Transcriptional regulation of miR-30a by YAP impacts PTPN13 and KLF9 levels and Schwann cell proliferation.

The Hippo pathway is a key regulatory pathway that is tightly regulated by mechanical cues such as tension, pressure, and contact with the extracellular matrix and other cells. At the distal end of the pathway is the yes-associated protein (YAP), a well-characterized transcriptional regulator. Through binding to transcription factors such as the TEA Domain TFs (TEADs) YAP regulates expression of several genes involved in cell fate, proliferation and death decisions. While the function of YAP as direct transcriptional regulator has been extensively characterized, only a small number of studies examined YAP function as a regulator of gene expression via microRNAs. We utilized bioinformatic approaches, including chromatin immunoprecipitation sequencing and RNA-Seq, to identify potential new targets of YAP regulation and identified miR-30a as a YAP target gene in Schwann cells. We find that YAP binds to the promoter and regulates the expression of miR-30a. Moreover, we identify several YAP-regulated genes that are putative miR-30a targets and focus on two of these, protein tyrosine pohosphatase non-receptor type 13 (PTPN13) and Kruppel like factor 9. We find that YAP regulation of Schwann cell proliferation and death is mediated, to a significant extent, through miR-30a regulation of PTPN13 in Schwann cells. These findings identify a new regulatory function by YAP, mediated by miR-30a, to downregulate expression of PTPN13 and Kruppel like factor 9. These studies expand our understanding of YAP function as a regulator of miRNAs and illustrate the complexity of YAP transcriptional functions.

Merlin in organ size control and tumorigenesis: Hippo versus EGFR?

The role of the NF2 gene as a tumor suppressor has been well established. In this issue of Genes & Development, Benhamouche and colleagues (pp. 1718-1730) demonstrate that NF2 is also involved in the regulation of organ size control in mammals. Conditional knockout of Nf2 in the mouse liver results in massive organ enlargement and eventual tumor development, which is attributed to the specific expansion of oval cells. Here we discuss these findings and the proposed molecular mechanisms involved within the context of our current understanding of the pathways regulated by NF2.

The path forward: 2015 International Children's Tumor Foundation conference on neurofibromatosis type 1, type 2, and schwannomatosis.

The Annual Children's Tumor Foundation International Neurofibromatosis Meeting is the premier venue for connecting discovery, translational and clinical scientists who are focused on neurofibromatosis types 1 and 2 (NF1 and NF2) and schwannomatosis (SWN). The meeting also features rare tumors such as glioma, meningioma, sarcoma, and neuroblastoma that occur both within these syndromes and spontaneously; associated with somatic mutations in NF1, NF2, and SWN. The meeting addresses both state of the field for current clinical care as well as emerging preclinical models fueling discovery of new therapeutic targets and discovery science initiatives investigating mechanisms of tumorigenesis. Importantly, this conference is a forum for presenting work in progress and bringing together all stakeholders in the scientific community. A highlight of the conference was the involvement of scientists from the pharmaceutical industry who presented growing efforts for rare disease therapeutic development in general and specifically, in pediatric patients with rare tumor syndromes. Another highlight was the focus on new investigators who presented new data about biomarker discovery, tumor pathogenesis, and diagnostic tools for NF1, NF2, and SWN. This report summarizes the themes of the meeting and a synthesis of the scientific discoveries presented at the conference in order to make the larger research community aware of progress in the neurofibromatoses.

FRAX597, a small molecule inhibitor of the p21-activated kinases, inhibits tumorigenesis of neurofibromatosis type 2 (NF2)-associated Schwannomas.

