Abrogation of BRAFV600E-induced senescence by PI3K pathway activation contributes to melanomagenesis

Division of Molecular Oncology, The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands.
Genes & development (Impact Factor: 12.64). 05/2012; 26(10):1055-69. DOI: 10.1101/gad.187252.112
Source: PubMed

ABSTRACT Human melanocytic nevi (moles) are benign lesions harboring activated oncogenes, including BRAF. Although this oncogene initially acts mitogenically, eventually, oncogene-induced senescence (OIS) ensues. Nevi can infrequently progress to melanomas, but the mechanistic relationship with OIS is unclear. We show here that PTEN depletion abrogates BRAF(V600E)-induced senescence in human fibroblasts and melanocytes. Correspondingly, in established murine BRAF(V600E)-driven nevi, acute shRNA-mediated depletion of PTEN prompted tumor progression. Furthermore, genetic analysis of laser-guided microdissected human contiguous nevus-melanoma specimens recurrently revealed identical mutations in BRAF or NRAS in adjacent benign and malignant melanocytes. The PI3K pathway was often activated through either decreased PTEN or increased AKT3 expression in melanomas relative to their adjacent nevi. Pharmacologic PI3K inhibition in melanoma cells suppressed proliferation and induced the senescence-associated tumor suppressor p15(INK4B). This treatment also eliminated subpopulations resistant to targeted BRAF(V600E) inhibition. Our findings suggest that a significant proportion of melanomas arise from nevi. Furthermore, these results demonstrate that PI3K pathway activation serves as a rate-limiting event in this setting, acting at least in part by abrogating OIS. The reactivation of senescence features and elimination of cells refractory to BRAF(V600E) inhibition by PI3K inhibition warrants further investigation into the therapeutic potential of simultaneously targeting these pathways in melanoma.

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Available from: Martin McMahon, Aug 29, 2015
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    • "instance, are frequently positive for activating BRaf V600E mutations; however, these cells bear a senescent phenotype [75]. Abrogation of such oncogene-induced senescence by PI3K activation allows for melanoma formation [76]. Induction of cellular senescence therefore has been recognized as promising therapeutic approach to prevent the proliferation of cancer cells. "
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    ABSTRACT: Lymphangiogenesis is a very early step in lymphatic metastasis. It is regulated and promoted not only by the tumor cells themselves, but also by cells of the tumor microenvironment, including cancer associated fibroblasts, mesenchymal stem cells, dendritic cells, or macrophages. Even the extracellular matrix as well as cytokines and growth factors are involved in the process of lymphangiogenesis and metastasis. The cellular and noncellular components influence each other and can be influenced by the tumor cells. The knowledge about mechanisms behind lymphangiogenesis in the tumor microenvironmental crosstalk is growing and offers starting points for new therapeutic approaches.
    BioMed Research International 09/2014; 2014:639058. DOI:10.1155/2014/639058 · 2.71 Impact Factor
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    • "In another context, mutant B-Raf induced cellular senescence rather than proliferation [23,24]. However, senescence can be overcome by phosphoinositide 3-kinase (PI3K)/AKT signaling [24] which is hyperactivated in RKO due to a PIK3CA mutation. By staining of senescence-associated β-galactosidase activity [25] we examined whether the differential proliferation rates observed upon serum deprivation were attributable to cellular senescence. "
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    ABSTRACT: Background Colorectal cancers carrying the B-Raf V600E-mutation are associated with a poor prognosis. The purpose of this study was to identify B-RafV600E-mediated traits of cancer cells in a genetic in vitro model and to assess the selective sensitization of B-RafV600E-mutant cancer cells towards therapeutic agents. Methods Somatic cell gene targeting was used to generate subclones of the colorectal cancer cell line RKO containing either wild-type or V600E-mutant B-Raf kinase. Cell-biologic analyses were performed in order to link cancer cell traits to the BRAF-mutant genotype. Subsequently, the corresponding tumor cell clones were characterized pharmacogenetically to identify therapeutic agents exhibiting selective sensitivity in B-RafV600E-mutant cells. Results Genetic targeting of mutant BRAF resulted in restoration of sensitivity to serum starvation-induced apoptosis and efficiently inhibited cell proliferation in the absence of growth factors. Among tested agents, the B-Raf inhibitor dabrafenib was found to induce a strong V600E-dependent shift in cell viability. In contrast, no differential sensitizing effect was observed for conventional chemotherapeutic agents (mitomycin C, oxaliplatin, paclitaxel, etoposide, 5-fluorouracil), nor for the targeted agents cetuximab, sorafenib, vemurafenib, RAF265, or for inhibition of PI3 kinase. Treatment with dabrafenib efficiently inhibited phosphorylation of the B-Raf downstream targets Mek 1/2 and Erk 1/2. Conclusion Mutant BRAF alleles mediate self-sufficiency of growth signals and serum starvation-induced resistance to apoptosis. Targeting of the BRAF mutation leads to a loss of these hallmarks of cancer. Dabrafenib selectively inhibits cell viability in B-RafV600E mutant cancer cells.
    Molecular Cancer 05/2014; 13(1):122. DOI:10.1186/1476-4598-13-122 · 5.40 Impact Factor
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    • "Several genetic events frequently occurring in melanoma have been described, including the loss of CDKN2A and ARF, amplification of CCND1 or CDK4 (Curtin et al., 2005; Kamb et al., 1994), alterations in MMP8, GRM3, ERBB4, GRIN2A, MITF (Garraway et al., 2005; Palavalli et al., 2009; Prickett et al., 2009; 2011; Wei et al., 2011; Yokoyama et al., 2011) and activation of the PI3K pathway (Stahl et al., 2004). We have demonstrated previously that the latter event, for example by reduction of the expression levels of PTEN, reflects a rate-limiting step in OIS abrogation on the path to oncogenic transformation (Vredeveld et al., 2012). Moreover, recent exome and whole genome sequencing studies reported melanoma frequently mutated genes including RAC1 and PREX2 (Berger et al., 2010; Hodis et al., 2012; Krauthammer et al., 2012). "
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    ABSTRACT: The activation of oncogenes in primary cells blocks proliferation by inducing oncogene-induced senescence (OIS), a highly potent in vivo tumor-suppressing program. A prime example is mutant BRAF, which drives OIS in melanocytic nevi. Progression to melanoma occurs only in the context of additional alteration(s) like the suppression of PTEN, which abrogates OIS. Here, we performed a near-genomewide short hairpin (sh)RNA screen for novel OIS regulators and identified by next generation sequencing and functional validation seven genes. While all but one were upregulated in OIS, their depletion abrogated BRAF(V) (600E) -induced arrest. With genome-wide DNA methylation analysis we found one of these genes, RASEF, to be hypermethylated in primary cutaneous melanomas compared to nevi. Bypass of OIS by depletion of RASEF was associated with suppression of several senescence biomarkers including senescence-associated (SA)-β-galactosidase activity, interleukins and tumor suppressor p15(INK) (4B) . Restoration of RASEF expression inhibited proliferation. These results illustrate the power of shRNA OIS bypass screens and identify a potential novel melanoma suppressor gene. This article is protected by copyright. All rights reserved.
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