2004 AACR Abstract

Development and Characterization of a Pre-clinical Model of Ovarian Carcinoma to Investigate Mechanisms of Resistance to the Anti-HER2/neu Antibody Herceptin™.

Leopoldo L. Luistro, Christian Tovar, Subal Bishayee, Pranela Rameshwar, Nicholas M. Ponzio, and Steve R. Ritland.

Hoffmann-La Roche Inc., Nutley, NJ, and UMDNJ - New Jersey Medical School and Graduate School of Biomedical Sciences, Newark, NJ.

The HER-2/neu (c-erbB2) gene encodes a 185-kDa transmembrane receptor tyrosine kinase that belongs to the epidermal growth factor receptor (EGFR) family, and is overexpressed in 15-30% of breast and ovarian carcinomas. Gene amplification of HER2 is associated with early metastatic relapse and shortened survival in breast cancer patients. Herceptin (trastuzumab) is a humanized monoclonal anti-HER2 antibody approved as a molecularly targeted therapy for metastatic breast cancer. In order to investigate mechanisms of acquired resistance to Herceptin, a SKOV-3 Herceptin-resistant (HR) pre-clinical human ovarian carcinoma model was developed in vivo. SKOV-3 cells which overexpress the HER2 protein were implanted subcutaneously (sc) into female athymic nu/nu mice. Generation of SKOV-3 HR cells was accomplished by passaging "break-through" tumors (initially responded to Herceptin but later lost efficacy) over 3 or 4 rounds of transplantation in naïve nu/nu mice. SKOV-3 HR cells were selected and sub-cultured in vitro in the continuous presence of Herceptin. Anti-proliferative assays (3H-thymidine incorporation, MTT, and Coulter counter cell counts) revealed SKOV-3 HR cells were not sensitive to the effects of Herceptin. The rate of doubling of SKOV-3 HR1 (clone 1) cells in culture is about 1.5 - 2x faster than parental SKOV-3 cells. Immunoblot analysis after 48 hrs of incubation with Herceptin exhibited a 59% downregulation of the HER2 protein in SKOV-3 cells. However, no such downregulation of HER2 occurred in SKOV-3 HR1 Herceptin treated cells. Transducers in the HER2 cell signaling pathway such as Akt and MAPK were likewise not downregulated in SKOV-3 HR1 cells. Cell surface HER2 receptor expression was examined by indirect fluorescence staining and FACS analysis. SKOV-3 HR1 cells expressed similar mean fluorescence intensity (MFI) as SKOV-3 cells. After Herceptin treatment for 96 hrs, MFI decreased in SKOV-3 cells by 31%, but was unaffected in SKOV-3 HR1 cells. Reintroduction in vivo of SKOV-3 HR cells in nu/nu mice and treatment of established sc tumors with Herceptin over 4 weeks demonstrated no significant antitumor efficacy. SKOV-3 sc tumors were growth inhibited by Herceptin at the end of study (T/C = 50%; p=0.054 vs. vehicle), while SKOV-3 HR1 tumors showed no growth inhibition (T/C = 92%; p=0.734 vs. vehicle). Thus, we have developed SKOV-3 HR clones through in vivo selection that still overexpress HER2 but are unresponsive to Herceptin. This model will be used to further investigate potential mechanisms of acquired therapeutic resistance to the anti-HER2/neu antibody Herceptin.