Abstract B081: Regulation and function of monocarboxylate transporters in osteosarcoma

Abstract

Abstract Background: Osteosarcoma (OS), the most common malignant bone tumor in humans and dogs, shares several features in both species including clinical presentation and molecular alterations. Despite numerous efforts there have been no improvements in outcome: 30% of people and 90% of dogs still die of metastasis. In both species, macroscopic metastasis exhibits inherent resistance to multiple agents (kinase inhibitors, chemotherapy, immunotherapy, among others). In both dogs and humans, the OS genomes exhibit low somatic mutational load and no clear molecular drivers. These data, along with a history of failed clinical trials suggest that contemporary approaches to therapeutic advancement (kinase inhibitors, immune checkpoint blockade) will likely have limited clinical impact, necessitating therapeutic innovation. Monocarboxylate transporters (MCTs) move lactic acid across the plasma membrane, providing a mechanism for tumor cells to meet their bioenergetic needs in a variety of limiting microenvironments. The objective of this work is credential MCTs (MCT1/MCT4) as therapeutic targets in OS and investigate mechanisms of synthetic lethality. Methods: STAT3, MCT1 and MCT4 expression and function were modulated in canine and human OS cell lines using a combination of CRISPR/cas9 techniques and pharmacologic agents (AZD3965, AstraZeneca; syrosingopine, Sigma Aldrich). Western blotting, luciferase reporter assays and chromatin immunoprecipitation (ChIP) were used to investigate the transcriptional regulation of MCT1/MCT4. The metabolic effects of STAT3 and MCT inhibition were investigated through measurement of lactate (Lactate-Glo, Promega) and respirometry (Seahorse XFe96 extracellular flux analyzer), while cell proliferation and invasion were evaluated using sulforhodamine B and Matrigel invasion assays. Results: MCT1/MCT4 and were found to be expressed in all OS cell lines, although at variable levels. Following knockdown of STAT3, MCT4 expression was downregulated in several OS cell lines. ChIP assays confirm enrichment of STAT3 and FOXM1 at the MCT4 promoter, and this was confirmed in STAT3 deficient OS lines that exhibited decreased MCT4-dependent luciferase reporter activity. The absence of MCT1 or MCT4 was associated with decreased basal and compensatory glycolysis and increased intracellular lactate. In addition, decreased proliferation and invasive capacity were noted concurrent with MCT loss. Dual MCT1/MCT4 inhibition exhibited synergy with doxorubicin, as determined by a combination index < 1. Conclusions: Loss of MCT1 or MCT4 function in OS cells decreases basal and compensatory glycolysis, cellular proliferation and invasive capacity. MCT4 is a direct transcriptional target of STAT3 and FOXM1, both of which exhibit constitutive activation in OS, supporting a link between MCT4/STAT3/FOXM1 and aerobic glycolysis. Despite promising results in vitro, rational drug combinations may be needed to maximize the effect of MCT1/4 inhibition. Together these studies will create a blueprint for clinical translational with the goal of optimizing strategies in dogs with OS prior to human trials. Citation Format: Heather L. Gardner, Joelle M. Fenger, Cheryl A. London. Regulation and function of monocarboxylate transporters in osteosarcoma [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B081. doi:10.1158/1535-7163.TARG-19-B081