Adenomatous Polyposis Coli Regulates ABC Transporters and DNA Repair Mechanisms to Cause Doxorubicin Resistance

Chemoresistance is a leading cause of breast cancer related deaths. Therefore, understanding the molecular basis for chemoresistance is essential for novel therapeutic advancement and improving patient outcome. The Adenomatous Polyposis Coli (APC) tumor suppressor is lost in up to 70% of sporadic breast cancer; however, little is known about how APC loss contributes to chemoresistance. Using mammary tumor cells isolated from the Apc^Min/+ mouse crossed to the Polyoma middle T antigen (PyMT) transgenic model, we made the novel observation that APC loss decreased doxorubicin (DOX)-induced apoptosis. We hypothesized that APC loss prevents DOX-mediated apoptosis through: 1) reduced intracellular DOX and 2) alterations in DNA damage and repair with the goal to discover new combination therapies for APC-deficient cancer.

To investigate the intracellular DOX accumulation, we measured the amount of DOX intracellular fluorescence and found decreased intracellular DOX retention with APC loss. The ATP-binding cassette (ABC) transporters, multidrug resistance 1 (MDR1) and multidrug resistance protein 1 (MRP1), are well established to contribute to drug resistance through drug efflux and are overexpressed in tumor initiating cells (TICs). We showed that MMTV-PyMT;Apc^Min/+ cells have an increased TIC population. To investigate this decreased intracellular DOX accumulation, we found increased expression of MDR1 and MRP1. MDR1 inhibition and MRP1 silencing restored DOX intracellular retention, decreased the TIC population, and increased DOX-induced cleaved caspase 3 expression in APC-deficient cells. Dual MDR1/MRP1 inhibition did not have an additive effect. Increased MDR1 activity was specific to APC loss, which was shown to be mediated through signal transducer and activator of transcription 3 (STAT3).

To investigate the effect of APC loss on DNA damage repair pathways, we measured the amount of DOX-induced DNA damage in APC-deficient cells. There was decreased DOX-induced DNA damage and damage signaling mediated through ataxia-telangiectasia mutated (ATM) activation with APC loss. Inhibiting ATM or DNA-PK increased DOX-induced apoptosis. In addition, we found that APC loss affects Topo IIα activity decreasing Topo IIα/DNA complexes.

Taken together, APC loss decreased intracellular DOX accumulation through increased efflux and decreased DOX-induced DNA damage. MDR1 and ATM inhibition restore DOX sensitivity specifically with APC loss demonstrating the potential use of combination therapy to overcome chemoresistance.