Transcriptional Coactivator YAP Research Articles

Amot and Yap1 regulate neuronal dendritic tree complexity and locomotor coordination in mice.

The angiomotin (Amot)–Yes-associated protein 1 (Yap1) complex plays a major role in regulating the inhibition of cell contact, cellular polarity, and cell growth in many cell types. However, the function of Amot and the Hippo pathway transcription coactivator Yap1 in the central nervous system remains unclear. We found that Amot is a critical mediator of dendritic morphogenesis in cultured hippocampal cells and Purkinje cells in the brain. Amot function in developing neurons depends on interactions with Yap1, which is also indispensable for dendrite growth and arborization in vitro. The conditional deletion of Amot and Yap1 in neurons led to a decrease in the complexity of Purkinje cell dendritic trees, abnormal cerebellar morphology, and impairments in motor coordination. Our results indicate that the function of Amot and Yap1 in dendrite growth does not rely on interactions with TEA domain (TEAD) transcription factors or the expression of Hippo pathway–dependent genes. Instead, Amot and Yap1 regulate dendrite development by affecting the phosphorylation of S6 kinase and its target S6 ribosomal protein.

Antibody-drug conjugate T-DM1 treatment for HER2+ breast cancer induces ROR1 and confers resistance through activation of Hippo transcriptional coactivator YAP1.

BackgroundA newly developed drug trastuzumab emtansine (T-DM1) has improved the survival of breast cancer (BC) patients. Despite an impressive initial clinical response, a subgroup of patient develop resistance and present therapeutic challenges. The underlying resistance mechanisms are not fully investigated. We report that T-DM1 treatment modulates the expression of ROR1 (type 1 receptor tyrosine kinase-like orphan receptor) and induces self-renewal of cancer stem cells (CSCs) leading to therapeutic resistance.MethodsUsing BC patient tumor samples, and BC cell lines we gained insight into the T-DM1 treatment induced ROR1 overexpression and resistance. In vitro sphere forming assays and in vivo extreme dilution assays were employed to analyze the stemness and self-renewal capacity of the cells. A series of molecular expression and protein assays including qRT-PCR, FACS-sorting, ELISA, immunostaining, Western blotting were used to provide evidence.FindingsExposure of cells to T-DM1 shifted ROR1 expression from low to high, enriched within the CSC subpopulation, coincident with increased Bmi1 and stemness factors. T-DM1 induced ROR1 cells showed high spheroid and tumor forming efficiency in vitro and in an animal model exhibiting shorter tumor-free time. Mechanistically, the overexpression of ROR1 is partly induced by the activation of YAP1 and its target genes. Silencing of ROR1 and YAP1 by pharmacologic inhibitors and/or sh/siRNA inhibited spheroid formation, the initiation of tumors and the capacity for self-renewal and ROR1 overexpression.InterpretationsThe results presented here indicate that simultaneous targeting of ROR1 and YAP1 may suppress CSC self-renewal efficacy and inhibit tumor progression in BC. In this manner such treatments may overcome the T-DM1 mediated therapeutic resistance and improve clinical outcome.FundThis study was supported by Neurogen Technologies for interdisciplinary research.

Identification of the kinase STK25 as an upstream activator of LATS signaling

The Hippo pathway maintains tissue homeostasis by negatively regulating the oncogenic transcriptional co-activators YAP and TAZ. Though functional inactivation of the Hippo pathway is common in tumors, mutations in core pathway components are rare. Thus, understanding how tumor cells inactivate Hippo signaling remains a key unresolved question. Here, we identify the kinase STK25 as an activator of Hippo signaling. We demonstrate that loss of STK25 promotes YAP/TAZ activation and enhanced cellular proliferation, even under normally growth-suppressive conditions both in vitro and in vivo. Notably, STK25 activates LATS by promoting LATS activation loop phosphorylation independent of a preceding phosphorylation event at the hydrophobic motif, which represents a form of Hippo activation distinct from other kinase activators of LATS. STK25 is significantly focally deleted across a wide spectrum of human cancers, suggesting STK25 loss may represent a common mechanism by which tumor cells functionally impair the Hippo tumor suppressor pathway.

