Comparison of tumor-associated YAP1 fusions identifies a recurrent set of functions critical for oncogenesis.

YAP1 is a transcriptional coactivator and the principal effector of the Hippo signaling pathway, which is causally implicated in human cancer. Several YAP1 gene fusions have been identified in various human cancers and identifying the essential components of this family of gene fusions has significant therapeutic value. Here, we show that the YAP1 gene fusions YAP1-MAMLD1, YAP1-FAM118B, YAP1-TFE3, and YAP1-SS18 are oncogenic in mice. Using reporter assays, RNA-seq, ChIP-seq, and loss-of-function mutations, we can show that all of these YAP1 fusion proteins exert TEAD-dependent YAP activity, while some also exert activity of the C'-terminal fusion partner. The YAP activity of the different YAP1 fusions is resistant to negative Hippo pathway regulation due to constitutive nuclear localization and resistance to degradation of the YAP1 fusion proteins. Genetic disruption of the TEAD-binding domain of these oncogenic YAP1 fusions is sufficient to inhibit tumor formation in vivo, while pharmacological inhibition of the YAP1-TEAD interaction inhibits the growth of YAP1 fusion-expressing cell lines in vitro. These results highlight TEAD-dependent YAP activity found in these gene fusions as critical for oncogenesis and implicate these YAP functions as potential therapeutic targets in YAP1 fusion-positive tumors.

αE-catenin inhibits a Src-YAP1 oncogenic module that couples tyrosine kinases and the effector of Hippo signaling pathway.

Cell-cell adhesion protein αE-catenin inhibits skin squamous cell carcinoma (SCC) development; however, the mechanisms responsible for this function are not completely understood. We report here that αE-catenin inhibits ß4 integrin-mediated activation of SRC tyrosine kinase.SRCis the first discovered oncogene, but the protein substrate critical for SRC-mediated transformation has not been identified. We found that YAP1, the pivotal effector of the Hippo signaling pathway, is a direct SRC phosphorylation target, and YAP1 phosphorylation at three sites in its transcription activation domain is necessary for SRC-YAP1-mediated transformation. We uncovered a marked increase in this YAP1 phosphorylation in human and mouse SCC tumors with low/negative expression of αE-catenin. We demonstrate that the tumor suppressor function of αE-catenin involves negative regulation of the ß4 integrin-SRC signaling pathway and that SRC-mediated phosphorylation and activation of YAP1 are an alternative to the canonical Hippo signaling pathway that directly connect oncogenic tyrosine kinase signaling with YAP1.

DLG5 connects cell polarity and Hippo signaling protein networks by linking PAR-1 with MST1/2.

Disruption of apical-basal polarity is implicated in developmental disorders and cancer; however, the mechanisms connecting cell polarity proteins with intracellular signaling pathways are largely unknown. We determined previously that membrane-associated guanylate kinase (MAGUK) protein discs large homolog 5 (DLG5) functions in cell polarity and regulates cellular proliferation and differentiation via undefined mechanisms. We report here that DLG5 functions as an evolutionarily conserved scaffold and negative regulator of Hippo signaling, which controls organ size through the modulation of cell proliferation and differentiation. Affinity purification/mass spectrometry revealed a critical role of DLG5 in the formation of protein assemblies containing core Hippo kinases mammalian ste20 homologs 1/2 (MST1/2) and Par-1 polarity proteins microtubule affinity-regulating kinases 1/2/3 (MARK1/2/3). Consistent with this finding, Hippo signaling is markedly hyperactive in mammalian Dlg5-/- tissues and cells in vivo and ex vivo and in Drosophila upon dlg5 knockdown. Conditional deletion of Mst1/2 fully rescued the phenotypes of brain-specific Dlg5 knockout mice. Dlg5 also interacts genetically with Hippo effectors Yap1/Taz Mechanistically, we show that DLG5 inhibits the association between MST1/2 and large tumor suppressor homologs 1/2 (LATS1/2), uses its scaffolding function to link MST1/2 with MARK3, and inhibits MST1/2 kinase activity. These data reveal a direct connection between cell polarity proteins and Hippo, which is essential for proper development of multicellular organisms.

Hepsin regulates TGFß signaling via fibronectin proteolysis.

