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Background: Cancer cells avidly consume glucose and convert it to lactate, resulting in a low pyruvate level. This phenomenon is known as the Warburg effect, and is important for cell proliferation. Although cMyc has often been described as an oncoprotein that preferentially contributes to the Warburg effect and tumor proliferation, mechanisms of action remain unclear. Histone deacetylase 3 (HDAC3) regulates gene expression by removing acetyl groups from lysine residues, as well as has an oncogenic role in apoptosis and contributes to the proliferation of many cancer cells including cholangiocarcinoma (CCA). HDAC inhibitors display antitumor activity in many cancer cell lines. Cancer cells maintain low levels of pyruvate to prevent inhibition of HDAC but the mechanisms remain elusive. The purpose of our study was to explore the role of cMyc in regulating pyruvate metabolism, as well as to investigate whether the inhibitory effect of pyruvate on HDAC3 could hold promise in the treatment of cancer cells. Methods: We studied pyruvate levels in CCA cell lines using metabolite analysis, and analyzed the relationship of pyruvate levels and cell proliferation with cell viability analysis. We cultivated CCA cell lines with high or low levels of pyruvate, and then analyzed the protein levels of HDAC3 and apoptotic markers via Western Blotting. We then explored the reasons of low levels of pyruvate by using seahorse analysis and 13C6 metabolites tracing analysis, and then confirmed the results using patient tissue protein samples through Western Blotting. Bioinformatics analysis and transfection assay were used to confirm the upstream target of the low levels of pyruvate status in CCA. The regulation of cMyc by HDAC3 was studied through immunoprecipitation and Western Blotting. Results: We confirmed downregulated pyruvate levels in CCA, and defined that high pyruvate levels correlated with reduced cell proliferation levels. Downregulated pyruvate levels decreased the inhibition to HDAC3 and consequently protected CCA cells from apoptosis. Synergistically upregulated LDHA, PKM2 levels resulted in low levels of pyruvate, as well as poor patient survival. We also found that low levels of pyruvate contributed to proliferation of CCA cells and confirmed that the upstream target is cMyc. Conversely, high activity of HDAC3 stabilized cMyc protein by preferential deacetylating cMyc at K323 site, which further contributed to the low pyruvate levels. Finally, this creates a positive feedback loop that maintained the low levels of pyruvate and promoted CCA proliferation. Conclusions: Collectively, our findings identify a role for promoting the low pyruvate levels regulated by c-Myc, and its dynamic acetylation in cancer cell proliferation. These targets, as markers for predicting tumor proliferation in patients undergoing clinical treatments, could pave the way towards personalized therapies. [ABSTRACT FROM AUTHOR]

Following the publication of the article, the authors have realized that Fig. 1A contained an error (essentially, the scale bars were drawn incorrectly). The corrected version of Fig. 1 is shown below, also including a modified version of the legend for Fig. 1A. Note that these revisions do not affect the overall conclusions reported in the paper. The authors apologize to the Editor of Oncology Reports and to the readership for any inconvenience caused. [the original article was published in Oncology Reports 39: 1957‑1965, 2018; DOI: 10.3892/or.2018.6275].

Cholangiocarcinoma (CCA) is a highly lethal malignancy with an often late diagnosis and consequent poor prognosis. Chemotherapy is the only therapeutic strategy for most patients. Compared to normal cells, tumor cells preferentially metabolize glucose to lactate, even in aerobic conditions. Such metabolic alterations not only support the growth and invasion of tumor cells, but also promote their chemoresistance. The purpose of our study was to explore the role of metformin in regulating the metabolism of CCA, as well as to investigate whether metformin could act as a chemosensitizer of the HDAC3 inhibitor BG45, and therefore have potential for the treatment of CCA. Through bioinformatic analysis, we found that aberrant metabolism contributed to the proliferation of CCA cells. Seahorse XF96 Extracellular Flux Analyzer analysis and lactate production analysis showed that metformin could act as a suppressor of the Warburg effect in CCA cells. Western blotting showed that metformin decreased the expression of LDHA, which plays a key role in the Warburg effect. However, suppression of the Warburg effect was not sufficient to induce CCA cellular apoptosis. According to our previous research, which showed that an HDAC3 inhibitor (MI192) was involved in CCA apoptosis, we observed that metformin combined with BG45 (a novel specific HDAC3 inhibitor) effectively induced the apoptosis of CCA cells in vitro. Furthermore, in vivo experiments revealed that the combined treatment with metformin and BG45 markedly reduced CCA growth in a CCA xenograft model. Our data revealed that reversing the Warburg effect with metformin sensitizes cells to the antitumor effects of HDAC3 inhibitors. This provides a rationale for using the combination of metformin and BG45 as a new therapeutic strategy in the treatment of CCA.