  Carolyn L. Smith,et al.  Intracellular signaling pathways: nongenomic actions of estrogens and ligand-independent activation of estrogen receptors. , 2001, Frontiers in bioscience : a journal and virtual library.

[4]  J. Slingerland,et al.  Constitutive MEK/MAPK Activation Leads to p27Kip1Deregulation and Antiestrogen Resistance in Human Breast Cancer Cells* , 2001, The Journal of Biological Chemistry.

[5]  J. Slingerland,et al.  Down-regulation of p21WAF1/CIP1 or p27Kip1 abrogates antiestrogen-mediated cell cycle arrest in human breast cancer cells. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[6]  H. Kurokawa,et al.  ErbB (HER) receptors can abrogate antiestrogen action in human breast cancer by multiple signaling mechanisms. , 2003, Clinical cancer research : an official journal of the American Association for Cancer Research.

[7]  James M. Roberts,et al.  CDK inhibitors: positive and negative regulators of G1-phase progression. , 1999, Genes & development.

[8]  C. Klinge,et al.  Estrogen receptor interaction with co-activators and co-repressors☆ , 2000, Steroids.

[9]  W Godolphin,et al.  Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer. , 1989, Science.

[10]  N. Anderson,et al.  Studies of epidermal growth factor receptor inhibition in breast cancer. , 2001, Endocrine-related cancer.

[11]  C. Arteaga,et al.  Herceptin-induced inhibition of phosphatidylinositol-3 kinase and Akt Is required for antibody-mediated effects on p27, cyclin D1, and antitumor action. , 2002, Cancer research.

[12]  J. Slingerland,et al.  Prognostic implications of expression of the cell cycle inhibitor protein p27Kip1 , 2004, Breast Cancer Research and Treatment.

[13]  J. Mester,et al.  Estrogen induction of the cyclin D1 promoter: involvement of a cAMP response-like element. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[14]  N. Koszewski,et al.  Estrogen receptor phosphorylation. Hormonal dependence and consequence on specific DNA binding. , 1992, The Journal of biological chemistry.

[15]  D. Lannigan.  Estrogen receptor phosphorylation , 2003, Steroids.

[16]  Christopher J. Barnes,et al.  The role of Shc and insulin-like growth factor 1 receptor in mediating the translocation of estrogen receptor α to the plasma membrane , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[17]  K. Ley,et al.  Interaction of oestrogen receptor with the regulatory subunit of phosphatidylinositol-3-OH kinase , 2000, Nature.

[18]  S. Cl,et al.  Intracellular signaling pathways: nongenomic actions of estrogens and ligand-independent activation of estrogen receptors. , 2001 .

[19]  Y. Chiew,et al.  Expression of c‐erbB receptors, heregulin and oestrogen receptor in human breast cell lines , 2000, International journal of cancer.

[20]  C. Osborne,et al.  Estrogen-dependent, tamoxifen-resistant tumorigenic growth of MCF-7 cells transfected with HER2/neu , 1992, Breast Cancer Research and Treatment.

[21]  John M. Daly,et al.  ErbB2 Potentiates Breast Tumor Proliferation through Modulation of p27Kip1-Cdk2 Complex Formation: Receptor Overexpression Does Not Determine Growth Dependency , 2000, Molecular and Cellular Biology.

[22]  W. McGuire,et al.  HER-2/neu amplification predicts poor survival in node-positive breast cancer. , 1990, Cancer research.

[23]  W. McGuire,et al.  Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene. , 1987, Science.

[24]  M. Roussel,et al.  Glycogen synthase kinase-3beta regulates cyclin D1 proteolysis and subcellular localization. , 1998, Genes & development.

[25]  Sutherland Rl,et al.  Cell cycle control by steroid hormones. , 1994 .

[26]  J. Slingerland,et al.  PKB/Akt phosphorylates p27, impairs nuclear import of p27 and opposes p27-mediated G1 arrest , 2002, Nature Medicine.

[27]  R. Blamey,et al.  Overexpression of cyclin D1 messenger RNA predicts for poor prognosis in estrogen receptor-positive breast cancer. , 1999, Clinical cancer research : an official journal of the American Association for Cancer Research.

[28]  F. Zindy,et al.  Inhibition of cyclin D1 phosphorylation on threonine-286 prevents its rapid degradation via the ubiquitin-proteasome pathway. , 1997, Genes & development.

[29]  G. Freiss,et al.  Effect of prolonged hydroxytamoxifen treatment of MCF‐7 cells on mitogen activated kinase cascade , 2002, International journal of cancer.

[30]  S. Franco,et al.  A phase II, open-label, multicenter study of GW572016 in patients with trastuzumab-refractory metastatic breast cancer. , 2004, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[31]  S. Reed,et al.  Cyclin E/cdk2 and cyclin A/cdk2 kinases associate with p107 and E2F in a temporally distinct manner. , 1992, Genes & development.

[32]  K. Lackey,et al.  The effects of the novel, reversible epidermal growth factor receptor/ErbB-2 tyrosine kinase inhibitor, GW2016, on the growth of human normal and tumor-derived cell lines in vitro and in vivo. , 2001, Molecular cancer therapeutics.

[33]  Wen-rong Gong,et al.  Activation of the Src/p21ras/Erk pathway by progesterone receptor via cross‐talk with estrogen receptor , 1998, The EMBO journal.