Transforming growth factor-beta (TGFß) is a multifunctional cytokine with a well-established role in mammary gland development and both oncogenic and tumor-suppressive functions. The extracellular matrix (ECM) indirectly regulates TGFß activity by acting as a storage compartment of latent-TGFß, but how TGFß is released from the ECM via proteolytic mechanisms remains largely unknown. In this study, we demonstrate that hepsin, a type II transmembrane protease overexpressed in 70% of breast tumors, promotes canonical TGFß signaling through the release of latent-TGFß from the ECM storage compartment. Mammary glands in hepsin CRISPR knockout mice showed reduced TGFß signaling and increased epithelial branching, accompanied by increased levels of fibronectin and latent-TGFß1, while overexpression of hepsin in mammary tumors increased TGFß signaling. Cell-free and cell-based experiments showed that hepsin is capable of direct proteolytic cleavage of fibronectin but not latent-TGFß and, importantly, that the ability of hepsin to activate TGFß signaling is dependent on fibronectin. Altogether, this study demonstrates a role for hepsin as a regulator of the TGFß pathway in the mammary gland via a novel mechanism involving proteolytic downmodulation of fibronectin.

YAP1 and its fusion proteins in cancer initiation, progression and therapeutic resistance.

YAP1 is a transcriptional co-activator whose activity is controlled by the Hippo signaling pathway. In addition to important functions in normal tissue homeostasis and regeneration, YAP1 has also prominent functions in cancer initiation, aggressiveness, metastasis, and therapy resistance. In this review we are discussing the molecular functions of YAP1 and its roles in cancer, with a focus on the different mechanisms of de-regulation of YAP1 activity in human cancers, including inactivation of upstream Hippo pathway tumor suppressors, regulation by intersecting pathways, miRNAs, and viral oncogenes. We are also discussing new findings on the function and biology of the recently identified family of YAP1 gene fusions, that constitute a new type of activating mutation of YAP1 and that are the likely oncogenic drivers in several subtypes of human cancers. Lastly, we also discuss different strategies of therapeutic inhibition of YAP1 functions.

Loss of Llgl1 in retinal neuroepithelia reveals links between apical domain size, Notch activity and neurogenesis.

To gain insights into the cellular mechanisms of neurogenesis, we analyzed retinal neuroepithelia deficient for Llgl1, a protein implicated in apicobasal cell polarity, asymmetric cell division, cell shape and cell cycle exit. We found that vertebrate retinal neuroepithelia deficient for Llgl1 retained overt apicobasal polarity, but had expanded apical domains. Llgl1 retinal progenitors also had increased Notch activity and reduced rates of neurogenesis. Blocking Notch function by depleting Rbpj restored normal neurogenesis. Experimental expansion of the apical domain, through inhibition of Shroom3, also increased Notch activity and reduced neurogenesis. Significantly, in wild-type retina, neurogenic retinal progenitors had smaller apical domains compared with proliferative neuroepithelia. As nuclear position during interkinetic nuclear migration (IKNM) has been previously linked with cell cycle exit, we analyzed this phenomenon in cells depleted of Llgl1. We found that although IKNM was normal, the relationship between nuclear position and neurogenesis was shifted away from the apical surface, consistent with increased pro-proliferative and/or anti-neurogenic signals associated with the apical domain. These data, in conjunction with other findings, suggest that, in retinal neuroepithelia, the size of the apical domain modulates the strength of polarized signals that influence neurogenesis.

Dlg5 maintains apical aPKC and regulates progenitor differentiation during lung morphogenesis.

Cell polarity plays an important role in tissue morphogenesis; however, the mechanisms of polarity and their role in mammalian development are still poorly understood. We show here that membrane-associated guanylate kinase protein Dlg5 is required for proper branching morphogenesis and progenitor differentiation in mammalian lung. We found that during lung development Dlg5 functions as an apical-basal polarity protein, which is necessary for the apical maintenance of atypical protein kinase C (aPKC). These results identify Dlg5 as a regulator of apical polarity complexes and uncover the critical function of Dlg5 in branching morphogenesis and differentiation of lung progenitor cells.

Cadherin-catenin proteins in vertebrate development.

