Xenobiotic transporter expression in breast cancer patients treated with neoadjuvant systemic therapy: implications for therapy

Three main xenobiotic transporters have been implicated in modulating breast cancer response to chemotherapy. These are Pgp (P-glycoprotein), MRP1 (multidrug resistance-associated protein 1), and BCRP (breast cancer resistance protein). My first aim was to investigate expression of these proteins before and after neoadjuvant chemotherapy (NAC) for breast cancer to determine whether their levels define response to NAC or subsequent survival. Immunohistochemistry was performed for Pgp, MRP1, and BCRP on paraffin embedded tissue representing matched pairs of core biopsy (pre-NAC) and resection specimens (post-NAC) from 39 breast cancer patients. Pgp and MRP1 were found to be significantly up-regulated after chemotherapy but levels did not relate to response or survival. High post-NAC BCRP expression independently predicted for poorer disease free survival (hazard ratio of 4.04; p=0.013). Evidence within the literature suggested that MRP1 up-regulation after chemotherapy may be driven by activated Notch1. My second aim was to determine whether activated Notch1 expression correlated with MRP1 expression in the same patient samples. Further immunohistochemistry to determine activated Notch1 expression revealed a significant correlation between post-NAC activated Notch1 and MRP1 expression (rho coefficient 0.6; p=0.0008). The hypothesis that inhibition of Notch signaling enhances killing of breast cancer cells by chemotherapeutics was developed. MTT assays were performed after treatment of breast cancer cells with combinations of doxorubicin and the Notch inhibitor DAPT. Minor additive inhibition of survival/proliferation was seen in the combination treatment, failing to provide strong support for the hypothesis. The BCRP promoter has an oestrogen response element. My third aim was to investigate whether BCRP expression within breast tumours is regulated by oestrogen and whether this impacts on cancer outcome. Immunohistochemistry was performed for BCRP in tumour samples from 51 patients receiving neoadjuvant endocrine therapy (NAET); matched core biopsy (pre-NAET) and resection specimens (post-NAET) were investigated. BCRP expression was significantly up-regulated after exposure to NAET (p<0.0001). High pre-NAET BCRP expression independently predicted for poorer DFS (hazard ratio of 17; p=0.014). Subsequent methylation analysis of cancer cell lines showed that the degree of methylation in the BCRP promoter region was potentially inversely correlated to the BCRP protein expression observed on immunoblotting. DNA was extracted from clinical sample and pyrosequencing analysis was performed. No such inverse correlation was observed in the clinical samples. My work demonstrates that analysis of tumour samples pre- and post-neoadjuvant therapies provides a powerful way of investigating therapy-dependent changes in expression of molecules of interest, and may be critical for determining the prognostic or predictive value of some markers. Given the relatively small sample size of the cohort examined, future higher powered studies are required to determine the prognostic significance of BCRP expression.