Abstract
Adenomatous Polyposis Coli (APC) is lost in approximately 70% of sporadic breast cancers, with an inclination towards triple negative breast cancer (TNBC). TNBC is treated with traditional chemotherapy, such as paclitaxel (PTX); however, tumors often develop drug resistance. We previously created APC knockdown cells (APC shRNA1) using the human TNBC cells, MDA-MB-157, and showed that APC loss induces PTX resistance. To understand the mechanisms behind APC-mediated PTX response, we performed cell cycle analysis and analyzed cell cycle related proteins. Cell cycle analysis indicated increased G2/M population in both PTX-treated APC shRNA1 and parental cells, suggesting that APC expression does not alter PTX-induced G2/M arrest. We further studied the subcellular localization of the G2/M transition proteins, cyclin B1 and CDK1. The APC shRNA1 cells had increased CDK1, which was preferentially localized to the cytoplasm, and increased baseline CDK6. RNA-sequencing was performed to gain a global understanding of changes downstream of APC loss and identified a broad mis-regulation of cell cycle-related genes in APC shRNA1 cells. Our studies are the first to show an interaction between APC and taxane response in breast cancer. The implications include designing combination therapy to re-sensitize APC-mutant breast cancers to taxanes using the specific cell cycle alterations.
Highlights
Breast cancer accounts for 30% of all new cancer diagnoses in women and is the leading cause of death for women between the ages of 20 and 59 years
Because PTX works by arresting cells during mitosis, we expected an increase in G2/M expression following PTX treatment in cells sensitive to the drug
Loss of the Adenomatous Polyposis Coli (APC) tumor suppressor in human triple negative breast cancer (TNBC) cells alters the response to taxane treatment, likely through cell cycle mediated changes
Summary
Cell culture and drug treatmentMDA-MB-157 breast cancer cells (ATCC, Manassas, VA) were maintained at 37 ̊C with 5% CO2 in RPMI 1640 media with 1:5000 plasmocin, 1% penicillin/streptomycin, 10% fetal bovine serum and 25 mM HEPES. The APC knockdown shRNA 1 and 2 cell lines were routinely maintained in media containing 1.5 μg/mL puromycin (MilliporeSigma, St Louis, MO). Reporter assays were performed as previously described [11]. Cells were plated in triplicate and transfected using Lipofectamine 2000 (Invitrogen) with either pTOPflash or pFOPflash as previously described, and co-transfected with pRL-TK (Renilla luciferase-thymidine kinase; Promega, Madison, WI). Cell lines treated at 50–70% confluence with 1 μM nocodazole, 0.078 μM paclitaxel, 16 μM cisplatin, or control DMSO for 24 hours. Nocodazole was used as a control for G2/M arrest, and cisplatin was used as a control because it effectively kills APC shRNA1 cells [7].
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