Joanne R. Doherty

Targeting lactate metabolism for cancer therapeutics

Lactate, once considered a waste product of glycolysis, has emerged as a critical regulator of cancer development, maintenance, and metastasis. Indeed, tumor lactate levels correlate with increased metastasis, tumor recurrence, and poor outcome. Lactate mediates cancer cell intrinsic effects on metabolism and has additional non-tumor cell autonomous effects that drive tumorigenesis. Tumor cells can metabolize lactate as an energy source and shuttle lactate to neighboring cancer cells, adjacent stroma, and vascular endothelial cells, which induces metabolic reprogramming. Lactate also plays roles in promoting tumor inflammation and in functioning as a signaling molecule that stimulates tumor angiogenesis. Here we review the mechanisms of lactate production and transport and highlight emerging evidence indicating that targeting lactate metabolism is a promising approach for cancer therapeutics.

Aurora kinases A and B are up-regulated by Myc and are essential for maintenance of the malignant state

Myc oncoproteins promote continuous cell growth, in part by controlling the transcription of key cell cycle regulators. Here, we report that c-Myc regulates the expression of Aurora A and B kinases (Aurka and Aurkb), and that Aurka and Aurkb transcripts and protein levels are highly elevated in Myc-driven B-cell lymphomas in both mice and humans. The induction of Aurka by Myc is transcriptional and is directly mediated via E-boxes, whereas Aurkb is regulated indirectly. Blocking Aurka/b kinase activity with a selective Aurora kinase inhibitor triggers transient mitotic arrest, polyploidization, and apoptosis of Myc-induced lymphomas. These phenotypes are selectively bypassed by a kinase inhibitor-resistant Aurkb mutant, demonstrating that Aurkb is the primary therapeutic target in the context of Myc. Importantly, apoptosis provoked by Aurk inhibition was p53 independent, suggesting that Aurka/Aurkb inhibitors will show efficacy in treating primary or relapsed malignancies having Myc involvement and/or loss of p53 function.

Blocking Lactate Export by Inhibiting the Myc Target MCT1 Disables Glycolysis and Glutathione Synthesis

Myc oncoproteins induce genes driving aerobic glycolysis, including lactate dehydrogenase-A that generates lactate. Here, we report that Myc controls transcription of the lactate transporter SLC16A1/MCT1 and that elevated MCT1 levels are manifest in premalignant and neoplastic Eμ-Myc transgenic B cells and in human malignancies with MYC or MYCN involvement. Notably, disrupting MCT1 function leads to an accumulation of intracellular lactate that rapidly disables tumor cell growth and glycolysis, provoking marked alterations in glycolytic intermediates, reductions in glucose transport, and in levels of ATP, NADPH, and ultimately, glutathione (GSH). Reductions in GSH then lead to increases in hydrogen peroxide, mitochondrial damage, and ultimately, cell death. Finally, forcing glycolysis by metformin treatment augments this response and the efficacy of MCT1 inhibitors, suggesting an attractive combination therapy for MYC/MCT1-expressing malignancies.

Tristetraprolin Impairs Myc-Induced Lymphoma and Abolishes the Malignant State

Myc oncoproteins directly regulate transcription by binding to target genes, yet this only explains a fraction of the genes affected by Myc. mRNA turnover is controlled via AU-binding proteins (AUBPs) that recognize AU-rich elements (AREs) found within many transcripts. Analyses of precancerous and malignant Myc-expressing B cells revealed that Myc regulates hundreds of ARE-containing (ARED) genes and select AUBPs. Notably, Myc directly suppresses transcription of Tristetraprolin (TTP/ZFP36), an mRNA-destabilizing AUBP, and this circuit is also operational during B lymphopoiesis and IL7 signaling. Importantly, TTP suppression is a hallmark of cancers with MYC involvement, and restoring TTP impairs Myc-induced lymphomagenesis and abolishes maintenance of the malignant state. Further, there is a selection for TTP loss in malignancy; thus, TTP functions as a tumor suppressor. Finally, Myc/TTP-directed control of select cancer-associated ARED genes is disabled during lymphomagenesis. Thus, Myc targets AUBPs to regulate ARED genes that control tumorigenesis.

Hrr25/CK1δ-directed release of Ltv1 from pre-40S ribosomes is necessary for ribosome assembly and cell growth

Cell growth relies on Hrr25/CK1δ-directed phosphorylation of Ltv1, which allows its release from nascent 40S ribosomal subunits and promotes subunit maturation.

Synthesis and Structure–Activity Relationships of Pteridine Dione and Trione Monocarboxylate Transporter 1 Inhibitors

Novel substituted pteridine-derived inhibitors of monocarboxylate transporter 1 (MCT1), an emerging target for cancer therapy, are reported. The activity of these compounds as inhibitors of lactate transport was confirmed using a 14C-lactate transport assay, and their potency against MCT1-expressing human tumor cells was established using MTT assays. The four most potent compounds showed substantial anticancer activity (EC50 37–150 nM) vs MCT1-expressing human Raji lymphoma cells.

