Association between ER±36 and PR A/B overexpression in trastuzumab resistant HER2+ breast cancer cell lines.

Abstract

e12501 Background: Clinical importance of the ERa36 isoform is poorly understood. It is present in 40% of the cells that lack the ERa66 and has also been linked to proliferation and invasion. It is also related to expression of phospho-src. Coincidently, phospho-src over-expression is associated to trastuzumab-resistance. We analyzed the possible link between trastuzumab-resistance and over expression of estrogen and progesterone receptors in HER2+ cell lines with trastuzumabTM chronic treatment. Methods: BT-474 and SKBR-3 cells were treated with trastuzumabTM with 4 ug/ml for 8 weeks, then 10 ug/ml for 2 weeks and with 20 ug/ml for the last 2 weeks. Protein extracts for western blot analysis where obtained from paternal cell lines and at 4, 8 and 12 weeks during treatment. Western blots were done for total and phosphorylated HER2, total and phosphorylated Src, aER and PR A/B. Densitometry analysis was applied to the western blots with the image-jTM software. Surface HER2 status was determined by flow citometry. Results: Treatment led to increase in T-HER but no changes in p-HER, T-Src and p-Src where increased, 5.27% and 18.01% respectively compared to baseline. 36KDa and 46KD aER isoforms increased with treatment. The most significant increase was in PR A expression. Western Blot for total HER2 and flow citometry for surface HER2 status revealed a link between HER2 and hormone receptor over-expression in TrastuzumabTM resistant BT-474 cells while both where absent in trastuzumabTM resistant SKBR-3 cells. Conclusions: A variety of trastuzumabTM-resistance mechanisms have been established, some of which include over-expression of p-Src. So far, none of them had been associated with expression of hormone receptors. We observed two molecular phenotypes associated to trastuzumabTM resistance: over-expression of HER2 linked to over-expression of hormone receptors in BT-474 cell and down-regulation of HER2 with no change in the hormone receptor status in the SKBR-3 cells. Our results suggest that over-expression of ERa36, ERa46 and PR A/B could represent a novel mechanism of trastuzumabTM resistance.