Potentiation of imatinib by cilostazol in sensitive and resistant gastrointestinal stromal tumor cell lines involves YAP inhibition.

Despite the introduction of tyrosine kinase inhibitors, gastrointestinal stromal tumors (GIST) resistance remains a major clinical challenge. We previously identified phosphodiesterase 3A (PDE3A) as a potential therapeutic target expressed in most GIST. The PDE3 inhibitor cilostazol reduced cell viability and synergized with the tyrosine kinase inhibitor imatinib (Gleevec™) in the imatinib-sensitive GIST882 cell line. Here, we found that cilostazol potentiated imatinib also in the imatinib-resistant GIST48 cell line. Cilostazol induced nuclear exclusion, hence inactivation, of the transcriptional co-activator YAP, in a cAMP-independent manner. Verteporfin, a YAP/TEAD interaction inhibitor, reduced by 90% the viability of both GIST882 and GIST48 cells. Our results highlight the potential use of compounds targeting PDE3A or YAP in combined multitherapy to tackle GIST resistance.

Abstract P2-02-02: The receptor tyrosine kinase EphA2 promotes glutamine metabolism in tumors by activating the transcriptional coactivators YAP and TAZ

Abstract Malignant tumors reprogram cellular metabolism to support cancer cell proliferation and survival. Although most cancers depend on a high rate of aerobic glycolysis, many cancer cells also display addiction to glutamine. Glutamine transporters and glutaminase activity are critical for glutamine metabolism in tumor cells, and upstream regulatory signaling pathways may represent a novel mechanism for targeted therapies in glutamine-addicted breast tumors such as Her2-overexpressing and triple-negative breast cancers (TNBC). We have found that the receptor tyrosine kinase EphA2 activated the transcriptional coactivators YAP and TAZ (YAP/TAZ), likely in a ligand-independent manner, to promote glutamine metabolism in cells and mouse models of breast cancer. Overexpression of EphA2 induced the nuclear accumulation of YAP and TAZ and increased the expression of YAP/TAZ target genes. Inhibition of the GTPase Rho or the kinase ROCK abolished EphA2-dependent YAP/TAZ nuclear localization. Silencing YAP or TAZ reduced the amount of intracellular glutamate through decreased expression of SLC1A5 and GLS, respectively, genes that encode proteins that promote glutamine uptake and metabolism. The regulatory DNA elements of both SLC1A5 and GLS contain the consensus sequence of the TEAD family of transcription factors that are closely associated with YAP/TAZ and were bound by TEAD4 in an EphA2-dependent manner. In patient breast cancer tissues, EphA2 expression positively correlated with that of YAP and TAZ, as well as that of GLS and SLC1A5. Although high expression of EphA2 predicted enhanced metastatic potential and poor patient survival, it also rendered breast cancer cells more sensitive to glutaminase inhibition. The findings define a previously unknown mechanism of EphA2-mediated glutaminolysis through YAP/TAZ activation in breast cancer and identify potential therapeutic targets in patients. Citation Format: Edwards DN, Ngwa VM, Wang S, Shiuan E, Brantley-Sieders DM, Kim LC, Reynolds AB, Chen J. The receptor tyrosine kinase EphA2 promotes glutamine metabolism in tumors by activating the transcriptional coactivators YAP and TAZ [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-02-02.

YAP and TAZ limit cytoskeletal and focal adhesion maturation to enable persistent cell motility.

Cell migration initiates by traction generation through reciprocal actomyosin tension and focal adhesion reinforcement, but continued motility requires adaptive cytoskeletal remodeling and adhesion release. Here, we asked whether de novo gene expression contributes to this cytoskeletal feedback. We found that global inhibition of transcription or translation does not impair initial cell polarization or migration initiation, but causes eventual migratory arrest through excessive cytoskeletal tension and over-maturation of focal adhesions, tethering cells to their matrix. The transcriptional coactivators YAP and TAZ mediate this feedback response, modulating cell mechanics by limiting cytoskeletal and focal adhesion maturation to enable persistent cell motility and 3D vasculogenesis. Motile arrest after YAP/TAZ ablation was partially rescued by depletion of the YAP/TAZ-dependent myosin phosphatase regulator, NUAK2, or by inhibition of Rho-ROCK-myosin II. Together, these data establish a transcriptional feedback axis necessary to maintain a responsive cytoskeletal equilibrium and persistent migration.

