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...

Summary A main limitation of therapies that selectively target kinase signaling pathways is the emergence of secondary drug resistance. Cetuximab, a monoclonal antibody that binds the extracellular domain of EGFR, is effective in a subset of KRAS wild type metastatic colorectal cancers1. After an initial response, secondary resistance invariably ensues, thereby limiting the clinical benefit of this drug2. The molecular bases of secondary resistance to cetuximab in colorectal cancer are poorly understood3-8. Here, we show for the first time that molecular alterations (in most instances point mutations) of KRAS are causally associated with the onset of acquired resistance to anti-EGFR treatment in colorectal cancers. Expression of mutant KRAS under the control of its endogenous gene promoter was sufficient to confer cetuximab resistance but resistant cells remained sensitive to combinatorial inhibition of EGFR and MEK. Analysis of metastases from patients who developed resistance to cetuximab or panitumumab showed the emergence of KRAS amplification in one sample and acquisition of secondary KRAS mutations in 60% (6/10) of the cases. KRAS mutant alleles were detectable in the blood of cetuximab treated patients as early as 10 months prior to radiographic documentation of disease progression. In summary, the results identify KRAS mutations as frequent drivers of acquired resistance to cetuximab in colorectal cancers, indicate that the emergence of KRAS mutant clones can be detected non-invasively months prior to radiographic progression and suggest early initiation of a MEK inhibitor as a rational strategy for delaying or reversing drug resistance.

Cancer is associated with mutated genes, and analysis of tumour-linked genetic alterations is increasingly used for diagnostic, prognostic and treatment purposes. The genetic profile of solid tumours is currently obtained from surgical or biopsy specimens; however, the latter procedure cannot always be performed routinely owing to its invasive nature. Information acquired from a single biopsy provides a spatially and temporally limited snap-shot of a tumour and might fail to reflect its heterogeneity. Tumour cells release circulating free DNA (cfDNA) into the blood, but the majority of circulating DNA is often not of cancerous origin, and detection of cancer-associated alleles in the blood has long been impossible to achieve. Technological advances have overcome these restrictions, making it possible to identify both genetic and epigenetic aberrations. A liquid biopsy, or blood sample, can provide the genetic landscape of all cancerous lesions (primary and metastases) as well as offering the opportunity to systematically track genomic evolution. This Review will explore how tumour-associated mutations detectable in the blood can be used in the clinic after diagnosis, including the assessment of prognosis, early detection of disease recurrence, and as surrogates for traditional biopsies with the purpose of predicting response to treatments and the development of acquired resistance.