Activation of endoplasmic reticulum stress response by enhanced polyamine catabolism is important in the mediation of cisplatin-induced acute kidney injury

Cisplatin-induced nephrotoxicity limits its use in many cancer patients. The expression of enzymes involved in polyamine catabolism, spermidine/spermine N1-acetyltransferase (SSAT) and spermine oxidase (SMOX) increase in the kidneys of mice treated with cisplatin. We hypothesized that enhanced polyamine catabolism contributes to tissue damage in cisplatin acute kidney injury (AKI). Using gene knockout and chemical inhibitors, the role of polyamine catabolism in cisplatin AKI was examined. Deficiency of SSAT, SMOX or neutralization of the toxic products of polyamine degradation, H2O2 and aminopropanal, significantly diminished the severity of cisplatin AKI. In vitro studies demonstrated that the induction of SSAT and elevated polyamine catabolism in cells increases the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) and enhances the expression of binding immunoglobulin protein BiP/GRP78) and CCAAT-enhancer-binding protein homologous protein (CHOP/GADD153). The increased expression of these endoplasmic reticulum stress response (ERSR) markers was accompanied by the activation of caspase-3. These results suggest that enhanced polyamine degradation in cisplatin AKI may lead to tubular damage through the induction of ERSR and the consequent onset of apoptosis. In support of the above, we show that the ablation of the SSAT or SMOX gene, as well as the neutralization of polyamine catabolism products modulate the onset of ERSR (e.g. lower BiP and CHOP) and apoptosis (e.g. reduced activated caspase-3). These studies indicate that enhanced polyamine catabolism and its toxic products are important mediators of ERSR and critical to the pathogenesis of cisplatin AKI.

Embryonic Expression and Function of the Xenopus Ink4d Cyclin D-Dependent Kinase Inhibitor.

Here we report the cloning and functional characterization of the cyclin D-dependent kinase 4 and 6 (Cdk4/6) inhibitory protein Cdkn2d/p19Ink4d of Xenopuslaevis (Xl-Ink4d). Xl-Ink4d is the only Ink4 family gene highly expressed during Xenopus development and its transcripts were detected maternally and during neurulation. The Xl-Ink4d protein has 63% identity to mouse and human Cdkn2d/p19Ink4d and its function as a negative regulator of cell cycle traverse is evolutionary conserved. Indeed, Xl-lnk4d can functionally substitute for mouse Cdkn2d in binding to mouse Cdk4 and inhibiting cyclin-D1-dependent CDK4 kinase activity. Further, enforced expression of Xl-lnk4d arrests mouse fibroblasts in the G1 phase of the cell cycle. These findings indicate that CDKN2d/p19Ink4d is conserved through vertebrate evolution and suggest Xl-lnk4d may contribute to the development of Xenopuslaevis.

Exploiting the co-reliance of tumours upon transport of amino acids and lactate: Gln and Tyr conjugates of MCT1 inhibitors

Glutamine and tyrosine-based amino acid conjugates of monocarboxylate transporter types 1 and 2 inhibitors (MCT1/2) were designed, synthesized and evaluated for their potency in blocking the proliferation of a human B lymphoma cell line that expresses the transporters Asct2, LAT1 and MCT1. Appropriate placement of an amino acid transporter recognition element was shown to augment anti-tumour efficacy vs. Raji cells. Amino acid conjugation also improves the pharmacodynamic properties of experimental MCT1/2 inhibitors.

Ribosome biogenesis factor Ltv1 chaperones the assembly of the small subunit head

The correct assembly of ribosomes from ribosomal RNAs (rRNAs) and ribosomal proteins (RPs) is critical, as indicated by the diseases caused by RP haploinsufficiency and loss of RP stoichiometry in cancer cells. Nevertheless, how assembly of each RP is ensured remains poorly understood. We use yeast genetics, biochemistry, and structure probing to show that the assembly factor Ltv1 facilitates the incorporation of Rps3, Rps10, and Asc1/RACK1 into the small ribosomal subunit head. Ribosomes from Ltv1-deficient yeast have substoichiometric amounts of Rps10 and Asc1 and show defects in translational fidelity and ribosome-mediated RNA quality control. These defects provide a growth advantage under some conditions but sensitize the cells to oxidative stress. Intriguingly, relative to glioma cell lines, breast cancer cells have reduced levels of LTV1 and produce ribosomes lacking RPS3, RPS10, and RACK1. These data describe a mechanism to ensure RP assembly and demonstrate how cancer cells circumvent this mechanism to generate diverse ribosome populations that can promote survival under stress.