CRISPR-Mediated Approaches to Regulate YAP/TAZ Levels.

The advent of CRISPR has revolutionized genomic engineering, and harnessing its power to regulate levels of the transcriptional co-activators YAP and TAZ represents an exciting new opportunity in the field of Hippo signaling. Initially repurposed from the microbial immune system to perform highly specific gene knockouts, CRISPR technology has now been expanded to modulate the transcriptional activity of any gene of interest in mammalian systems. Here, we describe strategies to employ CRISPR to genetically knock out the genes encoding for YAP (YAP1) or TAZ (WWTR1) in mammalian cell lines, as well as briefly outline an approach for utilizing CRISPR to transcriptionally modulate YAP/TAZ levels.

The SWI/SNF complex is a mechanoregulated inhibitor of YAP and TAZ.

Inactivation of ARID1A and other components of the nuclear SWI/SNF protein complex occurs at very high frequencies in a variety of human malignancies, suggesting a widespread role for the SWI/SNF complex in tumour suppression1. However, the underlying mechanisms remain poorly understood. Here we show that ARID1A-containing SWI/SNF complex (ARID1A-SWI/SNF) operates as an inhibitor of the pro-oncogenic transcriptional coactivators YAP and TAZ2. Using a combination of gain- and loss-of-function approaches in several cellular contexts, we show that YAP/TAZ are necessary to induce the effects of the inactivation of the SWI/SNF complex, such as cell proliferation, acquisition of stem cell-like traits and liver tumorigenesis. We found that YAP/TAZ form a complex with SWI/SNF; this interaction is mediated by ARID1A and is alternative to the association of YAP/TAZ with the DNA-binding platform TEAD. Cellular mechanotransduction regulates the association between ARID1A-SWI/SNF and YAP/TAZ. The inhibitory interaction of ARID1A-SWI/SNF and YAP/TAZ is predominant in cells that experience low mechanical signalling, in which loss of ARID1A rescues the association between YAP/TAZ and TEAD. At high mechanical stress, nuclear F-actin binds to ARID1A-SWI/SNF, thereby preventing the formation of the ARID1A-SWI/SNF-YAP/TAZ complex, in favour of an association between TEAD and YAP/TAZ. We propose that a dual requirement must be met to fully enable the YAP/TAZ responses: promotion of nuclear accumulation of YAP/TAZ, for example, by loss of Hippo signalling, and inhibition of ARID1A-SWI/SNF, which can occur either through genetic inactivation or because of increased cell mechanics. This study offers a molecular framework in which mechanical signals that emerge at the tissue level together with genetic lesions activate YAP/TAZ to induce cell plasticity and tumorigenesis.

YAP drives cutaneous squamous cell carcinoma formation and progression.

Squamous cell carcinoma (SCC) can progress to malignant metastatic cancer, including an aggressive subtype known as spindle cell carcinoma (spSCC). spSCC formation involves epithelial-to-mesenchymal transition (EMT), yet the molecular basis of this event remains unknown. The transcriptional co-activator YAP undergoes recurrent amplification in human SCC and overexpression of YAP drives SCC formation in mice. Here, we show that human spSCC tumours also feature strong nuclear localisation of YAP and overexpression of activated YAP (NLS-YAP-5SA) with Keratin-5 (K5-CreERt) is sufficient to induce rapid formation of both SCC and spSCC in mice. spSCC tumours arise at sites of epithelial scratch wounding, where tumour-initiating epithelial cells undergo EMT to generate spSCC. Expression of the EMT transcription factor ZEB1 arises upon wounding and is a defining characteristic of spSCC in mice and humans. Thus, the wound healing response synergises with YAP to drive metaplastic transformation of SCC to spSCC.

