Abstract P1-08-03: Targeting lactate metabolism as a novel therapeutic target in platinum-resistant triple negative breast cancer (TNBC)
Authors: Harada Narumi , Varghese Vidhya , Xie Lingmin , Kenny Laura M 
Published: May 2015

Introduction TNBC is a heterogeneous disease and is often associated with expression of a stem cell signature (CD44+, CD24-). Recently platinum agents have been shown to improve the pathological response rates in the neo-adjuvant setting; however a significant proportion of patients with TNBC will relapse. The study objective was to evaluate the potential of targeting glycolysis, mitochondrial function and lactate metabolism in platinum resistant TNBC cell lines.

Methods Stem cell signature and glycolytic phenotyping: CD44 and CD24 antibodies were used to measure the percentage of stem cells in each of the cell lines using flow cytometry(FACS). Protein expression of hexokinase, Glut1 transporter, lactate dehydrogenase A/B(LDHA and LDHB), and monocarboxylic transporter 1 and 4 (MCT1 and MCT4) was determined in MCF7(control), MDA-MB231, MDA-MB468, and HCC38 cells. Proliferation and apoptosis assays: IC50s for platinum, metformin, and MCT1 inhibitor AR-C155858, Tocris (MCT1I) were calculated by the sulforhodamine B (SRB) assay. Propidium iodide and AnnexinV staining intensity used to measure apoptosis by FACS. Metabolic analysis: The change in lactate and glucose levels in culture media post-treatment with metformin and MCT1I was quantified by 1H NMR. Intracellular lactate and ATP concentrations were determined by lactate(BioVision) and ATP determination assays(Invitrogen). Mitochondrial membrane potential and Reactive oxygen species (ROS) were measured by MitoTracker Red and MitoSOX Red. 2NBDG was used to assess cell glucose uptake. Combination index SRBs: Cells were treated with increasing concentrations of MCT1I and metformin for 72h. The combination index values were calculated using CalcuSyn® software.

Results MDA-MB231 and HCC38 cells were resistant to cisplatin and associated with a stem cell signature (CD44+, CD24-) which was not detected in the other cell lines. MCT1 was highly expressed in HCC38 cells but not at all in MDA-MB231. In contrast, MCT4 was abundantly expressed MDA-MB231 but minimally in HCC38 cells.

In these two cell lines, only MDA-MB231 was sensitive to metformin which reduced ATP production, induced ROS generation, and increased the percentage of apoptotic cells. HCC38 also produced ROS, but ATP production and apoptosis were unaffected.

HCC38 cells were more sensitive to MCT1I than other cells, which was associated with increased intracellular lactate.

The NMR data showed significant increases in lactate in culture media following treatment with metformin in MDA-MB231 and HCC38. Only HCC38 had increased 2NBDG uptake and reduced of glucose levels in culture medium by metformin.

Metformin significantly enhanced the anticancer efficacy of MCT1I in HCC38 cells, as a synergistic effect of the two agents was observed across the range of different concentrations; whilst an antagonistic effect was shown in MDA-MB231 cells with almost all concentrations.

Conclusions Targeting lactate metabolism selectively inhibits a platinum-resistant stem cell population of TNBC, this effect is enhanced when combined with metformin.

The inhibition of glycolysis and lactate metabolism may represent a new therapeutic strategy for this population and warrants further study.

Citation Format: Narumi Harada, Vidhya Varghese, Lingmin Xie, Laura M Kenny. Targeting lactate metabolism as a novel therapeutic target in platinum-resistant triple negative breast cancer (TNBC) [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P1-08-03.