The p21-activated kinases (PAKs) are immediate downstream effectors of the Rac/Cdc42 small G-proteins and implicated in promoting tumorigenesis in various types of cancer including breast and lung carcinomas. Recent studies have established a requirement for the PAKs in the pathogenesis of Neurofibromatosis type 2 (NF2), a dominantly inherited cancer disorder caused by mutations at the NF2 gene locus. Merlin, the protein product of the NF2 gene, has been shown to negatively regulate signaling through the PAKs and the tumor suppressive functions of Merlin are mediated, at least in part, through inhibition of the PAKs. Knockdown of PAK1 and PAK2 expression, through RNAi-based approaches, impairs the proliferation of NF2-null schwannoma cells in culture and inhibits their ability to form tumors in vivo. These data implicate the PAKs as potential therapeutic targets. High-throughput screening of a library of small molecules combined with a structure-activity relationship approach resulted in the identification of FRAX597, a small-molecule pyridopyrimidinone, as a potent inhibitor of the group I PAKs. Crystallographic characterization of the FRAX597/PAK1 complex identifies a phenyl ring that traverses the gatekeeper residue and positions the thiazole in the back cavity of the ATP binding site, a site rarely targeted by kinase inhibitors. FRAX597 inhibits the proliferation of NF2-deficient schwannoma cells in culture and displayed potent anti-tumor activity in vivo, impairing schwannoma development in an orthotopic model of NF2. These studies identify a novel class of orally available ATP-competitive Group I PAK inhibitors with significant potential for the treatment of NF2 and other cancers.

Synergistic effects of combined BET and FAK inhibition against Vestibular Schwannomas in NF2-related Schwannomatosis.

Neurofibromatosis type 2 (NF2) is a rare disorder that causes vestibular schwannomas (VS), meningiomas and ependymomas. To date, there is no FDA approved drug-based treatment for NF2. We have previously identified that BET inhibition can selectively reduce growth of the NF2-null schwannoma and Schwann cells in vitro and tumorigenesis in vivo and, separately, reported that inhibition of Focal Adhesion Kinase 1 (FAK1) via crizotinib has antiproliferative effects in NF2-null Schwann cells. The current study was aimed at determining whether combined BET and FAK inhibition can synergize and to identify the mechanisms of action. A panel of normal and NF2-null Schwann and schwannoma cell lines were used to characterize the effects of combined BET and FAK inhibition in vitro and in vivo using pharmacological and genetic approaches. The mechanism of action was explored by chromatin immunoprecipitation, ChIP-PCR, western blotting, and functional approaches. We find that combined BET and FAK inhibition are synergistic and inhibit the proliferation of NF2-null schwannoma and Schwann cell lines in vitro and in vivo, by arresting cells in the G1/S and G2/M phases of the cell cycle. Further, we identify the mechanism of action through the downregulation of FAK1 transcription by BET inhibition, which potentiates inhibition of FAK by 100-fold. Our findings suggest that combined targeting of BET and FAK1 may offer a potential therapeutic option for the treatment of NF2-related schwannomas.

HIF-2alpha deletion promotes Kras-driven lung tumor development.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer deaths worldwide. The oxygen-sensitive hypoxia inducible factor (HIF) transcriptional regulators HIF-1alpha and HIF-2alpha are overexpressed in many human NSCLCs, and constitutive HIF-2alpha activity can promote murine lung tumor progression, suggesting that HIF proteins may be effective NSCLC therapeutic targets. To investigate the consequences of inhibiting HIF activity in lung cancers, we deleted Hif-1alpha or Hif-2alpha in an established Kras(G12D)-driven murine NSCLC model. Deletion of Hif-1alpha had no obvious effect on tumor growth, whereas Hif-2alpha deletion resulted in an unexpected increase in tumor burden that correlated with reduced expression of the candidate tumor suppressor gene Scgb3a1 (HIN-1). Here, we identify Scgb3a1 as a direct HIF-2alpha target gene and demonstrate that HIF-2alpha regulates Scgb3a1 expression and tumor formation in human Kras(G12D)-driven NSCLC cells. AKT pathway activity, reported to be repressed by Scgb3a1, was enhanced in HIF-2alpha-deficient human NSCLC cells and xenografts. Finally, a direct correlation between HIF-2alpha and SCGB3a1 expression was observed in approximately 70% of human NSCLC samples analyzed. These data suggest that, whereas HIF-2alpha overexpression can contribute to NSCLC progression, therapeutic inhibition of HIF-2alpha below a critical threshold may paradoxically promote tumor growth by reducing expression of tumor suppressor genes, including Scgb3a1.

The L27 Domain of MPP7 enhances TAZ-YY1 Cooperation to Renew Muscle Stem Cells.