Cadherin-catenin adhesion is pivotal for the development of multicellular organisms. Features such as a large repertoire of homotypically interacting cadherins, rapid assembly and disassembly, and a connection to a force-generating actin cytoskeleton make cadherin-mediated junctions ideal structures for the execution of complex changes in cell and tissue morphology during development. Recent findings highlight the role of cadherin-catenin proteins as critical regulators of major developmental pathways. We re-evaluate the significance of cadherin-catenin adhesion structures and propose that in addition to intercellular adhesion, they may be used as biosensors of the external cellular environment that help adjust the behavior of individual cells to ensure survival of the entire organism.

Adherens junctions and cancer.

Cadherins and catenins are the central cell-cell adhesion molecules in adherens junctions (AJs). This chapter reviews the knowledge concerning the role of cadherins and catenins in epithelial cancer and examines the published literature demonstrating the changes in the expression and function of these proteins in human cancer and the association of these changes with patient outcomes. The chapter also covers the mechanistic studies aiming at uncovering the significance of changes in cadherin and catenin expression in cancer and potential molecular mechanisms responsible for the causal role of AJs in cancer initiation and progression.

Lethal giant larvae gene family ( Llgl1 and Llgl2 ) functions as a tumor suppressor in mouse skin epidermis.

Loss of cell polarity and tissue disorganization occurs in majority of epithelial cancers. Studies in simple model organisms identified molecular mechanisms responsible for the establishment and maintenance of cellular polarity, which play a pivotal role in establishing proper tissue architecture. The exact role of these cell polarity pathways in mammalian cancer is not completely understood. Here we analyzed the mammalian orthologs of drosophila apical-basal polarity gene lethal giant larvae ( lgl ), which regulates asymmetric stem cell division and functions as a tumor suppressor in flies. There are two mammalian orthologs of lgl ( Llgl1 and Llgl2 ). To determine the role of the entire lgl signaling pathway in mammals we generated mice with ablation of both Llgl1 and Llgl2 in skin epidermis using K14-Cre ( Llgl1/2 -/- cKO mice). Surprisingly, we found that ablation of Llgl1/2 genes does not impact epidermal polarity in adult mice. However, old Llgl1/2 cKO mice present with focal skin lesions which are missing epidermal layer and ripe with inflammation. To determine the role of lgl signaling pathway in cancer we generated Trp53 -/- /Llgl1/2 -/- cKO and Trp53 -/+ /Llgl1/2 -/- cKO mice. Loss of Llgl1/2 promoted squamous cell carcinoma (SCC) development in Trp53 -/- cKO and caused SCC in Trp53 -/+ cKO mice, while no cancer was observed in Trp53 -/+ cKO controls. Mechanistically, we show that ablation of Llgl1/2 causes activation of aPKC and upregulation of NF-kB signaling pathway, which may be necessary for SCC in Trp53 -/+ /Llgl1/2 -/- cKO mice. We conclude that Lgl signaling pathway functions as a tumor suppressor in mammalian skin epidermis.

SND1 binds to ERG and promotes tumor growth in genetic mouse models of prostate cancer.

SND1 and MTDH are known to promote cancer and therapy resistance, but their mechanisms and interactions with other oncogenes remain unclear. Here, we show that oncoprotein ERG interacts with SND1/MTDH complex through SND1's Tudor domain. ERG, an ETS-domain transcription factor, is overexpressed in many prostate cancers. Knocking down SND1 in human prostate epithelial cells, especially those overexpressing ERG, negatively impacts cell proliferation. Transcriptional analysis shows substantial overlap in genes regulated by ERG and SND1. Mechanistically, we show that ERG promotes nuclear localization of SND1/MTDH. Forced nuclear localization of SND1 prominently increases its growth promoting function irrespective of ERG expression. In mice, prostate-specific Snd1 deletion reduces cancer growth and tumor burden in a prostate cancer model (PB-Cre/Ptenflox/flox/ERG mice), Moreover, we find a significant overlap between prostate transcriptional signatures of ERG and SND1. These findings highlight SND1's crucial role in prostate tumorigenesis, suggesting SND1 as a potential therapeutic target in prostate cancer.

Cell fate determinant Llgl1 is required for propagation of acute myeloid leukemia.

