PHGDH Knockout Cell Lysate

ObjectiveTo conduct an epigenome-wide analysis of DNA methylation and obesity traits. MethodsDNA methylation was quantified in CD4+ T-cells using the Illumina Infinium HumanMethylation450 array in 991 participants of the Genetics of Lipid Lowering Drugs and Diet Network. Methylation at individual cytosine-phosphate-guanine (CpG) sites as a function of body mass index (BMI) and waist circumference (WC), adjusting for age, gender, study site, T-cell purity, smoking, and family structure, was modeled. ResultsEpigenome-wide significant associations between eight CpG sites and BMI and five CpG sites and WC, successfully replicating the top hits in whole blood samples from the Framingham Heart Study (n=2,377) and the Atherosclerosis Risk in Communities study (n=2,097), were found. Top findings were in CPT1A (meta-analysis P=2.7 x 10(-43) for BMI and 9.9 x 10(-23) for WC), PHGDH (meta-analysis P=2.0 x 10(-15) for BMI and 4.0 x 10(-9) for WC), CD38 (meta-analysis P=6.3 x 10(-11) for BMI and 1.6 x 10(-12) for WC), and long intergenic non-coding RNA 00263 (meta-analysis P=2.2 x 10(-16) for BMI and 8.9 x 10(-14) for WC), regions with biologically plausible relationships to adiposity. ConclusionsThis large-scale epigenome-wide study discovered and replicated robust associations between DNA methylation at CpG loci and obesity indices, laying the groundwork for future diagnostic and/or therapeutic applications.

Cancer cells adapt their metabolic processes to drive macromolecular biosynthesis for rapid cell growth and proliferation(1,2). RNA interference (RNAi)-based loss-of-function screening has proven powerful for the identification of new and interesting cancer targets, and recent studies have used this technology in vivo to identify novel tumour suppressor genes(3). Here we developed a method for identifying novel cancer targets via negative-selection RNAi screening using a human breast cancer xenograft model at an orthotopic site in the mouse. Using this method, we screened a set of metabolic genes associated with aggressive breast cancer and stemness to identify those required for in vivo tumorigenesis. Among the genes identified, phosphoglycerate dehydrogenase (PHGDH) is in a genomic region of recurrent copy number gain in breast cancer and PHGDH protein levels are elevated in 70% of oestrogen receptor (ER)-negative breast cancers. PHGDH catalyses the first step in the serine biosynthesis pathway, and breast cancer cells with high PHGDH expression have increased serine synthesis flux. Suppression of PHGDH in cell lines with elevated PHGDH expression, but not in those without, causes a strong decrease in cell proliferation and a reduction in serine synthesis. We find that PHGDH suppression does not affect intracellular serine levels, but causes a drop in the levels of alpha-ketoglutarate, another output of the pathway and a tricarboxylic acid (TCA) cycle intermediate. In cells with high PHGDH expression, the serine synthesis pathway contributes approximately 50% of the total anaplerotic flux of glutamine into the TCA cycle. These results reveal that certain breast cancers are dependent on increased serine pathway flux caused by PHGDH overexpression and demonstrate the utility of in vivo negative-selection RNAi screens for finding potential anticancer targets.