Treating triple negative breast cancer cells with erlotinib plus a select antioxidant overcomes drug resistance by targeting cancer cell heterogeneity
Among breast cancer patients, those diagnosed with the triple-negative breast cancer (TNBC) subtype have the worst prog-nosis. TNBC does not express estrogen receptor-alpha, progesterone receptor, or the HER2 oncogene; therefore, TNBC lacks targets for molecularly-guided therapies. The concept that EGFR oncogene inhibitor drugs could be used as targeted treatment against TNBC has been put forth based on estimates that 30–60% of TNBC express high levels of EGFR. However, results from clinical trials testing EGFR inhibitors, alone or in combination with cytotoxic chemotherapy, did not improve patient outcomes. Results herein offer an explanation as to why EGFR inhibitors failed TNBC patients and support how combining a select antioxidant and an EGFR-specific small molecule kinase inhibitor (SMKI) could be an effective, novel therapeutic strategy. Treatment with CAT-SKL—a re-engineered protein form of the antioxidant enzyme catalase—inhibited cancer stem-like cells (CSCs), and treatment with the EGFR-specific SMKI erlotinib inhibited non-CSCs. Thus, combining the antioxidant CAT-SKL with erlotinib targeted both CSCs and bulk cancer cells in cultures of EGFR-expressing TNBC-derived cells. We also report evidence that the mechanism for CAT-SKL inhibition of CSCs may depend on antioxidant-induced downregulation of a short alternative mRNA splicing variant of the methyl-CpG binding domain 2 gene, isoform MBD2c.We began our study by testing the effect of the combination treatment, CAT-SKL plus EGFR SMKI or HER2 SMKI, on cell viability across a panel of 8 cell lines. This included six EGFR-expressing, TNBC-derived cell lines and two HER2-amplified breast cancer-derived cell lines. Supplementary Table S1 outlines the molecular characteristics for each of these cell lines, which were previously reported14. Results of cell viability assays showed that CAT-SKL or SMKI alone had either a relatively modest effect or no effect, but a significant loss of viability due to three-day combined treatment was observed for four of the six TNBC cell lines, including MDA-MB-468, SUM-149, SUM-159, and HCC-70 (Fig. 1). Each of these four cell lines showed some level of lack of response to erlotinib treatment that was overcome by co-treatment with CAT-SKL. Treatment with CAT-SKL or erlotinib, alone or in combination, appeared to have no effect on TNBC cell lines MDA-MB-231 and HCC1937.For each of the HER2-amplified breast cancer cell lines tested— SUM-225 and SUM-190— loss of viability was observed for single agent treatment with CAT-SKL, and treatment with HER2-specific SMKI CP724,714 appeared to induce a maximum effect. The response to combination of CP724,714 and CAT-SKL was not significantly different compared to CP724,714 alone (Fig. 1). The results suggest that targeted inhibition of the HER2 oncogene by CP724,714 treatment superseded any potential benefit of CAT-SKL co-treatment.The previously established human triple negative breast cancer and HER2-positive breast cancer cell lines were recently acquired for this study from the American Type Culture Collection (ATCC, Manassas, VA) or from the Biobanking and Correlative Sciences Core (BCSC) at the Karmanos Cancer Institute (KCI, Detroit, MI). The BCSC provides a record of authenticity for each cell line lot based on short tandem repeat (STR) analysis. HCC-70 cells were maintained in 5% FBS RPMI-1640 media containing amphotercin B and gentamycin at 37 °C, 5% CO2. All other cells were maintained in 5% FBS DMEM media containing amphotercin B and gentamycin at 37 °C, 5% CO2. Erlotinib, CP724,714 and -(−)epicatechin were from Sigma-Aldrich (St. Louis, MO). We previously showed that CP724,714, which was originally developed by Pfizer Inc. (Groton, CT), specifically inhibits HER2 activity9. CAT-SKL is a reengineered form of the antioxidant enzyme catalase, formulated and prepared as described previously11,12,27,28,29.This work was funded in part by the institutional award ACS/IRG#11-053-01 (to ABF), QNRF NPRP7-363-3-089 (awarded to ABF and RM). Additional support came from NIH Center grant P30 CA022453 to the KCI.The authors declare no competing financial interests.Author Contributions A.B.F. wrote the manuscript. A.B.F., B.B. and S.R.T. designed experiments. B.B., P.W., E.G. and R.L.F. performed experiments. C.M., L.M. and G.D. ran microarray data and statistical analyses. S.R.T., J.L.B., B.B. and R.M. provided cell biological expertise and experimental insights, and edited the manuscript.