Stem cells regenerate differentiated cells to maintain and repair tissues and organs. They also replenish themselves, i.e. self-renewal, for the regenerative process to last a lifetime. How stem cells renew is of critical biological and medical significance. Here we use the skeletal muscle stem cell (MuSC) to study this process. Using a combination of genetic, molecular, and biochemical approaches, we show that MPP7, AMOT, and TAZ/YAP form a complex that activates a common set of target genes. Among these targets, Carm1 can direct MuSC renewal. In the absence of MPP7, TAZ can support regenerative progenitors and activate Carm1 expression, but not to a level needed for self-renewal. Facilitated by the actin polymerization-responsive AMOT, TAZ recruits the L27 domain of MPP7 to up-regulate Carm1 to the level necessary to drive MuSC renewal. The promoter of Carm1, and those of other common downstream genes, also contain binding site(s) for YY1. We further demonstrate that the L27 domain of MPP7 enhances the interaction between TAZ and YY1 to activate Carm1. Our results define a renewal transcriptional program embedded within the progenitor program, by selectively up-regulating key gene(s) within the latter, through the combination of protein interactions and in a manner dependent on the promoter context.

The L27 Domain of MPP7 enhances TAZ-YY1 Cooperation to Renew Muscle Stem Cells.

Stem cells regenerate differentiated cells to maintain and repair tissues and organs. They also replenish themselves, i.e. self-renewal, for the regenerative process to last a lifetime. How stem cells renew is of critical biological and medical significance. Here we use the skeletal muscle stem cell (MuSC) to study this process. Using a combination of genetic, molecular, and biochemical approaches, we show that MPP7, AMOT, and TAZ/YAP form a complex that activates a common set of target genes. Among these targets, Carm1 can direct MuSC renewal. In the absence of MPP7, TAZ can support regenerative progenitors and activate Carm1 expression, but not to a level needed for self-renewal. Facilitated by the actin polymerization-responsive AMOT, TAZ recruits the L27 domain of MPP7 to up-regulate Carm1 to the level necessary to drive MuSC renewal. The promoter of Carm1, and those of other common downstream genes, also contain binding site(s) for YY1. We further demonstrate that the L27 domain of MPP7 enhances the interaction between TAZ and YY1 to activate Carm1. Our results define a renewal transcriptional program embedded within the progenitor program, by selectively up-regulating key gene(s) within the latter, through the combination of protein interactions and in a manner dependent on the promoter context.

Cohesins localize with CTCF at the KSHV latency control region and at cellular c-myc and H19/Igf2 insulators.

Cohesins, which mediate sister chromatin cohesion, and CTCF, which functions at chromatin boundaries, play key roles in the structural and functional organization of chromosomes. We examined the binding of these two factors on the Kaposi's sarcoma-associated herpesvirus (KSHV) episome during latent infection and found a striking colocalization within the control region of the major latency transcript responsible for expressing LANA (ORF73), vCyclin (ORF72), vFLIP (ORF71), and vmiRNAs. Deletion of the CTCF-binding site from the viral genome disrupted cohesin binding, and crippled colony formation in 293 cells. Clonal instability correlated with elevated expression of lytic cycle gene products, notably the neighbouring promoter for K14 and vGPCR (ORF74). siRNA depletion of RAD21 from latently infected cells caused an increase in K14 and ORF74, and lytic inducers caused a rapid dissociation of RAD21 from the viral genome. RAD21 and SMC1 also associate with the cellular CTCF sites at mammalian c-myc promoter and H19/Igf2 imprinting control region. We conclude that cohesin subunits associate with viral and cellular CTCF sites involved in complex gene regulation and chromatin organization.

ß8 integrin and band 4.1B cooperatively regulate morphogenesis of the embryonic heart.

Morphogenesis of the heart is regulated by various cues, including growth factors and extracellular matrix (ECM) proteins. The mechanisms by which cardiac cells properly integrate these cues to regulate growth, differentiation, and migration remain poorly understood. Here we have used genetic strategies in mice to identify αvß8 integrin and its cytoskeletal adaptor protein, Band 4.1B, as essential regulators of cardiac morphogenesis. We demonstrate that approximately 60% of mouse embryos genetically null for ß8 integrin and Band 4.1B display cardiovascular phenotypes and die by E11.5. This premature death is due, in part, to defective development of the cardiac outflow tract (OFT), with reduced expression of smooth muscle α-actin (SMAα-actin) in OFT cells derived from the cardiac neural crest. These data are the first to identify cell adhesion and signaling pathways regulated by αvß8 integrin and Band 4.1B as essential for normal formation and function of the heart during embryogenesis.

