Diabetic Triple Negative Breast Cancer: Polyamine Enzymes, Ornithine decarboxylase and Spermine oxidase as Potential Targets

Abstract

Breast cancer (BC) is the second leading cause of death in women. Co‐existence of diabetes and BC, termed diabetic BC, can be lethal, leading to higher mortality (15‐40%) than BC alone. In triple negative breast cancer (TNBC) patients, chance of TNBC recurrence is about 3 months, and chance of mortality can be as high as 75%. TNBC is highly refractive, heterogenous, and resistant to most chemotherapies. Polyamines, which are ubiquitous in most cells, and play a role in cell growth, have been shown to be elevated in cancer, though their role in diabetic BC has not been explored. We hypothesized that high glucose/diabetic conditions increase TNBC cell proliferation through modification of the enzymes (ornithine decarboxylase and spermine oxidase) in the polyamine pathway. The objective was to determine potential targets in the polyamine pathway to mitigate TNBC advancement in diabetic conditions. For this purpose, MDA‐MB‐231 (TNBC) and MCF10‐A (normal breast epithelial) cell lines were used. Both were treated with low (5mM) and high glucose (25mM) concentrations and effects on polyamine levels, polyamine enzyme, and cell proliferation were monitored. Polyamine levels were assayed using reverse phase high performance liquid chromatography. Cell proliferation was assessed using a fluorescence‐based assay, and polyamine enzyme expression was monitored at the mRNA and protein level using RT‐PCR and Western Blots. Samples were sent to Johns Hopkins University for polyamine enzyme activity analysis. Further, an approved inhibitor of polyamine synthesis, alpha‐difluoromethylornithine (a‐DFMO, 5mM) was used in combination with glucose treatments. Under diabetic/high glucose conditions, putrescine levels were elevated in MDA‐MB‐231 cells (135%), which correlated with higher cell proliferation (123%). Polyamine enzyme activity of ODC increased (from ~75 pMolCO2/hr/mg protein to ~150 pMolCO2/hr/mg protein) and SMOX decreased (from ~0.4 pMolH202/min/mg protein to ~0.06 pMolH202/min/mg protein) in concert with changes observed at protein levels with high glucose exposure. DFMO was effective in reducing polyamine levels and cell proliferation, however re‐supplementation of polyamines (spermidine and spermine) restored cell proliferation to that, indicating that DFMO is only cytostatic. MCF‐10A cells showed marked elevation in cell proliferation with high glucose. DFMO prevented this increase; however no apparent change was observed in polyamine levels. The polyamine biosynthetic enzymes (ornithine decarboxylase and spermine oxidase) play an active role in increasing cell proliferation under high glucose conditions in TNBC. Since DFMO caused only cytostatic effects, there is a need for a more targeted approach to regulate polyamine levels and mitigate TNBC growth under diabetic conditions.