PKCι and YAP1 are crucial in promoting pancreatic tumorigenesis.

Pancreatic ductal adenocarcinoma (PDAC) is a fatal malignant disease with 5-year survival rate of less than 6%. Activating mutations of Kras (mu-Kras) are often detected in most of PDAC patients. Although it has been known that oncogenic Kras is the driver of pancreatic cancer initiation and development, the underlying mechanisms by which mu-Kras promotes PDAC remain poorly understood. Here, we identify that PKCι is one of the crucial factors for supporting the survival of pancreatic cancer cells expressing mu-Kras. Our study demonstrates that after the knockdown of PKCι, the expression of the transcriptional co-activator YAP1 is decreased, which hinders the expression of the downstream target gene Mcl-1, and subsequently sensitizes pancreatic cancer MiaPaCa and PANC-1 cells experssing mu-Kras to apoptosis. In comparison, the suppression of PKCι has little impact on the viability of non-neoplastic pancreatic HPDE6-C7 cells. Moreover, the transient overexpression of oncogenic Kras in HPDE6-C7 elevates the expression of PKCι and YAP1 concomitantly. The upregulated YAP1 in HPDE6-C7/ mu-Kras cells is abolished once PKCι is suppressed, suggesting the linear relationship among mu-Kras, PKCι and YAP1. This phenomenon is further proven by the co-upregulation of PKCι and YAP1 in HPDE6-C7 cells stably transfected with mu-Kras. Taken together, our findings suggest that PKCι acts through promoting YAP1 function to promote the survival of pancreatic cancer cells expressing mu-Kras. It appears that targeting PKCι-YAP1 signaling is a feasible strategy for developing new therapeutics for treating pancreatic cancer patients.

Cirrhotic stiffness affects the migration of hepatocellular carcinoma cells and induces sorafenib resistance through YAP.

A majority of hepatocellular carcinomas (HCCs) combine with liver cirrhosis. The cirrhotic liver has been implicated in interfering with the effects of HCC-targeted drugs, including sorafenib. Alterations in the tumor microenvironment of the cirrhotic liver include both biochemical and biomechanical factors. In this study, we induced sorafenib resistance in HCC cells. We observed changes in cell morphology, cytoskeletal architecture, and cellular stiffness in these sorafenib-resistant cells, resembling those adapted to stiffer substrates. To examine the contribution of mechanical factors in HCC cell growth and drug resistance, we used an in vitro cell culture system with adjustable stiffness mimicking the normal or cirrhotic liver tissues. We identified that mechanical adaptation conferred HCC cells with increased motility and sorafenib resistance. We further reported the mechanism underlying the involvement of the transcription coactivator YAP. Our results underline the important role of mechanical factors in the interaction between tumor cells and their microenvironment.

Loss of KIBRA function activates EGFR signaling by inducing AREG

The Hippo signaling pathway is a central regulator of organ size, tissue homeostasis, and tumorigenesis. KIBRA is a member of the WW domain-containing protein family and has recently been reported to be an upstream protein in the Hippo signaling pathway. However, the clinical significance of KIBRA deregulation and the underlying mechanisms by which KIBRA regulates breast cancer (BC) initiation and progression remain poorly understood. Here, we report that KIBRA knockdown in mammary epithelial cells induced epithelial-to-mesenchymal transition (EMT) and increased cell migration and tumorigenic potential. Mechanistically, we observed that inhibiting KIBRA induced growth factor-independent cell proliferation in 2D and 3D culture due to the secretion of amphiregulin (AREG), an epidermal growth factor receptor (EGFR) ligand. Also, we show that AREG activation in KIBRA-knockdown cells depended on the transcriptional coactivator YAP1. Significantly, decreased expression of KIBRA is correlated with recurrence and reduced BC patient survival. In summary, this study elucidates the molecular events that underpin the role of KIBRA in BC. As a result, our work provides biological insight into the role of KIBRA as a critical regulator of YAP1-mediated oncogenic growth, and may have clinical potential for facilitating patient stratification and identifying novel therapeutic approaches for BC patients.

