Abstract 1792: The potential role of targeting HER1-4 in bladder cancer
Authors: Christiana Kitromilidou, Douglas McDonald, Pedro Cutillas, Essam Ghazaly, Jackie Perry, Bodor Csaba, Simon Joel, Tom Powles
Journal: Cancer Research (2012)

Abstract Introduction: The survival of patients with invasive bladder cancer is short, suggesting an urgent need for new treatments. Given that HER family receptor tyrosine kinases (TK) are associated with tumor stage/grade and outcome in this malignancy (Chow et al, Clin Cancer Res, 2001), approaches using small molecule inhibitors of HER receptors may be of value. In the present study we have investigated the potential utility and mechanism of action of Gefitinib (HER1 inhibitor), CP-724714(HER2 inhibitor), Lapatinib(dual HER1/2 inhibitor) and Canertinib (pan-HER inhibitor) in a panel of human bladder cancer cell lines. Methods: Four bladder cancer cell lines were used (T24, J82, H1376, 647V). HER 1-4 receptor protein and transcript were determined by Western blot and RT-PCR respectively. An ATP assay was used to determine the cytotoxic effect of HER targeted TKIs, with effects on cell cycle distribution and apoptosis determined by flow cytometric methods. A cell line with high receptor expression was selected for RNAi-silencing experiments targeting HER2 using lentivectors to determine the effects of specific HER2 silencing compared to the effects of HER2 inhibition. The differential effects of HER2 inhibition or silencing on downstream signalling was studied using western blot analysis and LC-MS/MS based methods for Akt activity and phosphoprotein profiling. Results: The expression levels of HER1-4 members varied between the cell lines. Sensitivity to TKIs also varied and was not correlated with HER1-4 expression. However, the dual (Lapatinib) and pan (Canertinib) -HER inhibitors were more potent (up to 30-fold) than the single HER inhibitors Gefitinib and CP-724714. Cell cycle analysis typically showed an increase in the G2/M population with all treatments, with a clear increase in apoptotic cells (up to 70%) observed only with Lapatinib. RNAi mediated HER2-knockdown in T24 cells was confirmed by western blotting and RT-PCR. With the exception of Gefitinib, sensitivity to TKIs was not altered by HER2 knockdown, with EC50 values for the HER2 TKI CP-724714 in parental and HER2-knockdown cells of 36.4 ± 14.5 vs 31.5 ± 10.3 µM respectively. Both HER2 RNA-silencing and inhibition by TKIs resulted in decreased downstream signalling (40% and 70% respectively), based on PI3K activity and phosphorylation of AKT. Phosphoproteomic analysis of TKI inhibited or HER2-knockdown cells showed common phosphoproteins (AHNAK, ASF-1) that were altered with all treatments, and others that were specific for HER2 silencing, HER2 TKI or HER2 mAb (Trastuzumab) exposure. Similar studies are now being performed in a second cell line. Conclusions: TKIs targeting HER1-4 receptors had a clear effect on the viability of bladder cancer cells. Both HER2-RNA silencing and TKIs resulted in decreased PI3K pathway activity. Phosphoproteins altered by these treatments highlight specific kinases that might represent potential therapeutic targets. 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 1792. doi:1538-7445.AM2012-1792