Cancer Therapy: Preclinical Protein Kinase A Activation Confers Resistance to Trastuzumab in Human Breast Cancer Cell Lines

Purpose: Trastuzumab is a monoclonal antibody targeted to the Her2 receptor and approved for treatment of Her2-positive breast cancer. Among patients who initially respond to trastuzumab therapy, resistance typically arises within 1 year. BT/Her cells are trastuzumab-resistant variants of Her2-positive BT474 breast cancer cells. The salient feature of BT/Her cells is failure to downregulate phosphoinositide 3-kinase/ Akt signaling on trastuzumab binding. The current work addresses the mechanism of sustained signaling in BT/Her cells, focusing on the protein kinase A (PKA) pathway. Experimental Design: We performed microarray analysis on BT/Her and BT474 cell lines to identify genes that were upregulated or downregulated in trastuzumabresistant cells. Specific genes in the PKA pathway were quantified using reverse transcription-PCR and Western hybridization. Small interfering RNA transfection was used to determine the effects of gene knockdown on cellular response to trastuzumab. Electrophoretic mobility shift assays were used to measure cyclic AMP–responsive element binding activity under defined conditions. Immunohistochemistry was used to analyze protein expression in clinical samples. Results: BT/Her cells had elevated PKA signaling activity and several genes in the PKA regulatory network had altered expression in these cells. Downregulation of one such gene, the PKA-RIIα regulatory subunit, conferred partial trastuzumab resistance in Her2-positive BT474 and SK-Br-3 cell lines. Forskolin activation of PKA also produced significant protection against trastuzumab-mediated Akt dephosphorylation. In patient samples, PKA signaling appeared to be enhanced in residual disease remaining after trastuzumab-containing neoadjuvant therapy. Conclusions: Activation of PKA signaling may be one mechanism contributing to trastuzumab resistance in Her2-positive breast cancer. We propose a molecular model by which PKA confers its effects. (Clin Cancer Res 2009;15(23):7196–206) The Her2 (erbB2/neu) oncogene encodes a 185-kDa type I receptor tyrosine kinase that belongs to the epidermal growth factor receptor (EGFR) family (reviewed in ref. 1). It is overexpressed in 20% to 25% of invasive breast cancers and its levels correlate strongly with prognosis, thus making it an important therapeutic target in breast cancer (1–4). Trastuzumab (Herceptin; Genentech), approved for the treatment of Her2positive metastatic breast cancer, is a humanized monoclonal antibody that recognizes a juxtamembrane epitope in the extracellular domain of Her2 (5). Trastuzumab inhibits the growth of Her2-dependent breast cancer cell lines in culture (6–8) and in xenograft animal models (9, 10). The mechanisms by which trastuzumab inhibits growth of Her2-overexpressing cancer cells are not completely defined, but downmodulation of phosphoinositide 3-kinase (PI3K)/Akt and/or Ras/mitogenactivated protein kinase signaling pathways are essential features of trastuzumab response leading to eventual cell cycle arrest (11). Clinical data show that patient response rates to trastuzumab range from 12% to 34% when it is used as monotherapy in the metastatic setting (12, 13). This suggests that the majority of Her2-overexpressing tumors have intrinsic resistance to trastuzumab. Combining trastuzumab with paclitaxel (14, 15) or docetaxel (16) increases response rates, but most patients who achieve an initial response to trastuzumab-based regimens will develop resistance within 1 year (15). Efficacy can be further improved with the addition of platinum compounds in the advanced disease setting (17), but resistance likely occurs here as well. In the neoadjuvant setting, trastuzumab-containing regimens result in >50% complete pathologic response, implying that there remains a substantial proportion of Her2-overexpressing cancer cells that are resistant to such therapies (18). Authors' Affiliations: Division of Tumor Cell Biology and Departments of Anatomic Pathology and Medical Oncology and Therapeutics Research, City of Hope Comprehensive Cancer Center, Duarte, California Received 3/6/09; revised 8/17/09; accepted 9/9/09; published OnlineFirst