Housing and Husbandry Alternatives for Naked Mole Rat Colonies Used in Research Settings.

Naked mole rats (Heterocephalus glaber) are a unique rodent species originating in Africa and are increasingly being used in research. Their needs and characteristics differ from those of other rodents used in research. Unique housing systems are necessary to address the special macro- and microenvironmental requirements of NMRs. Naked mole rats are one of the 2 known eusocial mammalian species, are extremely long-living, are active burrowers, and are accustomed to a subterranean environment. Unlike typical rats and mice, naked mole rats need specific, unique housing systems that mimic their natural subterranean environment to support health and longevity. Here we provide an overview of naked mole rats and a housing method that can be used in research settings.

Validation of Bromodomain and Extraterminal proteins as therapeutic targets in neurofibromatosis type 2.

Background: Neurofibromatosis type 2 (NF2) is an autosomal dominant genetic disease characterized by development of schwannomas on the VIIIth (vestibular) cranial nerves. Bromodomain and extra-terminal domain (BET) proteins regulate gene transcription and their activity is required in a variety of cancers including malignant peripheral nerve sheath tumors. The use of BET inhibitors as a therapeutic option to treat NF2 schwannomas has not been explored and is the focus of this study. Methods: A panel of normal and NF2-null Schwann and schwannoma cell lines were used to characterize the impact of the BET inhibitor JQ1 in vitro and in vivo. The mechanism of action was explored by chromatin immunoprecipitation of the BET BRD4, phospho-kinase arrays and immunohistochemistry (IHC) of BRD4 in vestibular schwannomas. Results: JQ1 inhibited proliferation of NF2-null schwannoma and Schwann cell lines in vitro and in vivo. Further, loss of NF2 by CRISPR deletion or siRNA knockdown increased sensitivity of cells to JQ1. Loss of function experiments identified BRD4, and to a lesser extent BRD2, as BET family members mediating the majority of JQ1 effects. IHC demonstrated elevated levels of BRD4 protein in human vestibular schwannomas. Analysis of signaling pathways effected by JQ1 treatment suggests that the effects of JQ1 treatment are mediated, at least in part, via inhibition of PI3K/Akt signaling. Conclusions: NF2-deficient Schwann and schwannoma cells are sensitive to BET inhibition, primarily mediated by BRD4, which is overexpressed in human vestibular schwannomas. Our results suggest BRD4 regulates PI3K signaling and likely impedes NF2 schwannoma growth via this inhibition. These findings implicate BET inhibition as a therapeutic option for NF2-deficient schwannomas.

Organization of myelinated axons by Caspr and Caspr2 requires the cytoskeletal adapter protein 4.1B.

Caspr and Caspr2 regulate the formation of distinct axonal domains around the nodes of Ranvier. Caspr is required for the generation of a membrane barrier at the paranodal junction (PNJ), whereas Caspr2 serves as a membrane scaffold that clusters Kv1 channels at the juxtaparanodal region (JXP). Both Caspr and Caspr2 interact with protein 4.1B, which may link the paranodal and juxtaparanodal adhesion complexes to the axonal cytoskeleton. To determine the role of protein 4.1B in the function of Caspr proteins, we examined the ability of transgenic Caspr and Caspr2 mutants lacking their 4.1-binding sequence (d4.1) to restore Kv1 channel clustering in Caspr- and Caspr2-null mice, respectively. We found that Caspr-d4.1 was localized to the PNJ and is able to recruit the paranodal adhesion complex components contactin and NF155 to this site. Nevertheless, in axons expressing Caspr-d4.1, Kv1 channels were often detected at paranodes, suggesting that the interaction of Caspr with protein 4.1B is necessary for the generation of an efficient membrane barrier at the PNJ. We also found that the Caspr2-d4.1 transgene did not accumulate at the JXP, even though it was targeted to the axon, demonstrating that the interaction with protein 4.1B is required for the accumulation of Caspr2 and Kv1 channels at the juxtaparanodal axonal membrane. In accordance, we show that Caspr2 and Kv1 channels are not clustered at the JXP in 4.1B-null mice. Our results thus underscore the functional importance of protein 4.1B in the organization of peripheral myelinated axons.

