Abstract 774: Role of t-Darpp in a shift to EGFR signaling in trastuzumab-resistant cells
Authors: Erin Denny, Manoj Kanagaraj, Susan E. Kane
Journal: Cancer Research (2012)

Abstract Elucidating the molecular mechanism of both primary and acquired resistance to trastuzumab is vital to gaining insight into ways to improve therapeutic outcomes in breast cancer patients. We have previously shown that levels of the 32kDa dopamine and cAMP-regulated phosphoprotein Darpp-32 (Dp32) and its amino-truncated isoform t-Darpp (tDp) are altered in trastuzumab-resistant BT474 cells. Specifically, tDp is upregulated in models of trastuzumab resistance and over-expression of the protein is sufficient to confer resistance. We have further demonstrated that trastuzumab-resistant BT474 cells are more sensitive to the EGFR tyrosine kinase inhibitor (TKI), AG1478, in the presence of trastuzumab than in its absence (1), suggesting that these cells gain a dependence on EGFR when HER2 signaling is shut down. We have now examined the mechanism by which tDp confers resistance in HER2+ breast cancer cells. We used SK-Br-3 cells stably expressing an empty vector, tDp or flag-tDp, as well as two trastuzumab-resistant BT474 clones, BT1.0C and BT1.0E, that endogenously over-express moderate and very high levels of tDp, respectively. Cells with elevated tDp levels had a higher basal level of p-EGFR (Y1068) and we observed an increase in p-EGFR upon trastuzumab treatment in all of the HER2+ cells. To further investigate if EGFR signaling plays a role in tDp's resistance mechanism, we performed SRB assays to test the sensitivity of tDp over-expressing cells to EGFR TKIs, AG1478 and Gefitinib. We found that cells expressing high tDp are 2.5- to 7.5-fold more sensitive to EGFR inhibition compared to controls (p&amp;lt;0.01). Furthermore, the combination treatment of AG1478 and trastuzumab was synergistic in inhibiting the trastuzumab-resistant BT1.0E cell line that highly over-expresses tDp at an ED50, ED75 and ED90 CI of 0.61, 0.46, and 0.35, respectively. The parental BT474 cells and BT1.0C clone, which expresses lower levels of tDp, showed antagonism at the ED50 but synergism at the ED75 and ED90. Using siRNA knock-down of tDp, we will show whether tDp is required in causing an increased dependence on EGFR signaling. We will also report on similar combination experiments using tDp-transfected SK-Br-3 cells, to determine if tDp expression is sufficient for conferring synergism, and with a HER2 TKI in combination with EGFR TKI. Taken together, our results suggest that tDp is a novel molecular target that can mediate resistance to trastuzumab by inducing a compensatory shift to EGFR signaling when HER2 is inhibited. 1. Chan CT, Metz MZ, &amp; Kane SE (2005) Differential sensitivities of trastuzumab (Herceptin)-resistant human breast cancer cells to phosphoinositide-3 kinase (PI-3K) and epidermal growth factor receptor (EGFR) kinase inhibitors. Breast Cancer Res Treat 91(2):187-201. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 774. doi:1538-7445.AM2012-774