Raf-1 and Bcl-2 Induce Distinct and Common Pathways That Contribute to Breast Cancer Drug Resistance 1 Julianne M. Davis, Patrick M. Navolanic, Caroline R. Weinstein-Oppenheimer, Linda S. Steelman, Wei Hu, Marina Konopleva, Mikhail V. Blagosklonny, and James A. McCubrey 2 Department of Microbiology and Immunology [J. M. D., P. M. N., C. R. W-O., L. S. S., J. A. M.] and Leo Jenkins Cancer Center [J. A. M.], Brody School of Medicine at East Carolina University, Greenville, North Carolina 27858; Departamento de Ciencias Farmace´uticas y Nutricio´n, Facultad de Farmacia, Universidad de Valparaı ´so, Valparaı ´so, Chile [C. R. W-O.]; Section of Molecular Hematology and Therapy Laboratory, Department of Blood and Bone Marrow Transplantation, University of Texas, M. D. Anderson Cancer Center, Houston, Texas 77030 [W. H., M. K.]; and Medicine Branch, National Cancer Institute, NIH, Bethesda, Maryland 20892 [M. V. B.] ABSTRACT Overexpression of Bcl-2 plays a role in the development of drug resistance in leukemia and other apoptosis-prone tumors. Raf isoforms are serine/threonine kinases that act as signal transducers in cascades initiated by many growth factors and mitogens. Raf isoform activation has been linked to drug resistance in leukemia. In this study we investigated effects of Bcl-2 and Raf-1 on doxorubicin-induced growth inhibition of MCF-7 breast cancer cells. In the absence of doxorubicin, overexpression of Bcl-2 or a constitutively ac- tive form of Raf-1 in MCF-7 cells did not affect proliferation rate. Overexpression of Bcl-2 increased resistance of MCF-7 cells to doxorubicin in 2-day, 5-day, and 8-week assays. Analysis of doxorubicin sensitivity of individual MCF/Bcl-2 clones showed that doxorubicin resistance was positively correlated with level of Bcl-2 overexpression. Overexpres- sion of constitutively active Raf-1 also increased resistance to doxorubicin. Induction of Raf-1 activity in MCF-7 cells overexpressing Bcl-2 resulted in greater doxorubicin resist- ance than induction of Raf-1 activity in MCF-7 cells lacking Bcl-2 overexpression. Furthermore, levels of P-glycoprotein mRNA were increased in MCF-7 cells overexpressing a constitutively active Raf-1. MCF-7 cells overexpressing con- stitutively active Raf-1 were also more resistant to paclitaxel, which, like doxorubicin, is a substrate of P-glycoprotein. These observations suggest both independent and overlap- ping roles for Raf-1 and Bcl-2 oncogenes in the resistance to growth inhibition by doxorubicin. INTRODUCTION The development of resistance by cancer cells to a wide variety of chemotherapeutic agents poses a major obstacle in the successful treatment of cancer. Drug resistance is observed in a broad range of cell types from breast and prostate to leukemic blasts (1). Many mechanisms contribute to drug resistance, including drug inactivation, extrusion of the drug by cell mem- brane pumps, mutations of drug targets, and failure to initiate apoptosis (2–5). Prevention of apoptosis can result from a va- riety of conditions, including retention of the mitochondrial membrane potential and cytokine stimulation (4, 6, 7). A more detailed understanding of drug resistance mechanisms in breast cancer may improve the success rate for many already devel- oped chemotherapeutic agents by forming a basis for the design of adjuvant therapy. The search for proteins responsible for drug-resistant phe- notypes has implicated the antiapoptotic molecule Bcl-2. Bcl-2 belongs to a family of proteins regulating apoptosis that includes both proapoptotic and antiapoptotic members (8, 9). These proteins are believed to modulate apoptosis by forming ho- modimers or heterodimers with other Bcl-2 family members (10 –15). Association of Bcl-2 family proteins with proteins outside the Bcl-2 family is another mechanism by which these proteins are regulated (16). Although a precise understanding of how Bcl-2 exerts its antiapoptotic effects remains elusive, it has been found to be overexpressed in many cancers including colorectal, prostate, and 70% of all breast cancers (12, 17, 18). In addition to Bcl-2, other proteins have been linked with resistance to chemotherapeutic drugs. Raf isoforms are interme- diates in signal transduction cascades initiated by growth factors (19 –21). They exert their effects in part through the highly conserved Raf/MEK 3 /ERK pathway (22–25). A cascade of ki- nase activation occurs after the cognate receptor is ligated (19, 23, 25–27). Raf isoforms have been reported to increase expres- sion of certain proteins, including p21 Cip1 (M r 21,000 cyclin- dependent kinase-interacting protein) and c-Myc, when acti- vated (28). Some reports have suggested that this signal transduction pathway may be involved in the regulation of Received 12/27/01; revised 10/9/02; accepted 10/25/02. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported in part by grants (to J. A. M) from the National Cancer Institute (R01CA512025 and R01CA98195) and the North Carolina Biotechnology Center (2000-ARG-0003). J. A. M. was also supported in part by the East Carolina University Interdisciplinary Program Sup- porting Breast Cancer Research. 2 To whom requests for reprints should be addressed. Phone: (252) 744- 2704; Fax: (252) 744-3104; E-mail: mccubreyj@mail.ecu.edu. 3 The abbreviations used are: MEK, mitogen-activated protein/extracel- lular signal-regulated kinase kinase; ERK, extracellular signal-regulated kinase; P-gp, P-glycoprotein; FBS, fetal bovine serum; AR, androgen receptor; RT-PCR, reverse transcription-PCR; GAPDH, glyceralde- hyde-3-phosphate dehydrogenase; TBST, 25 mM Tris (pH 8.0), 125 mM NaCl, and 0.025% Tween; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-di- phenyltetrazolium bromide; NCI/ADR-RES, National Cancer Institute/ Adriamycin-resistant; PMA, phorbol 12-myristate 13-acetate. 1161 Vol. 9, 1161–1170, March 2003 Clinical Cancer Research Cancer Research. on December 2, 2021. © 2003 American Association for clincancerres.aacrjournals.org Downloaded from