Abstract 1452: The receptor tyrosine kinase EphA2 promotes glutamine metabolism in tumors by activating the transcriptional coactivators YAP and TAZ

Abstract Malignant tumors reprogram cellular metabolism to support cancer cell proliferation and survival. Although most cancers depend on a high rate of aerobic glycolysis, many cancer cells also display addiction to glutamine. Glutamine transporters and glutaminase activity are critical for glutamine metabolism in tumor cells. We found that the receptor tyrosine kinase EphA2 activated the TEAD family transcriptional coactivators YAP and TAZ (YAP/TAZ), likely in a ligand-independent manner, to promote glutamine metabolism in cells and mouse models of HER2-positive breast cancer. Overexpression of EphA2 induced the nuclear accumulation of YAP and TAZ and increased the expression of YAP/TAZ target genes. Inhibition of the GTPase Rho or the kinase ROCK abolished EphA2-dependent YAP/TAZ nuclear localization. Silencing YAP or TAZ substantially reduced the amount of intracellular glutamate through decreased expression of SLC1A5 and GLS, respectively, genes that encode proteins that promote glutamine uptake and metabolism. The regulatory DNA elements of both SLC1A5 and GLS contain TEAD binding sites and were bound by TEAD4 in an EphA2-dependent manner. In patient breast cancer tissues, EphA2 expression positively correlated with that of YAP and TAZ, as well as that of GLS and SLC1A5. Although high expression or EphA2 predicted enhanced metastatic potential and poor patient survival, it also rendered HER2-positive breast cancer cells more sensitive to glutaminase inhibition. The findings define a previously unknown mechanism of EphA2-mediated glutaminolysis through YAP/TAZ activation in HER2-positive breast cancer and identify therapeutic targets in patients. Citation Format: Deanna N. Edwards, Verra M. Ngwa, Shan Wang, Eileen Shiuan, Dana Brantley-Sieders, Laura Kim, Albert B. Reynolds, Jin Chen. The receptor tyrosine kinase EphA2 promotes glutamine metabolism in tumors by activating the transcriptional coactivators YAP and TAZ [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1452.

Abstract 4134: HELLS is upregulated in Sonic hedgehog-associated medulloblastoma and proliferating cerebellar progenitors in a YAP-dependent manner

Abstract Aberrant hedgehog signaling has been implicated in many human cancers, including medulloblastoma (MB), the most common malignant pediatric brain tumor. In fact, 30% of MB tumors are driven by aberrant activity of the Sonic hedgehog (SHH) signaling pathway. Our efforts focus on unraveling the downstream components of this pathway to better understand medulloblastoma and better inform therapeutic treatment development and decisions. While some of the signaling alterations in SHH MB are due to gene mutations or amplifications, others may have their roots in epigenetics. Therefore, we examined expression levels of several candidate epigenetic regulators in our systems and identified lymphoid-specific helicase (HELLS) as a gene whose expression is markedly induced by SHH. HELLS is a unique member of the SNF2 family of chromatin remodelers with multiple epigenetic functions in DNA methylation, histone acetylation and methylation, and chromatin remodeling. Additional roles in transcription activation and DNA repair have also been reported. Of interest, HELLS was shown to delay senescence by inhibiting the expression of CDKN2A, the genetic locus of P16INK4A, a key tumor suppressor whose inactivation has recently been reported as critical to MB progression. Confirming our initial finding, we observed considerably higher levels of HELLS in primary cultures of cerebellar granule neuron precursors (CGNPs) treated with SHH and in SmoA1 mouse MB tumor tissue when compared to adjacent normal cerebellum. Our preliminary analysis of a large cohort of MB patients also indicates overexpression of HELLS in human SHH MB. Bioinformatic promoter analysis and experiments with SHH pathway inhibitors suggest regulation of HELLS expression through members of the SHH proliferation program. Downstream effectors of SHH include GLI1/2 and YAP1. Inhibition of GLI1 and GLI2 with Gant61 in both CGNPs and cultured mouse MB cells resulted in a reduction of HELLS, but an increase of apoptosis markers may indicate this reduction is due to cell death rather than specific downregulation of HELLS. In contrast, verteporfin, an inhibitor of the interaction between the transcriptional co-activator YAP1 and the transcription factor TEAD1, resulted in a reduction of HELLS at the mRNA and protein level without a concomitant increase of apoptosis markers. These results suggest that transcriptional upregulation of HELLS in mouse cerebellar progenitors and MB tumors is mediated by the SHH effector YAP1. Experiments to confirm direct involvement of YAP1 in HELLS gene transcription are underway. Future studies to understand the role of HELLS in SHH MB have the potential to provide a better understanding of the disease and new avenues for development of targeted treatments to improve the lives of these patients. Citation Format: M. Hope Robinson, Anna M. Kenney. HELLS is upregulated in Sonic hedgehog-associated medulloblastoma and proliferating cerebellar progenitors in a YAP-dependent manner [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4134.

