Exploring mechanisms of acquired taxane resistance in breast cancer

Abstract

Breast cancer is the most commonly diagnosed cancer globally and, despite advances in detection and improved treatment, it remains the leading cause of cancer death among women. Chemotherapy regimens include taxanes in combination, or in sequence, with anthracyclines as first-line treatment with proven survival benefits. However, the development of chemoresistance is a major clinical challenge in the treatment of breast cancer with limited treatment options available thereafter. Identification of mechanisms driving taxane resistance is therefore critical to developing new treatment strategies to improve patient survival. The primary aim of this thesis was to model acquired taxane resistance and identify actionable pathways associated with the progression to a chemoresistant phenotype.

Isogenic models of acquired taxane resistance were developed from the breast cancer cell lines MDA-MB-231 and MCF7 by continuous exposure to either paclitaxel or docetaxel. STR profiling confirmed the parental lineage of the derived taxane resistant isogenic cell lines and assessment of biomarkers by western blotting confirmed retention of the triple negative phenotype of MDA-MB-231. MCF7 25PACR cells exhibited a slight decrease in ERα expression but otherwise retained the protein expression profile of its parent. The resistant cell lines exhibited the ability to proliferate and progress through the cell cycle in the presence of taxanes, unlike the parental cell lines which arrested at the G2/M phase. This was reflected in the absence of apoptosis in the resistant cell lines when exposed to concentrations of taxanes which induced cell death in the parental counterpart. Low level cross resistance was observed to anthracyclines and other commonly used chemotherapeutic agents. Expression of p-gyp protein encoded by MDR1 was assessed by western blotting and shown to be slightly elevated in both MCF7 25PACR and MDA-MB-231 25DOCR cell lines. However knockdown of MDR1 by siRNA did not result in reversion to the same level of taxane sensitivity as observed in the parental cell lines, suggesting other mechanisms are involved in the resistance observed in the isogenic cell lines derived for this study.

To gain insight in to the pathways driving taxane resistance in the cell line models, a multi-omic approach was taken. Targeted exome sequencing of the isogenic taxane resistant cell lines revealed few resistance-associated genomic changes. Transcriptomic analysis of the parental and isogenic taxane resistant cell lies was performed by Nanostring®. Bioinformatic analysis revealed deregulation of the PI3- Akt, MAPK, and transcriptional regulation pathways in the isogenic cell lines, suggesting that inhibition of these pathways may be an effective treatment strategy for taxane resistant breast cancer. RPPA and Western blotting confirmed these findings and a small molecule kinase screen identified several clinically relevant inhibitors with nanomolar potency in the resistant cell line models. The PI3K inhibitors, PIK75 and BKM120, and the CDK inhibitors, dinaciclib and alvocidib, were selected for further investigation via a combination of in vitro techniques. Confirmation of their potency against all taxane resistant cell line models was established and combination assays to assess synergy with other chemotherapeutics performed.

Together, these results extend our knowledge of the drivers of taxane resistance in breast cancer and support the use of PI3K and CDK inhibition as candidate therapeutics in taxane resistant breast cancer.