Interrogating the regulation, function and therapeutic potential of monocarboxylate transporters in osteosarcoma

A distinguishing feature of cancer cells is their ability to undergo aerobic glycolysis (known as the Warburg effect), allowing them to thrive in a variety of microenvironments. Monocarboxylate transporters (MCTs) are key facilitators of aerobic glycolysis, moving lactate across the plasma membrane, and are critical for growth and metastasis of glycolytic tumors. Lactate enters cells through MCT1 and MCT4 and is converted by LDH to pyruvate. Forkhead box protein M1 (FOXM1), a transcription factor implicated in survival, metastasis, and chemotherapy resistance of cancer cells also influences cellular metabolism through transactivation of the LDH gene. Interestingly, another transcription factor, STAT3, directly regulates FOXM1 transcription and LDH expression. FOXM1 has been shown to bind to the STAT3 promoter in cancer cells, altering basal STAT3 expression, indicating potential circular regulation of FOXM1/STAT3 gene expression that modulates cellular metabolic capacity. Our laboratory has been investigating the biology of osteosarcoma (OS) for the past 10 years using canine OS as a spontaneous model of the human disease, identifying constitutive activation of STAT3 in OS cell lines and primary tumor samples. Both MCT1 and MCT4 are expressed in OS cell lines and tumor samples, and MCT4 expression increases in response to lactate. Furthermore, our data show that combining MCT1/4 inhibition with doxorubicin promotes synergistic inhibition of OS cell growth. The purpose of this proposal is to expand upon our initial work and interrogate the mechanisms through which STAT3 and FOXM1 regulate MCT1/4 expression and function in OS cell lines. We hypothesize that FOXM1 is over-expressed in OS cells and regulates key features of aerobic glycolysis through STAT3, ultimately modulating MCT1/4 expression and function. We further predict that combined inhibition of MCT1/4 and STAT3 will exhibit synergistic activity with chemotherapy. The specific aims of this work are to:

1. Determine the impact of MCT1/4 on aerobic glycolysis in OS lines: We will use CRISPR-Cas9 to knockout MCT1 and MCT4 genes in OS lines and then assess the effects on metabolic phenotype using the Seahorse assay as well as other key biologic features of glycolysis.
2. Characterize the effects of STAT3 activation on MCT1/4 expression and function. We will determine the impact of STAT3 activation on MCT1/4 expression and function and assess how FOX1M regulates this process in in the context of STAT3 phosphorylation.
3. Assess consequences of combined doxorubicin, MCT1/MCT4 and STAT3 inhibition. We will evaluate the utility of combined STAT3 and MCT inhibition with doxorubicin chemotherapy to induce synthetic lethality in OS cell lines.

Impact: Understanding the biology that drives aerobic glycolysis in OS cells will facilitate the design of future studies combining MCT1/4 inhibitors with chemotherapy in dogs with OS as a prelude to subsequent human trials.