Abstract 2052: A novel PHD2/VHL- mediated regulation of YAP1 and its role in vascular mimicry and tumor angiogenesis

Abstract Non-small cell lung cancer (NSCLC) is the leading cause of cancer related death worldwide. In this study, we show that transcriptional co-activator YAP1, the oncogenic component of the Hippo pathway, may contribute to the progression of NSCLC by supporting tumor angiogenesis and vascular mimicry of cancer stem-like cells (CSCs). NSCLC CSCs were found to have higher mRNA expression of VEGF receptor II (KDR) and Angiopoietin-2 (AngPT-2); depletion of YAP1 inhibited the promoter activity as well as the mRNA expression of VEGF, KDR and AngPT-2, with a corresponding reduction in vascular mimicry as well as tumor growth in a mouse xenograft studies. These results suggest a possible mechanism by which YAP1 regulates tumor angiogenesis. A role for YAP1 in tumor angiogenesis was further supported by the finding that NSCLC cells grown in hypoxic conditions showed higher levels of YAP1. There was little to no changes in the canonical Hippo pathway proteins like LATS1/2, MST1/2, SAV and MOB as well as in the expression of TAZ, an orthologue of YAP1. Elevated YAP1 was found to associate with HIF1α under hypoxic conditions and enhance its transcriptional activity; YAP1 could increase HIF1α-mediated induction of the VEGF promoter confirmed by chromatin immunoprecipitations and transient transfection assays. Elevated levels of YAP1 and HIF1α interaction was detected in lung tumor tissues compared to normal lung tissue, as detected by proximity ligation assay (PLA), suggesting that the higher association of YAP1 with HIF1α and resulting transcriptional activity might have contributed to tumor growth. An examination of the underlying mechanism by which YAP1 levels are elevated under hypoxic conditions revealed a novel regulation of YAP1 protein by prolyl hydroxylase PHD2 and E3 ubiquitin ligase VHL, which are mainly known to regulate HIF1α under normoxia. PHD2 was found to hydroxylate proline residue(s) of YAP1 between aa 284 to aa 289 as seen by mutational studies. YAP1 was found to directly associate with PHD2 as well as with VHL. Depletion of PHD2 or treatment with DMOG, an inhibitor of prolyl hydroxylases, reduced YAP1 association with VHL ligase. This further elevated YAP1 levels in the nucleus. Interestingly, disruption of the YAP1-PHD2 interaction using a domain specific peptide enhanced the angiogenic tubule formation by endothelial cells. Our data therefore identifies a novel non-canonical pathway of regulation of YAP1 that supports angiogenesis and tumor growth. Citation Format: Namrata Bora Singhal, Biswarup Saha, Srikumar Chellappan. A novel PHD2/VHL- mediated regulation of YAP1 and its role in vascular mimicry and tumor angiogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2052.