What's new in neurofibromatosis? Proceedings from the 2009 NF Conference: new frontiers.

The NF Conference is the largest annual gathering of researchers and clinicians focused on neurofibromatosis and has been convened by the Children's Tumor Foundation for over 20 years. The 2009 NF Conference was held in Portland, Oregon from June 13 to June 16, 2009 and co-chaired by Kathryn North from the University of Sydney and The Children's Hospital at Westmead, Sydney, Australia; and Joseph Kissil from the Wistar Institute, Philadelphia. The Conference included 80 platform presentations in 9 sessions over 4 days; over 100 abstracts presented as posters; and three Keynote presentations. To date, there have been tremendous advances in basic research in the pathogenesis of neurofibromatosis, and more recently in progress toward identifying effective drug therapies and the commencement of neurofibromatosis clinical trials. The NF Conference attendees have significantly increased (doubling from 140 in 2005 to 280 attending in 2009) with a significant increase in attendance of physicians and clinical researchers. Correspondingly the NF Conference scope has expanded to include translational research, clinical trials and clinical management issues while retaining a core of basic research. These themes are reflected in the highlights from the 2009 NF Conference presented here.

The use of non-traditional models in the study of cancer resistance-the case of the naked mole rat.

Non-traditional model organisms are typically defined as any model the deviates from the typical laboratory animals, such as mouse, rat, and worm. These models are becoming increasingly important in human disease research, such as cancer, as they often display unusual biological features. Naked mole rats (NMRs) are currently one of the most popular non-traditional model, particularly in the longevity and cancer research fields. NMRs display an exceptionally long lifespan (~30 years), yet have been observed to display a low incidence of cancer, making them excellent candidates for understanding endogenous cancer resistance mechanisms. Over the past decade, many potential resistance mechanisms have been characterized. These include unique biological mechanisms involved in genome stability, protein stability, oxidative metabolism, and other cellular mechanisms such as cell cycle regulation and senescence. This review aims to summarize the many identified cancer resistance mechanisms to understand some of the main hypotheses that have thus far been generated. Many of these proposed mechanisms remain to be fully characterized or confirmed in vivo, giving the field a direction to grow and further understand the complex biology displayed by the NMR.

YAP-Mediated Recruitment of YY1 and EZH2 Represses Transcription of Key Cell-Cycle Regulators.

The Hippo pathway regulates cell proliferation and organ size through control of the transcriptional regulators YAP (yes-associated protein) and TAZ. Upon extracellular stimuli such as cell-cell contact, the pathway negatively regulates YAP through cytoplasmic sequestration. Under conditions of low cell density, YAP is nuclear and associates with enhancer regions and gene promoters. YAP is mainly described as a transcriptional activator of genes involved in cell proliferation and survival. Using a genome-wide approach, we show here that, in addition to its known function as a transcriptional activator, YAP functions as a transcriptional repressor by interacting with the multifunctional transcription factor Yin Yang 1 (YY1) and Polycomb repressive complex member enhancer of zeste homologue 2 (EZH2). YAP colocalized with YY1 and EZH2 on the genome to transcriptionally repress a broad network of genes mediating a host of cellular functions, including repression of the cell-cycle kinase inhibitor p27, whose role is to functionally promote contact inhibition. This work unveils a broad and underappreciated aspect of YAP nuclear function as a transcriptional repressor and highlights how loss of contact inhibition in cancer is mediated in part through YAP repressive function. SIGNIFICANCE: This study provides new insights into YAP as a broad transcriptional repressor of key regulators of the cell cycle, in turn influencing contact inhibition and tumorigenesis.

Requirement for Rac1 in a K-ras induced lung cancer in the mouse.

