Role for IGFBP7 in Senescence Induction by BRAF

In their recent Matters Arising, Scurr et al. (2010) questioned several of our conclusions regarding the role of IGFBP7 in BRAFV600E-mediated senescence induction. In our original study in Cell (Wajapeyee et al., 2008), we used a genome-wide RNA interference (RNAi) screen to identify 17 genes required for an activated BRAF oncogene (BRAFV600E) to block proliferation of primary melanocytes and melanoma cells. One of these genes encodes a secreted protein, IGFBP7, which we showed has a central role in BRAFV600E-mediated senescence and apoptosis. Here, we reproduce several of the key findings of our earlier study and present new results that substantiate our original claims. In our original study (Wajapeyee et al., 2008), we showed that expression of BRAFV600E in primary melanocytes increases synthesis and secretion of IGFBP7, which then acts through an auto-crine/paracrine pathway to induce senes-cence. BRAFV600E-mediated induction of IGFBP7 expression was directly demonstrated in six independent experiments. By contrast, Scurr et al. (2010) claim in their Matters Arising that BRAFV600E results in decreased IGFBP7. We introduced BRAFV600E into cultured human melanocytes by retroviral transduction, and in a series of new experiments now also show that transient transfection of a BRAFV600E-expression plasmid results in increased IGFBP7 (Figure S1A available online). Furthermore, BRAFV600E-mediated induction of IGFBP7 is, as expected, lost following addition of the MEK inhibi-tor U0126, which blocks BRAF-MEK-ERK signaling. Transfection of a BRAFV600E-expression plasmid into melanocytes also induces expression of a cotransfected IGFBP7 reporter plasmid (Figure S1B). Originally, we showed that BRAFV600E transcriptionally activates other genes involved in senescence or apopto-sis, including PEA15, SMARCB1, and BNIP3L (Wajapeyee et al., 2008). By contrast, Scurr et al. (2010) claim that following introduction of BRAFV600E into primary melanocytes, PEA15, SMARCB1, BNIP3L, and p53 protein levels are reduced. Their p53 result is particularly surprising because activated oncogenes induce a DNA-damage response, which is expected to elevate p53 levels. Indeed, in direct contrast to Scurr et al. (2010), another study has shown that BRAFV600E increases p53 levels in melanocytes (Yu et al., 2009). To confirm our original conclusions, we performed a new immunoblot experiment, which shows that BRAFV600E activates the DNA-damage response and markedly upregulates expression of PEA15, SMARCB1, BNIP3L, and p53 in primary melanocytes (Figure S1C). Following the primary screen, we performed 11 independent experiments demonstrating that the BRAFV600E-mediated block to cellular proliferation requires IGFBP7 (Wajapeyee et al., 2008). These experiments involved two different cell types, two unrelated IGFBP7 short-hairpin RNAs (shRNAs), and …