Abstract 3166: The HU177 cryptic collagen epitope selectively regulates melanoma cell behavior by a CDK5 and PTPN12 associated mechanism

Abstract Previous studies indicate that selectively inhibiting cellular interactions with the HU177 cryptic collagen epitope, which can be generated in vivo during structural remodeling of the extracellular matrix (ECM), inhibits tumor growth and metastasis. However, a detailed understanding of the mechanisms by which antagonists of the HU177 collagen epitope inhibit these pathological processes is not fully understood. Uncovering the molecular mechanisms by which cellular interactions with this collagen epitope regulate tumor cell behavior may allow the development of more effective clinical strategies targeting tumor cell-ECM interactions. Here we provide evidence for the first time, that specific targeting of the HU177 cryptic collagen epitope reduces phosphorylation of cyclin-dependent kinase (CDK5) and in addition, reduces the expression of the phosphatase PTPN12 in melanoma cells attached to denatured collagen. Importantly, both CDK5 < PTPN12 play a functional role in the ability of the HU177 collagen epitope to regulate migration of melanoma cells as reducing expression of these molecules inhibited the anti-migratory activity of the anti-HU177 antibody. Surprisingly, reduction in either CDK5 or PTPN12 also inhibited nuclear accumulation of YAP. These novel findings help define a previously unknown signaling mechanism by which both CDK5 < PTPN12 may play a selective role in controlling nuclear accumulations of the transcriptional co-activator YAP following melanoma cell interactions with the HU177 cryptic collagen epitope. Taken together, these studies provide new molecular insight into the role of the HU177 collagen epitope in controlling melanoma cell behavior. Given that direct targeting of integrin receptors that mediate tumor cellular interactions with the ECM have demonstrated only limited anti-tumor activity in human clinical trials, a more detailed molecular understanding of how generation of biologically relevant cryptic elements of the ECM control melanoma cell behavior will likely facilitate the development of more effective new clinical strategies to control tumor progression. Citation Format: Xianghua Han, Jennifer M. Caron, Peter C. Brooks. The HU177 cryptic collagen epitope selectively regulates melanoma cell behavior by a CDK5 and PTPN12 associated mechanism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3166.

Abstract 4373: AR signaling in concert with PP2A/B regulates YAP1 expression in prostate cancer cells

Abstract The transcriptional coactivator YAP1 is a key nuclear effector of the Hippo pathway. STK4/MST1 and LATS1/2 are the core kinase components of the Hippo pathway, which restricts organ size and prevents tumorigenesis by attenuating cell proliferation and inducing cell death. Previously, we have reported that STK4/Hippo-YAP1 signaling could play a critical role in prostate cancer progression and therapeutic relapse. Here, we investigated the effects of androgen hormone signaling on YAP1 expression and post-transcriptional modifications in prostate cancer cells. We demonstrated that androgen exposure reduced Ser127 phosphorylation on YAP1 in a time-dependent manner in the castration-sensitive prostate cancer cell line, LNCaP, but without altering the levels of YAP1-Ser127 phosphorylation in the C4-2 cell line, a castration-resistant subline of LNCaP cells. As demonstrated by imaging and cell fractionation methods, androgen exposure promoted the nuclear accumulation of YAP1 in LNCaP; however, regardless of androgen exposure, YAP1 was accumulated in the nucleus of C4-2 cells. In addition, we demonstrated that androgen exposure reduced YAP1-Ser127 phosphorylation that was induced by okadaic acid, a potent activator of Ser/Thr phosphatases PP1 and PP2A. Also, we demonstrated that androgen exposure increased PP2A/B protein expression. Moreover, reduction of phospho-Ser127-YAP1 was correlated with increases in total YAP1 protein, which coincided with androgen receptor (AR) nuclear accumulation by androgen. Consistent with these observation, the genetic (siRNA) or the pharmacologic (enzalutamide) inhibition of AR signaling attenuated the expression of YAP1 protein. Furthermore, our analysis of the publicly available TCGA (The Cancer Genome Atlas) set indicated that YAP1 and AR mRNA expressions were positively correlated in prostate clinical samples. These observations suggest that YAP1 is a direct target of androgen hormone signaling, implicating that the AR-PP2A/B-YAP1 axis is a viable cancer drug target. Citation Format: Bekir Cinar, Carlos S. Moreno. AR signaling in concert with PP2A/B regulates YAP1 expression in prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4373.

