Unknown,Transcriptomics,Genomics,Proteomics

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Mitochondrial and nuclear genomic response to loss of LRPPRC expression

ABSTRACT: Rapid advances in genotyping and sequencing technology have dramatically accelerated the discovery of genes underlying human disease. Elucidating the function of such genes and understanding their role in pathogenesis, however, remains challenging. Here, we introduce a genomic strategy to functionally characterize such genes, and apply it to LRPPRC (leucine-rich PPR-motif containing), a poorly studied gene that is mutated in Leigh Syndrome, French Canadian type (LSFC). We utilize RNAi to engineer an allelic series of cellular models in which LRPPRC has been stably silenced to different levels of knockdown efficiency. Using expression profiling, we discovered a specific role for LRPPRC in the expression of all mitochondrial DNA (mtDNA)-encoded mRNAs, but not the rRNAs, without affecting nuclear genes encoding mitochondrial proteins. We designed seven shRNAs targeting the LRPPRC cDNA sequence to silence its expression in MCH58 immortalized human fibroblasts. The LRPPRC expression level in these cells ranged from 9% to 100%. We demonstrated that knockdown cells carried stable silencing of the target gene and associated biochemical phenotypes. Our goal was to engineer stable knockdown cells which recapitulate the LSFC disease phenotype and subject them to expression profiling using Affymetric microarrays to identify genesets and biochemical pathways that are altered.

ORGANISM(S): Homo sapiens

SUBMITTER: Vamsi Mootha

PROVIDER: E-GEOD-20847 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Many cancers rely on glycolytic metabolism to fuel rapid proliferation. This has spurred interest in designing drugs that target tumor glycolysis such as AZD3965, a small molecule inhibitor of Monocarboxylate Transporter 1 (MCT1) currently undergoing Phase I evaluation for cancer treatment. Since MCT1 mediates proton-linked transport of monocarboxylates such as lactate and pyruvate across the plasma membrane (Halestrap and Meredith, 2004), AZD3965 is thought to block tumor growth through disruption of lactate transport and glycolysis. Here we show that MCT1 inhibition impairs proliferation of glycolytic breast cancer cells that express MCT4 via disruption of pyruvate rather than lactate export. We found that MCT1 expression is elevated in glycolytic breast tumors and cell lines as well as in malignant breast and lung tissues. High MCT1 expression predicts poor prognosis in breast and lung cancer patients. Stable knockdown and AZD3965-mediated inhibition of MCT1 promote oxidative metabolism. Acute inhibition of MCT1 reduces pyruvate export rate but does not consistently alter lactate transport or glycolytic flux in breast cancer cells that also express MCT4. Despite the lack of glycolysis impairment, MCT1 loss-of-function decreases breast cancer cell proliferation and blocks growth of mammary fat pad xenograft tumors. Our data suggest that MCT1 expression is elevated in glycolytic cancers to promote pyruvate export, which when inhibited enhances oxidative metabolism and reduces proliferation. This study presents an alternative molecular consequence of MCT1 inhibitors that further supports their use as anti-cancer therapeutics. Since MCT1 levels are elevated in glycolytic and malignant breast tumors, we hypothesized that MCT1 may contribute to the Warburg effect metabolic phenotype. To test this hypothesis, we generated whole genome microarray data from breast cancer cell lines either a) expressing a short hairpin (sh)RNA-mediated stable knockdown of MCT1; or b) treated for 24 hours with an MCT1 inhibitor (AZD3965). Scramble shRNA or DMSO were used as controls, and all conditions were analzed in triplicate. The cell lines used â HS578T, SUM149PT, and SUM159PT â are among the most glycolytic in a panel of 31 breast cancer cell lines.

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Mitochondrial and nuclear genomic response to loss of LRPPRC expression

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