Pilot: Role Of Succinate In Regulating Cancer Cell Plasticity And Stemness

Project Summary Triple-negative breast cancer (TNBC) remains a significant clinical challenge due to high incidences of metastasis and post-treatment relapse. Tumor cells in TNBC tissue exhibit enhanced cell plasticity and stemness, which is crucial for cancer metastasis. Metabolic and epigenetic reprogramming plays crucial roles in the regulation of cell plasticity. Succinate is an intermediate metabolite of the tricarboxylic acid (TCA) cycle. Aberrant accumulation of succinate has been detected in many cancers. However, the cellular function and regulation of succinate in breast cancer development and progression are not well-defined. Using unbiased stable isotope- resolved metabolomics (SIRM) analysis, we showed that the epithelial-to-mesenchymal transition (EMT) was associated with profound changes in metabolites, including elevation of cytoplasmic succinate. Succinate is an intermediate metabolite of the tricarboxylic acid (TCA) cycle and a product inhibitor of TET family of 5- methylcytosine hydroxylases. We found that treatment with the membrane permeable succinate was sufficient to induce mesenchymal phenotypes, enhance cancer cell stemness and colonization, and reduce DNA hydroxymethylation and gene transcription in mammary epithelial cells. These results indicate the crucial roles of succinate in breast cancer progression. By analyzing the gene expression profiles and ChIP-seq data, we identified DDR1 as a potential target of the succinate/5hMC axis. We will define roles of the succinate/5hMC axis in regulating DDR1 expression and cancer cell stemness. We will also test the hypothesis that succinate enhances cancer cell plasticity and stemness by repressing DNA hydroxymethylation-dependent DDR1 transcription. Aim 1. Define molecular mechanism by which succinate represses DDR1 expression and DNA hydroxymethylation. Succinate treatment reduced 5hMC accumulation in chromatin, which is associated with EMT. We will test the hypothesis that succinate represses DDR1 expression by reducing DNA hydroxymethylation at the CpG island, and subsequently enhances cancer cell plasticity and stemness. We will also identify potential pathways and transcription factors that mediate succinate function by integrating RNA-seq, ChIP-seq, and position weight matrix analyses. Aim 2. Define roles of the succinate/DDR1 axis in regulating cancer cell colonization and metastasis. Using the in vivo dilution assay and the orthotopic mammary tumor model, we define roles of the succinate/DDR1 axis in regulating cancer cell colonization and TNBC metastasis. Using the tissue microarray generated at the Markey Cancer Center We will also determine whether 5hMC levels in human breast cancer tissue is associated with cancer subtypes and expression of DDR1 and EMT markers. Use of COBRE core: We have worked with Metabolism Core to quantify metabolite changes during the EMT. In this pilot project, we will utilize the Imaging Core to analyze cancer cell colonization in lungs and expression of epithelial and mesenchymal markers in mammary epithelial cells. Statement of extramural funding application. This study will reveal novel roles of succinate-associated epigenetic reprogramming in cancer metastasis. Successfully completion of this project will provide necessary data for the external funding application. We have identified a collagen hydroxylation enzyme as a potential regulator of succinate accumulation in TNBC cells. An NCI R01 has been developed. In the R01 project, we will define the function and regulation of succinate in TNBC using genetic engineering mouse model of mammary tumor and evaluate the potential of targeting the collagen hydroxylation enzyme to inhibit succinate accumulation and TNBC progression. The A0 application was scored at 23 percentile. The data generated from this proposal will be critical for addressing reviewers’ comments to resubmit the application.