Given the prevalence of Ras mutations in human cancer, it is critical to understand the effector pathways downstream of oncogenic Ras leading to transformation. To directly assess the requirement for Rac1 in K-ras-induced tumorigenesis, we employed a model of lung cancer in which an oncogenic allele of K-ras could be activated by Cre-mediated recombination in the presence or absence of conditional deletion of Rac1. We show that Rac1 function is required for tumorigenesis in this model. Furthermore, although Rac1 deletion alone was compatible with cell viability and proliferation, when combined with K-ras activation in primary epithelial cells, loss of Rac1 caused a profound reduction in proliferation. These data show a specific requirement for Rac1 function in cells expressing oncogenic K-ras.

The NF2 tumor suppressor merlin interacts with Ras and RasGAP, which may modulate Ras signaling.

Inactivation of the tumor suppressor NF2/merlin underlies neurofibromatosis type 2 (NF2) and some sporadic tumors. Previous studies have established that merlin mediates contact inhibition of proliferation; however, the exact mechanisms remain obscure and multiple pathways have been implicated. We have previously reported that merlin inhibits Ras and Rac activity during contact inhibition, but how merlin regulates Ras activity has remained elusive. Here we demonstrate that merlin can directly interact with both Ras and p120RasGAP (also named RasGAP). While merlin does not increase the catalytic activity of RasGAP, the interactions with Ras and RasGAP may fine-tune Ras signaling. In vivo, loss of RasGAP in Schwann cells, unlike the loss of merlin, failed to promote tumorigenic growth in an orthotopic model. Therefore, modulation of Ras signaling through RasGAP likely contributes to, but is not sufficient to account for, merlin's tumor suppressor activity. Our study provides new insight into the mechanisms of merlin-dependent Ras regulation and may have additional implications for merlin-dependent regulation of other small GTPases.

An Immunocompetent Mouse Model of HPV16(+) Head and Neck Squamous Cell Carcinoma.

The incidence of human papilloma virus (HPV)-associated head and neck squamous cell carcinoma (HNSCC) is increasing and implicated in more than 60% of all oropharyngeal carcinomas (OPSCCs). Although whole-genome, transcriptome, and proteome analyses have identified altered signaling pathways in HPV-induced HNSCCs, additional tools are needed to investigate the unique pathobiology of OPSCC. Herein, bioinformatics analyses of human HPV(+) HNSCCs revealed that all tumors express full-length E6 and identified molecular subtypes based on relative E6 and E7 expression levels. To recapitulate the levels, stoichiometric ratios, and anatomic location of E6/E7 expression, we generated a genetically engineered mouse model whereby balanced expression of E6/E7 is directed to the oropharyngeal epithelium. The addition of a mutant PIK3CAE545K allele leads to the rapid development of pre-malignant lesions marked by immune cell accumulation, and a subset of these lesions progress to OPSCC. This mouse provides a faithful immunocompetent model for testing treatments and investigating mechanisms of immunosuppression.

Loss of the putative tumor suppressor band 4.1B/Dal1 gene is dispensable for normal development and does not predispose to cancer.

The band 4.1 proteins are cytoskeletal proteins, harboring a conserved FERM domain highly homologous to the N-terminal FERM domain of ezrin, radixin, moesin, and merlin. Recently, a truncated form of the 4.1B protein, termed Dal-1, was identified in a screen as down regulated in adenocarcinoma of the lung and was mapped to chromosome 18p11.3, which is lost in 38% of primary non-small cell lung carcinoma tumors. Analysis of several meningiomas has shown that Dal-1 expression was lost in 76% of the tumors. To further elucidate the function of the 4.1B/Dal-1 gene in development and tumorigenesis we generated mice deficient for this allele. The 4.1B/Dal-1 null mice develop normally and are fertile. Rates of cellular proliferation and apoptosis in brain, mammary, and lung tissues from the 4.1B/Dal-1 null mice were indistinguishable from those seen with wild-type mice. Aging studies indicate that these mice do not have a propensity to develop tumors. Analysis of fibroblasts from these mice demonstrated that the growth characteristics and kinetics of these cells were not different from those of cells from the wild-type mice. These findings indicate that the 4.1B gene is not required for normal development and that 4.1B/Dal-1 does not function as a tumor suppressor gene.