Scribble complex proteins can influence cell fate decisions and self-renewal capacity of hematopoietic cells. While specific cellular functions of Scribble complex members are conserved in mammalian hematopoiesis, they appear to be highly context dependent. Using CRISPR/Cas9-based genetic screening, we have identified Scribble complex-related liabilities in AML including LLGL1. Despite its reported suppressive function in HSC self-renewal, inactivation of LLGL1 in AML confirms its relevant role for proliferative capacity and development of AML. Its function was conserved in human and murine models of AML and across various genetic backgrounds. Inactivation of LLGL1 results in loss of stemness-associated gene-expression including HoxA-genes and induces a GMP-like phenotype in the leukemia stem cell compartment. Re-expression of HoxA9 facilitates functional and phenotypic rescue. Collectively, these data establish LLGL1 as a specific dependency and putative target in AML and emphasizes its cell-type specific functions.

Failure of epithelial tube maintenance causes hydrocephalus and renal cysts in Dlg5-/- mice.

Epithelial tubes represent fundamental building blocks of metazoan organisms; however, the mechanisms responsible for their formation and maintenance are not well understood. Here, we show that the evolutionarily conserved coiled-coil MAGUK protein Dlg5 is required for epithelial tube maintenance in mammalian brain and kidneys. We demonstrate that Dlg5(-/-) mice develop fully penetrant hydrocephalus and kidney cysts caused by a deficiency in membrane delivery of cadherin-catenin adhesion complexes and loss of cell polarity. Dlg5 travels with cadherin-containing vesicles and binds to syntaxin 4, a t-SNARE protein that regulates fusion of transport vesicles with the lateral membrane domain. We propose that Dlg5 functions in plasma membrane delivery of cadherins by linking cadherin-containing transport vesicles with the t-SNARE targeting complex. These findings show that Dlg5 is causally involved in hydrocephalus and renal cysts and reveal that targeted membrane delivery of cadherin-catenin adhesion complexes is critical for cell polarity and epithelial tube maintenance.

Hepsin promotes prostate cancer progression and metastasis.

The majority of cancer-related deaths are associated with metastasis; however, little is known about the mechanisms of this process. Hepsin is a cell surface serine protease that is markedly upregulated in human prostate cancer; however, the functional significance of this upregulation is unknown. We report here that hepsin overexpression in prostate epithelium in vivo causes disorganization of the basement membrane. Overexpression of hepsin in a mouse model of nonmetastasizing prostate cancer has no impact on cell proliferation, but causes disorganization of the basement membrane and promotes primary prostate cancer progression and metastasis to liver, lung, and bone. We provide in vivo evidence that upregulation of a cell surface serine protease in a primary tumor promotes cancer progression and metastasis.

Planar cell polarity planes the inconveniences of cell division into a smooth morphogenetic process.

Cell divisions are necessary, but also very disruptive for morphogenesis. Dividing cells lose many intercellular contacts and polarized features. This breaks the magnificent topology of the developing embryo and, if left unrepaired, can lead to severe tissue disorganization. A recent study demonstrated that cells use the planar cell polarity pathway to reestablish polarity and reintegrate daughter cells into developing tissue.

A causal role for ERG in neoplastic transformation of prostate epithelium.

A significant proportion of human prostate cancers carry a chromosomal rearrangement resulting in the overexpression of the ETS transcription factor, ERG; however, the functional significance of this event is poorly understood. We report here that up-regulation of ERG transcript is sufficient for the initiation of prostate neoplasia. In agreement with measurements of ERG transcripts, we found that ERG protein is expressed in neoplastic human prostate epithelium. Overexpression of ERG in prostate cell lines increased cell invasion. Moreover, targeted expression of this transcript in vivo in luminal prostate epithelial cells of transgenic mice results in initiation of prostate neoplasia observed as the development of focal precancerous prostatic intraepithelial neoplasia (PIN). Similar to human cancers, luminal epithelial cells in these PIN lesions displace diminishing in numbers basal epithelial cells and establish direct contact with the stromal cell compartment. Loss of basal cells is considered to be one of the critical hallmarks of human prostate cancer; however, the mechanisms responsible for this event were unknown. We propose that up-regulation of ERG in human prostate cancer activates cell invasion programs that subsequently displace basal cells by neoplastic epithelium. Our data demonstrate that ERG plays an important causal role in the transformation of prostate epithelium and should be considered as a target for prevention or early therapeutic intervention.

Mechanical instability of adherens junctions overrides intrinsic quiescence of hair follicle stem cells.

