Abstract PD2-05: Evaluating the role of recurrent ESR1-CCDC170 in breast cancer endocrine resistance
Authors: Y Hu, J Veeraraghavan, X Wang, Y Tan, J Kim, R Schiff, X-S Wang
Journal: Cancer Research (2016)

Abstract Background Recurrent gene fusions resulting from chromosome translocations are critical genetic aberrations causing cancer. In our previous study, we identified recurrent rearrangements between ESR1 and its neighbor, CCDC170, in 6-8% of luminal B tumors. Luminal B subtype is a more aggressive ER+ breast cancer, with a higher risk of early relapse after endocrine therapy. These rearrangements enable the expression of N-terminally truncated CCDC170 (ΔCCDC170) under ESR1 promoter. Consistent with the behavior of luminal B tumors, ectopic ΔCCDC170 expression in ER+ breast cancer cells, led to markedly increased cell motility, invasion, anchorage-independent growth, and reduced endocrine sensitivity in vitro, as well as enhanced xenograft growth in vivo. In the present study, we studied the role of ESR1-CCDC170 in breast cancer endocrine resistance in vivo and explored the potential mechanism. Methods To study endocrine resistance in vivo, we transplanted T47D cells stably overexpressing (OE) control (empty) construct or 2 ΔCCDC170 fusion variants (E2-E7 and E2-E10) bilaterally to 4-6 week old female athymic nude mice (supplemented with 17β-estradiol pellets). The tumor growth was monitored biweekly and tumor volume was measured by the formula 1/2(length × width2). When the tumors reach 150–200 mm3, mice were randomly allocated to vehicle or tamoxifen (tam) treatment groups. For ERE luciferase assay, cells were co-transfected with ERE luciferase reporter (ERE-TK-Luc) and pCMV β-galactosidase. The luciferase levels were measured and normalized to β-gal activity. For immunoblot analysis, T47D OE cells were estrogen-deprived, serum-starved, and treated with vehicle, estrogen (E2) or tam. Reverse Phase Protein Array (RPPA) analysis was performed using ∼200 validated antibodies against an array of key signaling molecules in cancer. Results Our in vivo endocrine sensitivity study showed that, while T47D vector control tumors mostly regressed after tam treatment, the regression of E2-E7 tumors was significantly slower. Moreover, E2-E10 tumors continued to grow despite tam treatment. These observations suggest that ΔCCDC170 may render the T47D xenografts less sensitive to tam in vivo. Kaplan–Meier analysis revealed a significantly worse progression-free survival (defined by tumor doubling time) for E2-E7 (p&amp;lt;0.01) and E2-E10 (p&amp;lt;0.001) tumors treated with tam compared to control tumors. ΔCCDC170 expression in T47D cells enhanced the ER transcriptional activity in the presence of E2 but not tam, suggesting that the fusion-mediated endocrine-sensitivity changes is unlikely due to restoration of classic ER activity. Immunoblot analysis of T47D OE cells revealed hyperactive growth factor signaling even after serum withdrawal, which was not significantly affected by tam treatment. Preliminary RPPA analysis revealed upregulation of key signaling molecules in T47D cells expressing ΔCCDC170, such as Her3, AMPK, Akt, Erk, c-Myc, and Src-3. Conclusion These data suggest a potential role of ESR1-CCDC170 in mediating breast cancer endocrine resistance, presumably due to hyperactive growth factor signaling endowed by this fusion. Further studies are required to elucidate the role of endogenous ESR1-CCDC170 in breast cancer endocrine resistance, and discover the precise engaged mechanisms. Citation Format: Hu Y, Veeraraghavan J, Wang X, Tan Y, Kim J, Schiff R, Wang X-S. Evaluating the role of recurrent ESR1-CCDC170 in breast cancer endocrine resistance. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr PD2-05.