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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.

Background: 5-Fluorouracil (5-FU) is one of the most commonly used chemotherapy drugs for the treatment of colorectal cancer in the adjuvant and metastatic disease settings. One of the main targets of 5-FU is the enzyme thymidylate synthase (TS). High expression levels of TS in colorectal tumors removed from patients have been associated with drug resistance. The Forkhead box transcription factor M1 (FOXM1) has been demonstrated to regulate several aspects in cancer cells including: cell cycle progression, apoptosis, and cell survival. FOXM1 is over expressed in many types of cancers, including colorectal, and has been implicated in drug resistance in breast cancer. Thiostrepton is a thiazole antibiotic which selectively inhibits FOXM1. The relationship between FOXM1/TS and 5-FU resistance has not previously been established and may present a new treatment strategy. Purpose: In this study we sought to determine 1) the role of FOXM1 in regulation of TS, 2) whether inhibition of FOXM1 using thiostrepton or transient knockdown using a small interfering RNA would inhibit the TS expression in colorectal cancer cells, 3) whether combined therapy with 5-FU and thiostrepton would have a synergistic effect on cell survival, and 4) the effect of treatment with thiostrepton/ 5-FU/ combined thiostrepton and 5-FU on cellular migration and colony formation. Methods and results: HCT116, DLD1, and HT29 cells were treated with 0.5ug/ml 5-FU. TS and FOXM1 protein and mRNA expression were determined using Western blot and RTPCR. 5-FU treatment resulted in an initial increase (6h) followed by a decrease (48h) of both TS and FOXM1 in p53 wild type HCT116 cells, in p53 mutant DLD1 and HT29 cells whilst an initial increase was observed in both FOXM1 and TS there was no subsequent decrease. SRB assay was used to quantify the IC50 of 5-FU in HCT116, DLD1 and HT29 and was found to be 0.67±.23, 0.68±.22 and 3.09±.39 respectively. Transient knockdown of FOXM1 led to a significant decrease of TS in mRNA but not protein in HCT116 cells. For thiostrepton the IC50 was found to be 0.60uM, 1.07uM, and 3.54uM, in HCT116, DLD1 and HT29 cells, respectively. Combination of 5-FU and thiostrepton showed synergistic effects in all 3 colon cancer cell lines. Chromatin immunoprecipitation (ChIP) was performed in HCT116 and DLD1 cell lines to determine if FOXM1 binds to the TS promoter region (0 - 2000bp upstream of the TS transcription start site). 3 negative primers were designed further to 2000bp upstream region. ChIP data revealed enrichment for FOXM1 at the TS promoter (0-1500 bp upstream) and no enrichment observed in the negative control. Inhibition of FOXM1 by thiostrepton led to a drop in enrichment in the TS promoter region. These results confirmed the role of FOXM1 in TS regulation. Chip Sequencing (ChIP-seq) was then performed to study further FOXM1 targets and binding sites of FOXM1 to other well-known 5-FU targets such as thymidine phosphorylase (TP) and thymidine kinase 1 (TK-1). The FOXM1 specific ChIP-seq libraries and corresponding input controls were sequenced on the Illumina Hi Seq 2000. ChIP-seq confirmed that FOXM1 significantly binds to both these factors. FOXM1 binding to cell cycle regulatory genes E2F2 and E2F3 was also shown in both cell lines. Combination treatment of 5-FU and thiostrepton significantly decreases colony formation in HCT116, DLD1 and HT29 cell lines. 5-FU and thiostrepton combination also decreases migration in HCT116 cells. Conclusion: FOXM1 regulates the 5-FU target gene TS, and inhibition of FOXM1 acts enhances the cytotoxic effect of 5-FU in colorectal cancer cells and warrants further investigation as a new treatment strategy. Citation Format: Vidhya Varghese, Luca Magnani, Narumi Harada, Lam W. Eric, Laura Kenny. Inhibition of FOXM1 by thiostrepton increases sensitivity to 5-fluorouracil (5-FU) by downregulating thymidylate synthase (TS) in colorectal cancer. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr A54.

Background: Tumor resistance to chemotherapy is a major challenge in cancer therapeutics today. 5-Fluorouracil (5-FU) is one of the most commonly used cancer therapeutic agents. The Forkhead box transcription factor M1 (FOXM1) has been shown to play vital roles in cell cycle progression, apoptosis, and cell survival. FOXM1 is over expressed in many types of cancers, including colorectal carcinoma and has been implicated in drug resistance in breast cancer. Thymidylate synthase (TS) enzyme is an important precursor in DNA synthesis. The mechanism of action of 5-FU has been associated with TS. High TS expression levels in tumors are generally related to 5-FU resistance. In this study we investigated the role of FOXM1 in 5-FU resistance and the therapeutic potential of the FOXM1 inhibitor thiostrepton (a thiazole antibiotic) in combination with 5-FU in colorectal cancer. We further analyzed the relation between TS and FOXM1 gene expressions in colorectal cancer. Methods and Results: HCT116, DLD1, and HT29 cells were treated with 5-FU to detect the protein, mRNA expression of FOXM1 and TS. This resulted in a decrease in TS and FOXM1 expression in p53 wild type HCT116 cells but not in p53 mutant DLD1 or HT29. We also observed that FOXM1 expression was significantly correlated with TS expression by thiostrepton treatment. The IC50 of 5-FU was determined by SRB assay and was found to be 0.92ug/mL, 0.45ug/mL and 2.8ug/mL, while thiostrepton IC50 was found to be 0.60uM, 1.07uM, and 3.54uM, in HCT116, DLD1, and HT29 cells respectively. Combination of 5-FU and thiostrepton showed synergistic effect in all 3 colorectal cancer cells. Chromatin immunoprecipitation (ChIP) was performed in HCT116 and DLD1 cells to determine if FOXM1 binds to the TS promoter region (0 - 2000bp upstream of the TS transcription start site). Three negative primers were designed further to 2000bp upstream region. ChIP data revealed enrichment for FOXM1 at the TS promoter (0 - 1500 bp upstream) and no enrichment observed in the negative control. Inhibition of FOXM1 by thiostrepton led to a decrease in enrichment in the TS promoter region. These results confirmed the role of FOXM1 in TS regulation. Chip Sequencing (ChIP-seq) was then performed to study further FOXM1 targets and binding sites of FOXM1 to other well-known 5-FU targets such as thymidine phosphorylase (TP) and thymidine kinase 1 (TK1). The FOXM1 specific ChIP-seq libraries and corresponding input controls were sequenced on the Illumina Hi Seq 2000. ChIP-seq confirmed that FOXM1 significantly binds to both these factors. FOXM1 binding to cell cycle regulatory genes E2F1, E2F2 and E2F3 was also shown in both cell lines. Conclusion: FOXM1 plays a critical role in 5-FU drug resistance by regulating 5-FU target genes such as TS, TK1. Inhibition of FOXM1 acts synergistically with 5-FU in colorectal cancer and warrants further investigation as a new treatment strategy. Citation Format: Vidhya Varghese, Luca Magnani, Narumi Harada, Francesco A. Mauri, Eric W. Lam, Laura M. Kenny. Forkhead box transciption factor M1 (FOXM1) plays a critical role in colorectal cancer resistance by regulating thymidylate synthase (TS). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-220. doi:10.1158/1538-7445.AM2014-LB-220