During malignant transformation, cancer cells acquire genetic mutations that override the normal mechanisms controlling cellular proliferation. Primary rodent cells are efficiently converted into tumorigenic cells by the coexpression of cooperating oncogenes. However, similar experiments with human cells have consistently failed to yield tumorigenic transformants, indicating a fundamental difference in the biology of human and rodent cells. The few reported successes in the creation of human tumour cells have depended on the use of chemical or physical agents to achieve immortalization, the selection of rare, spontaneously arising immortalized cells, or the use of an entire viral genome. We show here that the ectopic expression of the telomerase catalytic subunit (hTERT) in combination with two oncogenes (the simian virus 40 large-T oncoprotein and an oncogenic allele of H-ras) results in direct tumorigenic conversion of normal human epithelial and fibroblast cells. These results demonstrate that disruption of the intracellular pathways regulated by large-T, oncogenic ras and telomerase suffices to create a human tumor cell.

This is an author version of the article published on:Questa è la versione dell'autore dell'articolo: Unresponsiveness of colon cancer to BRAF(V600E) inhibition through feedback activation of EGFR.Prahallad A, Sun C, Huang S, Di Nicolantonio F, Salazar R, Zecchin D, Beijersbergen RL, Bardelli A, Bernards R. Nature. 2012 Jan 26;483(7387):100-3. doi: 10.1038/nature10868. The final version is available at: La versione definitiva è disponibile alla URL:http://www.nature.com/nature/journal/v483/n7387/full/nature10868.html . We therefore set out to screen a short hairpin RNA (shRNA) library representing the full complement of 518 human kinases 12 (the "kinome") and 17 additional kinase-related genes (Table S1) for genes whose inhibition confers sensitivity to PLX4032 in BRAF V600E mutant CRC. WiDr cells were infected with the lentiviral kinome shRNA collection and cultured in the absence or presence of PLX4032 for 10 and 18 days, respectively. After this, the relative abundance of shRNA vectors was determined by next generation sequencing of the bar code identifiers present in each shRNA vector (Fig. 1C, see methods). We arbitrarily considered only shRNA vectors that had been sequenced at least 300 times and which were depleted at least five-fold by the drug treatment. Fig. 1D shows that only very few of the 3388 shRNA vectors in the library met this stringent selection criterion, among which were three independent shRNA vectors targeting the Epidermal Growth FactorReceptor (EGFR, see Table S2 for all selected shRNAs). This suggested that suppression of EGFR synergizes with BRAF inhibition in these CRC cells. To validate this finding, we infected WiDr cells with each of these three EGFR shRNA vectors (all of which reduced EGFR levels (Fig. 1F)) and cultured these cells with or without PLX4032 for two weeks. . We therefore began by investigating a potential role of CDC25C in the activation of EGFR. We suppressed CDC25C in WiDr cells by shRNA and monitored levels of p-EGFR.We found that two independent shCDC25C vectors caused an increase in p-EGFR (Fig. 2E).Moreover, treatment of WiDr cells with PLX4032 inhibited phosphorylation of CDC25C at Thr48 (Fig. 2F), which has been shown to be required for its phosphatase activity 15 .Together, these data are consistent with a model in which BRAF inhibition leads to inhibition of MEK and ERK kinases, which in turn leads to a reduced activation of CDC25C. Inhibition of CDC25C in turn causes an increase in p-EGFR due to decreased dephosphorylation (Fig. 2E). Our data do not exclude that the related CDC25A and B or other phosphatases are also involved in this feedback regulation of EGFR.The EGFR is expressed primarily in epithelial cancers 17. Since melanomas are derived from the neural crest, we reasoned that the favourable response of melanomas to vemurafenib might result from the paucity of EGF receptors on these tumours and hence the 6 absence of the feedback activation of EGFR by BRAF inhibition. We compared EGFR expression in a panel of BRAF V600E mutant melanoma, colo...

Telomerase, the ribonucleoprotein enzyme that elongates telomeres, is repressed in normal human somatic cells but is reactivated during tumor progression. We report the cloning of a human gene, hEST2, that shares significant sequence similarity with the telomerase catalytic subunit genes of lower eukaryotes. hEST2 is expressed at high levels in primary tumors, cancer cell lines, and telomerase-positive tissues but is undetectable in telomerase-negative cell lines and differentiated telomerase-negative tissues. Moreover, the message is up-regulated concomitant with the activation of telomerase during the immortalization of cultured cells and down-regulated during in vitro cellular differentiation. Taken together, these observations suggest that the induction of hEST2 mRNA expression is required for the telomerase activation that occurs during cellular immortalization and tumor progression.

A large-scale RNA interference screen to discover genes involved in trastuzumab resistance in breast cancer identified only PTEN as a modulator of drug sensitivity. Oncogenic mutants of PIK3CA (activator of the same pathway and frequently mutated in breast cancer) also conferred resistance to trastuzumab in cell culture. In a cohort of 55 breast cancer patients, activation of the PI3K pathway, as judged by the presence of oncogenic PIK3CA mutations or low PTEN expression, was associated with poor prognosis after trastuzumab therapy, and the combined analysis of PTEN and PIK3CA identified twice as many patients at increased risk for progression compared to PTEN alone. Thus, assessment of PI3K pathway activation may provide a biomarker to identify patients unlikely to respond to trastuzumab-based therapy.

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Telomerase is a ribonucleoprotein enzyme that maintains the protective structures at the ends of eukaryotic chromosomes, called telomeres. In most human somatic cells, telomerase expression is repressed, and telomeres shorten progressively with each cell division. In contrast, most human tumors express telomerase, resulting in stabilized telomere length. These observations indicate that telomere maintenance is essential to the proliferation of tumor cells. We show here that expression of a mutant catalytic subunit of human telomerase results in complete inhibition of telomerase activity, reduction in telomere length and death of tumor cells. Moreover, expression of this mutant telomerase eliminated tumorigenicity in vivo. These observations demonstrate that disruption of telomere maintenance limits cellular lifespan in human cancer cells, thus validating human telomerase reverse transcriptase as an important target for the development of anti-neoplastic therapies.

RNA interference (RNAi) has become a powerful technique for reverse genetics and drug discovery and, in both of these areas, large-scale high-throughput RNAi screens are commonly performed. The statistical techniques used to analyze these screens are frequently borrowed directly from smallmolecule screening; however small-molecule and RNAi data characteristics differ in meaningful ways. We examine the similarities and differences between RNAi and small-molecule screens, highlighting particular characteristics of RNAi screen data that must be addressed during analysis. Additionally, we provide guidance on selection of analysis techniques in the context of a sample workflow.