[34]  A. Bianco,et al.  c-erbB2 expression predicts tamoxifen efficacy in breast cancer patients , 2004, Breast Cancer Research and Treatment.

[35]  Carlos L Arteaga,et al.  ErbB-targeted therapeutic approaches in human cancer. , 2003, Experimental cell research.

[36]  J. Carroll,et al.  A Pure Estrogen Antagonist Inhibits Cyclin E-Cdk2 Activity in MCF-7 Breast Cancer Cells and Induces Accumulation of p130-E2F4 Complexes Characteristic of Quiescence* , 2000, The Journal of Biological Chemistry.

[37]  Y. Geng,et al.  Specific protection against breast cancers by cyclin D1 ablation , 2001, Nature.

[38]  S. Parsons,et al.  Src family kinases and HER2 interactions in human breast cancer cell growth and survival , 2001, Oncogene.

[39]  K. Korach,et al.  Peptide growth factors elicit estrogen receptor-dependent transcriptional activation of an estrogen-responsive element. , 1993, Molecular endocrinology.

[40]  A. Lenferink,et al.  Blockade of the epidermal growth factor receptor tyrosine kinase suppresses tumorigenesis in MMTV/Neu + MMTV/TGF-alpha bigenic mice. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[41]  R. Schiff,et al.  Mechanisms of tamoxifen resistance: increased estrogen receptor-HER2/neu cross-talk in ER/HER2-positive breast cancer. , 2004, Journal of the National Cancer Institute.

[42]  V. Jordan,et al.  “Studies on the estrogen receptor in breast cancer” — 20 years as a target for the treatment and prevention of cancer , 2004, Breast Cancer Research and Treatment.

[43]  R. Sutherland,et al.  Cyclin D1 protein is overexpressed in hyperplasia and intraductal carcinoma of the breast. , 1998, Clinical cancer research : an official journal of the American Association for Cancer Research.

[44]  Herman Yeger,et al.  Decreased levels of the cell-cycle inhibitor p27Kip1 protein: Prognostic implications in primary breast cancer , 1997, Nature Medicine.

[45]  S. Tsutsui,et al.  Prognostic value of epidermal growth factor receptor (EGFR) and its relationship to the estrogen receptor status in 1029 patients with breast cancer , 2004, Breast Cancer Research and Treatment.

[46]  James M. Roberts,et al.  Expression of cell-cycle regulators p27Kip1 and cyclin E, alone and in combination, correlate with survival in young breast cancer patients , 1997, Nature Medicine.

[47]  M. Pagano,et al.  Regulation of the Cdk inhibitor p27 and its deregulation in cancer , 2000, Journal of cellular physiology.

[48]  Y. Yarden,et al.  Untangling the ErbB signalling network , 2001, Nature Reviews Molecular Cell Biology.

[49]  Simak Ali,et al.  Phosphatidylinositol 3-Kinase/AKT-mediated Activation of Estrogen Receptor α , 2001, The Journal of Biological Chemistry.

[50]  Daniel Metzger,et al.  Activation of the Estrogen Receptor Through Phosphorylation by Mitogen-Activated Protein Kinase , 1995, Science.

[51]  Alfonso Bellacosa,et al.  Cytoplasmic relocalization and inhibition of the cyclin-dependent kinase inhibitor p27Kip1 by PKB/Akt-mediated phosphorylation in breast cancer , 2002, Nature Medicine.

[52]  R. Nicholson,et al.  Elevated levels of epidermal growth factor receptor/c-erbB2 heterodimers mediate an autocrine growth regulatory pathway in tamoxifen-resistant MCF-7 cells. , 2003, Endocrinology.

[53]  A. Stead,et al.  Clinical summary of 67 heavily pre-treated patients with metastatic carcinomas treated with GW572016 in a phase Ib study. , 2004, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[54]  A. Bilancio,et al.  PI3‐kinase in concert with Src promotes the S‐phase entry of oestradiol‐stimulated MCF‐7 cells , 2001, The EMBO journal.

[55]  Hong Ma,et al.  Anti-tumor activity of GW572016: a dual tyrosine kinase inhibitor blocks EGF activation of EGFR/erbB2 and downstream Erk1/2 and AKT pathways , 2002, Oncogene.

[56]  T. Willson,et al.  Circumventing tamoxifen resistance in breast cancers using antiestrogens that induce unique conformational changes in the estrogen receptor. , 2001, Cancer research.

[57]  R. Sutherland,et al.  Cell cycle control by steroid hormones. , 1994, Seminars in cancer biology.

[58]  M. Sliwkowski,et al.  HER-2 tyrosine kinase pathway targets estrogen receptor and promotes hormone-independent growth in human breast cancer cells. , 1995, Oncogene.

[59]  R. Sutherland,et al.  Inducible overexpression of cyclin D1 in breast cancer cells reverses the growth-inhibitory effects of antiestrogens. , 1997, Clinical cancer research : an official journal of the American Association for Cancer Research.

[60]  A. Harris,et al.  Epidermal growth factor receptor (EGFr) as a marker for poor prognosis in node-negative breast cancer patients: Neu and tamoxifen failure , 1990, The Journal of Steroid Biochemistry and Molecular Biology.

[61]  Antony W Burgess,et al.  Epidermal growth factor receptor: mechanisms of activation and signalling. , 2003, Experimental cell research.