Abstract P5-04-05: Targeting the pH regulatory mechanisms of breast cancer cells
Authors: J Meehan, C Ward, E Jarman, C Xintaropoulou, C Martinez-Perez, A Turnbull, C Supuran, M Dixon, I Kunkler, SP Langdon
Journal: Cancer Research (2016)

Abstract Background: The abnormal regulation of H+ ions, leading to a reversed pH gradient in tumor cells in comparison to normal cells, is considered to be one of the hallmarks of cancer. This feature, however, has yet to be exploited as a therapeutic target. The aim of this study was to assess whether targeting proteins (CAIX, NHE1 and V-ATPase) that permit hypoxic cancer cell adaptation to acidosis in the tumor microenvironment can produce an effective therapeutic response in breast cancer, using 2D and 3D models. Method: Western blotting and gene expression analysis were performed on MCF-7, MDA-MB-231 and HBL-100 cancer cells to assess target protein expression in differing O2 conditions in 2D, while IHC was used to measure protein levels in 3D using multicellular tumor spheroids. Sulforhodamine B assays were executed to analyze the effects of inhibitors targeting CAIX, NHE1 and V-ATPase on breast cancer cell proliferation in 2D. 3D invasion assays were performed with MDA-MB-231 spheroids and explant tissue derived from human patients to see if CAIX inhibition had any effect on cancer cell invasion. An MDA-MB-231 xenograft model was used to investigate the effects of CAIX inhibition in vivo. Clonogenic assays were performed with MDA-MB-231 spheroids to evaluate whether any of the drugs combined effectively with irradiation. Results: 2D and 3D expression analysis showed that CAIX levels were extremely responsive to changes in O2 conditions in each of the cell lines, with HBL100 cells exhibiting the largest changes in both mRNA (42-fold increase) and protein (78-fold increase) levels at low (0.5%) O2 concentrations. NHE1 and V-ATPase mRNA/protein levels were, however, much more consistently expressed across the cell lines in different O2 conditions. Drugs targeting CAIX, NHE1 and V-ATPase had anti-proliferative effects on the breast cancer cells in 2D. Normoxic cancer cells were the most sensitive to drug treatment, acute hypoxic cancer cells showed increased resistance to the anti-proliferative effects of these drugs, while chronic hypoxic cells had IC50 values more similar to the normoxic cells. The results for the CAIX inhibitor were unexpected, as we had predicted that the increased levels of CAIX in the acute hypoxic cells would make them more sensitive to treatment. CAIX inhibition did, however, significantly reduce the invasion of cancer cells from both MDA-MB-231 spheroids (p≤0.01) and explant tissue (p≤0.001). Targeting pH regulation was also shown to have an effect in vivo on MDA-MB-231 xenografts, with CAIX inhibition significantly reducing the growth (p≤0.05) and proliferation (p≤0.05) of tumors within mice. Finally, clonogenic assays showed that drugs targeting both CAIX and NHE1 led to a significant reduction in colony number when combined with radiation (p≤0.05), compared to either drug individually or radiation treatment alone. Conclusions: This study shows that drugs targeting pH regulation molecules have potential in the treatment of breast cancer. This is highlighted by their ability to affect the proliferation and invasion of breast cancer cells, along with their ability to be combined with radiation. Of the 3 pH regulatory molecules, CAIX represents the target with the most promise. Citation Format: Meehan J, Ward C, Jarman E, Xintaropoulou C, Martinez-Perez C, Turnbull A, Supuran C, Dixon M, Kunkler I, Langdon SP. Targeting the pH regulatory mechanisms of breast cancer cells. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-04-05.