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(R) cells are trastuzumab-resistant variants of Her2-positive BT474 breast cancer cells. The salient feature of BT/Her(R) 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(R) cells, focusing on the protein kinase A (PKA) pathway.
Experimental design: We performed microarray analysis on BT/Her(R) and BT474 cell lines to identify genes that were upregulated or downregulated in trastuzumab-resistant 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(R) 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-RIIalpha 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.