Cross-Cohort Analysis Identifies a TEAD4-MYCN Positive Feedback Loop as the Core Regulatory Element of High-Risk Neuroblastoma.

High-risk neuroblastomas show a paucity of recurrent somatic mutations at diagnosis. As a result, the molecular basis for this aggressive phenotype remains elusive. Recent progress in regulatory network analysis helped us elucidate disease-driving mechanisms downstream of genomic alterations, including recurrent chromosomal alterations. Our analysis identified three molecular subtypes of high-risk neuroblastomas, consistent with chromosomal alterations, and identified subtype-specific master regulator proteins that were conserved across independent cohorts. A 10-protein transcriptional module-centered around a TEAD4-MYCN positive feedback loop-emerged as the regulatory driver of the high-risk subtype associated with MYCN amplification. Silencing of either gene collapsed MYCN-amplified (MYCNAmp) neuroblastoma transcriptional hallmarks and abrogated viability in vitro and in vivo Consistently, TEAD4 emerged as a robust prognostic marker of poor survival, with activity independent of the canonical Hippo pathway transcriptional coactivators YAP and TAZ. These results suggest novel therapeutic strategies for the large subset of MYCN-deregulated neuroblastomas.Significance: Despite progress in understanding of neuroblastoma genetics, little progress has been made toward personalized treatment. Here, we present a framework to determine the downstream effectors of the genetic alterations sustaining neuroblastoma subtypes, which can be easily extended to other tumor types. We show the critical effect of disrupting a 10-protein module centered around a YAP/TAZ-independent TEAD4-MYCN positive feedback loop in MYCNAmp neuroblastomas, nominating TEAD4 as a novel candidate for therapeutic intervention. Cancer Discov; 8(5); 582-99. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 517.

The history and regulatory mechanism of the Hippo pathway.

How the organ size is adjusted to the proper size during development and how organs know that they reach the original size during regeneration remain long-standing questions. Based on studies using multiple model organisms and approaches for over 20 years, a consensus has been established that the Hippo pathway plays crucial roles in controlling organ size and maintaining tissue homeostasis. Given the significance of these processes, the dysregulation of the Hippo pathway has also implicated various diseases, such as tissue degeneration and cancer. By regulating the downstream transcriptional coactivators YAP and TAZ, the Hippo pathway coordinates cell proliferation and apoptosis in response to a variety of signals including cell contact inhibition, polarity, mechanical sensation and soluble factors. Since the core components and their functions of the Hippo pathway are evolutionarily conserved, this pathway serves as a global regulator of organ size control. Therefore, further investigation of the regulatory mechanisms will provide physiological insights to better understand tissue homeostasis. In this review, the historical developments and current understandings of the regulatory mechanism of Hippo signaling pathway are discussed.

Mechanical strain regulates the Hippo pathway in Drosophila.

ABSTRACTAnimal cells are thought to sense mechanical forces via the transcriptional co-activators YAP (or YAP1) and TAZ (or WWTR1), the sole Drosophila homolog of which is named Yorkie (Yki). In mammalian cells in culture, artificial mechanical forces induce nuclear translocation of YAP and TAZ. Here, we show that physiological mechanical strain can also drive nuclear localisation of Yki and activation of Yki target genes in the Drosophila follicular epithelium. Mechanical strain activates Yki by stretching the apical domain, reducing the concentration of apical Crumbs, Expanded, Kibra and Merlin, and reducing apical Hippo kinase dimerisation. Overexpressing Hippo kinase to induce ectopic activation in the cytoplasm is sufficient to prevent Yki nuclear localisation even in flattened follicle cells. Conversely, blocking Hippo signalling in warts clones causes Yki nuclear localisation even in columnar follicle cells. We find no evidence for involvement of other pathways, such as Src42A kinase, in regulation of Yki. Finally, our results in follicle cells appear generally applicable to other tissues, as nuclear translocation of Yki is also readily detectable in other flattened epithelial cells such as the peripodial epithelium of the wing imaginal disc, where it promotes cell flattening.