Near‐genomewide RNAi screening for regulators of BRAF^V600E‐induced senescence identifies RASEF, a gene epigenetically silenced in melanoma

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, depletion of each of them 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 but not 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.

The Conundrum of Genetic “Drivers” in Benign Conditions

“…In each of these examples, the importance of the alterations to the tumor is beyond doubt, as demonstrated by responses to cognate therapies and other experimental evidence. The presence of these “drivers” at higher rates in benign or premalignant lesions and, in the case of benign melanocytic nevi, their frequent failure to regress with BRAF inhibitor therapy suggest that there is an endogenous inhibitor and/or one or more oncogenic cofactor alterations that are required for malignant evolution to occur (42,43,47,49,50,59,60). …”

“…Unbiased, genome-wide shRNA library screening for genes capable of reversing BRAF V600E-induced senescence identified PTEN as the most potent, and pharmacologic inhibition of PI3K signaling restored p15 INK4B -dependent senescence (49). The same group identified seven genes that, when silenced, could revert BRAF -associated senescence (60). However, only RAS and RASEF were differentially expressed in melanocytic nevi vs melanoma.…”

“…However, only RAS and RASEF were differentially expressed in melanocytic nevi vs melanoma. Functional studies suggested that RASEF acts in a Rb-dependent fashion and affects expression of the senescence-associated secretome (60). …”

A large Rab GTPase encoded by CRACR2A is a component of subsynaptic vesicles that transmit T cell activation signals

“…S10). Numerous gene linkage analyses of acute and chronic myeloid leukemia, and melanoma identified Rab45 (alternatively RASEF [RAS and EF-hand domain containing] or FLJ31614) as a potential tumor suppressor gene (37, 38). However, the molecular mechanism underlying its relationship with human diseases is not clearly understood.…”

A large Rab GTPase family in a small GTPase world

“…Numerous gene linkage analyses of acute and chronic myeloid leukemia, and melanoma identified Rab45 (alternatively RASEF [RAS and EF-hand domain containing] or FLJ31614) as a potential tumor suppressor gene. 25,26 However, the molecular mechanism underlying its relationship with human diseases is not clearly understood. Therefore, studies of CRACR2A-a can uncover possible roles of these large Rab GTPases in intracellular signaling and vesicle trafficking in the future.…”