Faculty Bibliography

Cancer cells adapt their metabolic processes to drive macromolecular biosynthesis for rapid cell growth and proliferation. 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. 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 upon increased serine pathway flux caused by PHGDH overexpression and demonstrate the utility of in vivo negative-selection RNAi screens for finding potential anticancer targets.

Constitutive expression of telomerase in human cells prevents the onset of senescence and crisis by maintaining telomere homeostasis. However, accumulating evidence suggests that the human telomerase reverse transcriptase catalytic subunit (TERT) contributes to cell physiology independently of its ability to elongate telomeres. Here we show that TERT interacts with the RNA component of mitochondrial RNA processing endoribonuclease (RMRP), a gene that is mutated in the inherited pleiotropic syndrome cartilage-hair hypoplasia. Human TERT and RMRP form a distinct ribonucleoprotein complex that has RNA-dependent RNA polymerase (RdRP) activity and produces double-stranded RNAs that can be processed into small interfering RNA in a Dicer (also known as DICER1)-dependent manner. These observations identify a mammalian RdRP composed of TERT in complex with RMRP.

POT1 is a 3' telomeric single-stranded overhang binding protein that has been implicated in chromosome end protection, the regulation of telomerase function, and defining the 5' chromosome terminus. In human cancer cells that exhibit constitutive hTERT activity, hPOT1 exerts control over telomere length. Primary human fibroblasts express low levels of catalytically active hTERT in an S-phase-restricted manner that fails to counteract telomere attrition with cell division. Here, we show that diploid human fibroblasts in which hPOT1 expression has been suppressed harbor telomeres that are longer than control cells. This difference in telomere length delays the onset of replicative senescence and is dependent on S-phase-restricted hTERT expression. These findings are consistent with the view that hPOT1 promotes a nonextendable telomere state resistant to extension by S-phase-restricted telomerase. Manipulating this function of hPOT1 may thus hasten the cytotoxic effects of telomerase inhibition.

Constitutive expression of telomerase prevents senescence and crisis by maintaining telomere homeostasis. However, recent evidence suggests that telomerase is dynamically regulated in normal cells and also contributes to transformation independently of net telomere elongation. Here, we show that suppression of the telomerase catalytic subunit [human telomerase reverse transcriptase (hTERT)] expression abrogates the cellular response to DNA double strand breaks. Loss of hTERT does not alter short-term telomere integrity but instead affects the overall configuration of chromatin. Cells lacking hTERT exhibit increased radiosensitivity, diminished capacity for DNA repair, and fragmented chromosomes, demonstrating that loss of hTERT impairs the DNA damage response.

The SV40 small t antigen (ST) interacts with the serine-threonine protein phosphatase 2A (PP2A). To investigate the role of this interaction in transformation, we suppressed the expression of the PP2A B56gamma subunit in human embryonic kidney (HEK) epithelial cells expressing SV40 large T antigen, hTERT, and H-RAS. Suppression of PP2A B56gamma expression inhibited PP2A-specific phosphatase activity similar to that achieved by ST and conferred the ability to grow in an anchorage-independent fashion and to form tumors. Overexpression of PP2A B56gamma3 in tumorigenic HEK cells expressing ST or human lung cancer cell lines partially reversed the tumorigenicity of these cells. These observations identify specific PP2A complexes involved in human cell transformation.