R-Ras is required for murine dendritic cell maturation and CD4+ T-cell priming.

R-Ras is a member of the RAS superfamily of small GTP-binding proteins. The physiologic function of R-Ras has not been fully elucidated. We found that R-Ras is expressed by lymphoid and nonlymphoid tissues and drastically up-regulated when bone marrow progenitors are induced to differentiate into dendritic cells (DCs). To address the role of R-Ras in DC functions, we generated a R-Ras-deficient mouse strain. We found that tumors induced in Rras(-/-) mice formed with shorter latency and attained greater tumor volumes. This finding has prompted the investigation of a role for R-Ras in the immune system. Indeed, Rras(-/-) mice were impaired in their ability to prime allogeneic and antigen-specific T-cell responses. Rras(-/-) DCs expressed lower levels of surface MHC class II and CD86 in response to lipopolysaccharide compared with wild-type DCs. This was correlated with a reduced phosphorylation of p38 and Akt. Consistently, R-Ras-GTP level was increased within 10 minutes of lipopolysaccharide stimulation. Furthermore, Rras(-/-) DCs have attenuated capacity to spread on fibronectin and form stable immunologic synapses with T cells. Altogether, these findings provide the first demonstration of a role for R-Ras in cell-mediated immunity and further expand on the complexity of small G-protein signaling in DCs.

ROCK1 mechano-signaling dependency of human malignancies driven by TEAD/YAP activation.

Rho family mechano-signaling through the actin cytoskeleton positively regulates physiological TEAD/YAP transcription, while the evolutionarily conserved Hippo tumor suppressor pathway antagonizes this transcription through YAP cytoplasmic localization/degradation. The mechanisms responsible for oncogenic dysregulation of these pathways, their prevalence in tumors, as well as how such dysregulation can be therapeutically targeted are not resolved. We demonstrate that p53 DNA contact mutants in human tumors, indirectly hyperactivate RhoA/ROCK1/actomyosin signaling, which is both necessary and sufficient to drive oncogenic TEAD/YAP transcription. Moreover, we demonstrate that recurrent lesions in the Hippo pathway depend on physiological levels of ROCK1/actomyosin signaling for oncogenic TEAD/YAP transcription. Finally, we show that ROCK inhibitors selectively antagonize proliferation and motility of human tumors with either mechanism. Thus, we identify a cancer driver paradigm and a precision medicine approach for selective targeting of human malignancies driven by TEAD/YAP transcription through mechanisms that either upregulate or depend on homeostatic RhoA mechano-signaling.

Characterization of R-ras3/m-ras null mice reveals a potential role in trophic factor signaling.

R-Ras3/M-Ras is a member of the RAS superfamily of small-molecular-weight GTP-binding proteins. Previous studies have demonstrated high levels of expression in several regions of the central nervous system, and a constitutively active form of M-Ras promotes cytoskeletal reorganization, cellular transformation, survival, and differentiation. However, the physiological functions of M-Ras during embryogenesis and postnatal development have not been elucidated. By using a specific M-Ras antibody, we demonstrated a high level of M-Ras expression in astrocytes, in addition to neurons. Endogenous M-Ras was activated by several trophic factors in astrocytes, including epidermal growth factor (EGF), basic fibroblast growth factor, and hepatocyte growth factor. Interestingly, M-Ras activation by EGF was more sustained compared to prototypic Ras. A mouse strain deficient in M-Ras was generated to investigate its role in development. M-Ras null mice appeared phenotypically normal, and there was a lack of detectable morphological and neurological defects. In addition, primary astrocytes derived from Mras(-/-) mice did not appear to display substantial alterations in the activation of both the mitogen-activated protein kinase and phosphatidylinositol 3-kinase pathways in response to trophic factors.

An ATF2-derived peptide sensitizes melanomas to apoptosis and inhibits their growth and metastasis.

Melanomas are among the aggressive tumor types because of their notorious resistance to treatment and their high capacity to metastasize. ATF2 is among transcription factors implicated in the progression of melanoma and its resistance to treatment. Here we demonstrate that the expression of a peptide spanning amino acids 50-100 of ATF2 (ATF2(50-100)) reduces ATF2 transcriptional activities while increasing the expression and activity of c-Jun. Altering the balance of Jun/ATF2 transcriptional activities sensitized melanoma cells to apoptosis, an effect that could be attenuated by inhibiting c-Jun. Inhibition of ATF2 via RNA interference likewise increased c-Jun expression and primed melanoma cells to undergo apoptosis. Growth and metastasis of SW1 and B16F10 mouse melanomas were inhibited by ATF2(50-100) to varying degrees up to a complete regression, depending on the mode (inducible, constitutive, or adenoviral delivery) of its expression.

Targeted inhibition of the KLF6 splice variant, KLF6 SV1, suppresses prostate cancer cell growth and spread.

