Summary The lactate dehydrogenase-A (LDH-A) enzyme catalyzes the inter-conversion of pyruvate and lactate, is upregulated in human cancers and is associated with aggressive tumor outcomes. Here we use a novel inducible murine model and demonstrate that inactivation of LDH-A in mouse models of NSCLC driven by oncogenic K-RAS or EGFR leads to decreased tumorigenesis and disease regression in established tumors. We also show that abrogation of LDH-A results in reprogramming of pyruvate metabolism, with decreased lactic fermentation in vitro, in vivo, and ex vivo. This was accompanied by re-activation of mitochondrial function in vitro but not in vivo or ex vivo. Finally, using a specific small molecule LDH-A inhibitor, we demonstrated that LDH-A is essential for cancer initiating cell survival and proliferation. Thus, LDH-A can be a viable therapeutic target for NSCLC including cancer stem cell-dependent drug resistant tumors.

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BackgroundMost normal cells in the presence of oxygen utilize glucose for mitochondrial oxidative phosphorylation. In contrast, many cancer cells rapidly convert glucose to lactate in the cytosol, a process termed aerobic glycolysis. This glycolytic phenotype is enabled by lactate dehydrogenase (LDH), which catalyzes the inter-conversion of pyruvate and lactate. The purpose of this study was to identify and characterize potent and selective inhibitors of LDHA.MethodsHigh throughput screening and lead optimization were used to generate inhibitors of LDHA enzymatic activity. Effects of these inhibitors on metabolism were evaluated using cell-based lactate production, oxygen consumption, and 13C NMR spectroscopy assays. Changes in comprehensive metabolic profile, cell proliferation, and apoptosis were assessed upon compound treatment.Results3-((3-carbamoyl-7-(3,5-dimethylisoxazol-4-yl)-6-methoxyquinolin-4-yl) amino) benzoic acid was identified as an NADH-competitive LDHA inhibitor. Lead optimization yielded molecules with LDHA inhibitory potencies as low as 2 nM and 10 to 80-fold selectivity over LDHB. Molecules in this family rapidly and profoundly inhibited lactate production rates in multiple cancer cell lines including hepatocellular and breast carcinomas. Consistent with selective inhibition of LDHA, the most sensitive breast cancer cell lines to lactate inhibition in hypoxic conditions were cells with low expression of LDHB. Our inhibitors increased rates of oxygen consumption in hepatocellular carcinoma cells at doses up to 3 microM, while higher concentrations directly inhibited mitochondrial function. Analysis of more than 500 metabolites upon LDHA inhibition in Snu398 cells revealed that intracellular concentrations of glycolysis and citric acid cycle intermediates were increased, consistent with enhanced Krebs cycle activity and blockage of cytosolic glycolysis. Treatment with these compounds also potentiated PKM2 activity and promoted apoptosis in Snu398 cells.ConclusionsRapid chemical inhibition of LDHA by these quinoline 3-sulfonamids led to profound metabolic alterations and impaired cell survival in carcinoma cells making it a compelling strategy for treating solid tumors that rely on aerobic glycolysis for survival.

Ror2 is an orphan receptor tyrosine kinase that plays crucial roles in developmental morphogenesis, particularly of the skeleton. We have identified human Ror2 as a novel regulator of canonical Wnt signaling in osteoblastic (bone-forming) cells with selective activities, enhancing Wnt1 but antagonizing Wnt3. Immunoprecipitation studies demonstrated physical interactions between human Ror2 and mammalian Wnt1 and Wnt3. Functionally, Ror2 antagonized Wnt1- and Wnt3-mediated stabilization of cytosolic beta-catenin in osteoblastic cells. However, Ror2 had opposing effects on a more distal step of canonical Wnt signaling: it potentiated Wnt1 activity but inhibited Wnt3 function as assessed by changes in Wnt-responsive reporter gene activity. Despite binding to Ror2, neither Wnt1 nor Wnt3 altered receptor activity as assessed by levels of Ror2 autophosphorylation. The ability of Ror2 to regulate canonical Wnt signaling in osteoblastic cells should have physiological consequences in bone, because Wnt signaling is known to modulate osteoblast survival and differentiation. Expression of Ror2 mRNA was highly regulated in a biphasic manner during human osteoblast differentiation, being virtually undetectable in pluripotent stem cells, increasing 300-fold in committed preosteoblasts, and disappearing again in osteocytes. Furthermore, Ror2 expression in osteoblasts was suppressed by the Wnt antagonist, secreted frizzled-related protein 1. The regulated expression of Ror2 during osteoblast differentiation, its inverse expression pattern with secreted frizzled-related protein 1, and its ability to modulate Wnt signaling in osteoblastic cells suggest that Ror2 may regulate bone formation.

Mechanisms controlling human bone formation remain to be fully elucidated. We have used differential display-polymerase chain reaction analysis to characterize osteogenic pathways in conditionally immortalized human osteoblasts (HOBs) representing distinct stages of differentiation. We identified 82 differentially expressed messages and found that the Wnt antagonist secreted frizzled-related protein (sFRP)-1 was the most highly regulated of these. Transient transfection of HOBs with sFRP-1 suppressed canonical Wnt signaling by 70% confirming its antagonistic function in these cells. Basal sFRP-1 mRNA levels increased 24-fold during HOB differentiation from pre-osteoblasts to pre-osteocytes, and then declined in mature osteocytes. This expression pattern correlated with levels of cellular viability such that the preosteocytes, which had the highest levels of sFRP-1 mRNA, also had the highest rate of cell death. Basal sFRP-1 mRNA levels also increased 29-fold when primary human mesenchymal stem cells were differentiated to osteoblasts supporting the developmental regulation of the gene. Expression of sFRP-1 mRNA was induced 38-fold following prostaglandin E 2 (PGE 2 ) treatment of pre-osteoblasts and mature osteoblasts that had low basal message levels. In contrast, sFRP-1 expression was down-regulated by as much as 80% following transforming growth factor (TGF)-b1 treatment of preosteocytes that had high basal mRNA levels. Consistent with this, treatment of pre-osteoblasts and mature osteoblasts with PGE 2 increased apoptosis threefold, while treatment of pre-osteocytes with TGF-b1 decreased cell death by 50%. Likewise, over-expression of sFRP-1 in HOBs accelerated the rate of cell death threefold. These results establish sFRP-1 as an important negative regulator of human osteoblast and osteocyte survival.