Prostate cancer is a leading cause of cancer death in men. Risk prognostication, treatment stratification, and the development of rational therapeutic strategies lag because the molecular mechanisms underlying the initiation and progression from primary to metastatic disease are unknown. Multiple lines of evidence now suggest that KLF6 is a key prostate cancer tumor suppressor gene including loss and/or mutation in prostate cancer tumors and cell lines and decreased KLF6 expression levels in recurrent prostate cancer samples. Most recently, we identified a common KLF6 germ line single nucleotide polymorphism that is associated with an increased relative risk of prostate cancer and the increased production of three alternatively spliced, dominant-negative KLF6 isoforms. Here we show that although wild-type KLF6 (wtKLF6) acts as a classic tumor suppressor, the single nucleotide polymorphism-increased splice isoform, KLF6 SV1, displays a markedly opposite effect on cell proliferation, colony formation, and invasion. In addition, whereas wtKLF6 knockdown increases tumor growth in nude mice >2-fold, short interfering RNA-mediated KLF6 SV1 inhibition reduces growth by approximately 50% and decreases the expression of a number of growth- and angiogenesis-related proteins. Together, these findings begin to highlight a dynamic and functional antagonism between wtKLF6 and its splice variant KLF6 SV1 in tumor growth and dissemination.

USP7 Enforces Heterochromatinization of p53 Target Promoters by Protecting SUV39H1 from MDM2-Mediated Degradation.

The H3K9me3 repressive histone conformation of p53 target promoters is abrogated in response to p53 activation by MDM2-mediated SUV39H1 degradation. Here, we present evidence that the USP7 deubiquitinase protects SUV39H1 from MDM2-mediated ubiquitination in the absence of p53 stimulus. USP7 occupies p53 target promoters in unstressed conditions, a process that is abrogated with p53 activation associated with loss of the H3K9me3 mark on these same promoters. Mechanistically, USP7 forms a trimeric complex with MDM2 and SUV39H1, independent of DNA, and modulates MDM2-dependent SUV39H1 ubiquitination. Furthermore, we show that this protective function of USP7 on SUV39H1 is independent of p53. Finally, USP7 blocking cooperates with p53 in inducing apoptosis by enhancing p53 promoter occupancy and dependent transactivation of target genes. These results uncover a layer of the p53 transcriptional program mediated by USP7, which restrains relaxation of local chromatin conformation at p53 target promoters.

Suppression of glioblastoma tumorigenicity by the Kruppel-like transcription factor KLF6.

The Kruppel-like transcription factor KLF6 is a novel tumor-suppressor gene mutated in a significant fraction of human prostate cancer. It is localized to human chromosome 10p14-15, a region that displays frequent loss of heterozygosity in glioblastoma multiforme (GBM). Indeed, mutations of the KLF6 gene have recently been reported in this tumor type. In this study, we report that the expression of KLF6 is attenuated in human GBM when compared with primary astrocytes. Expression of KLF6 in GBM cells reverts their tumorigenicity both in vitro and in vivo, which is correlated with its transactivation of the p21/CIP1/WAF1 promoter. Additionally, KLF6 inhibits cellular transformation induced by several oncogenes (c-sis/PDGF-B, v-src, H-Ras, and EGFR) that are components of signaling cascades implicated in GBM. Our results provide the first evidence of functional tumor suppression by KFL6, and its loss may contribute to glial tumor progression.

Effects on proliferation and melanogenesis by inhibition of mutant BRAF and expression of wild-type INK4A in melanoma cells.

Activating BRAF mutations and loss of wild-type INK4A expression both occur at high frequencies in melanomas. Here, we present evidence that BRAF and INK4A have different effects on melanogenesis, a marker of melanocytic differentiation. Human melanoma cell line 624Mel harbors mutations in both BRAF and INK4A. The in vitro and in vivo growth of these cells was inhibited by either reduced expression of mutant BRAF using stable retroviral RNA interference (RNAi) or retrovirus-mediated stable expression of wild-type INK4A cDNA. Consistent with the observed growth inhibition, phosphorylation of S780 and S795 in pRB, both CDK4/6 targets, was suppressed in cells expressing either mutant BRAF RNAi or wild-type INK4A. Interestingly, melanoma cells expressing mutant BRAF RNAi had increased pigmentation, produced more mature melanosomes and melanin and expressed higher levels of tyrosinase and tyrosinase-related protein-1, whereas melanogenesis was not induced by wild-type INK4A. We found that the melanocyte lineage-specific master control protein microphthalmia-associated transcription factor was upregulated by inhibition of mutant BRAF, which may be the cause for the melanogenic effect of BRAF RNAi. The results suggest that, although both BRAF and INK4A lesions promote cell growth and tumor formation, mutant BRAF may also induce dedifferentiation in melanoma cells.