Vinculin, a mechanotransducer associated with both adherens junctions (AJs) and focal adhesions (FAs), plays a central role in force transmission through cell-cell and cell-substratum contacts. We generated the conditional knockout (cKO) of vinculin in murine skin that results in the loss of bulge stem cell (BuSC) quiescence and promotes continual cycling of the hair follicles. Surprisingly, we find that the AJs in vinculin cKO cells are mechanically weak and impaired in force generation despite increased junctional expression of E-cadherin and α-catenin. Mechanistically, we demonstrate that vinculin functions by keeping α-catenin in a stretched/open conformation, which in turn regulates the retention of YAP1, another potent mechanotransducer and regulator of cell proliferation, at the AJs. Altogether, our data provide mechanistic insights into the hitherto-unexplored regulatory link between the mechanical stability of cell junctions and contact-inhibition-mediated maintenance of BuSC quiescence.

Hepsin cooperates with MYC in the progression of adenocarcinoma in a prostate cancer mouse model.

BACKGROUND: Hepsin is a cell surface protease that is over-expressed in more than 90% of human prostate cancer cases. The previously developed Probasin-hepsin/Large Probasin-T antigen (PB-hepsin/LPB-Tag) bigenic mouse model of prostate cancer demonstrates that hepsin promotes primary tumors that are a mixture of adenocarcinoma and neuroendocrine (NE) lesions, and metastases that are NE in nature. However, since the majority of human prostate tumors are adenocarcinomas, the contribution of hepsin in the progression of adenocarcinoma requires further investigation. METHODS: We crossed the PB-hepsin mice with PB-Hi-myc transgenic mouse model of prostate adenocarcinoma and characterized the tumor progression in the resulting PB-hepsin/PB-Hi-myc bigenic mice. RESULTS: We report that PB-hepsin/PB-Hi-myc bigenic mice develop invasive adenocarcinoma at 4.5 months. Further, histological analysis of the 12- to 17-month-old mice revealed that the PB-hepsin/PB-Hi-myc model develops a higher grade adenocarcinoma compared with age-matched tumors expressing only PB-Hi-myc. Consistent with targeting hepsin to the prostate, the PB-hepsin/PB-Hi-myc tumors showed higher hepsin expression as compared to the age-matched myc tumors. Furthermore, endogenous expression of hepsin increased in the PB-Hi-myc mice as the tumors progressed. CONCLUSIONS: Although we did not detect any metastases from the prostates in either the PB-hepsin/PB-Hi-myc or the PB-Hi-myc mice, our data suggests that hepsin and myc cooperate during the progression to high-grade prostatic adenocarcinoma.

Rearranged ERG confers robustness to prostate cancer cells by subverting the function of p53.

OBJECTIVE: ERG rearrangements are frequent and early events in prostate cancer. The functional role of rearranged ERG, however, is still incompletely understood. ERG rearrangements are maintained during prostate cancer progression suggesting that they may confer a selective advantage. The molecular basis of this notion is the subject of this study. METHODS: A variety of immunological methods were used to characterize the effects of rearranged ERG on p53. Consequences of an overexpression of N-terminally deleted ERG on p53 function were interrogated by measuring apoptosis and cellular senescence in the presence or absence of exogenous DNA damage. Effects of N-terminally deleted ERG on the transactivation function of p53 were analyzed by qRT-PCR. RESULTS: We show that overexpression of ERG leads to an increased basal level of DNA damage and a stabilization of p53 that involves a sequestration of its E3 ubiquitin ligase, MDM2, into nucleoli. A higher p53 expression was also observed in vivo in an ERG-overexpressing prostatic intraepithelial neoplasia mouse model. The correlation between ERG and p53 expression was corroborated in 163 patients with prostate cancer. ERG overexpression was found to inhibit both apoptosis and cellular senescence induced by exogenous DNA damage. Mechanistically, this protective effect of ERG involved an abrogation of the DNA damage-induced expression of p53 target genes. CONCLUSIONS: By protecting tumor cells from the antiproliferative consequences of genotoxic stress, ERG may allow the survival and proliferation of genomically unstable tumor cells. Targeting ERG may therefore represent a promising strategy to suppress such adverse features during prostate cancer progression.

Actin cable dynamics and Rho/Rock orchestrate a polarized cytoskeletal architecture in the early steps of assembling a stratified epithelium.

To enable stratification and barrier function, the epidermis must permit self-renewal while maintaining adhesive connections. By generating K14-GFP-actin mice to monitor actin dynamics in cultured primary keratinocytes, we uncovered a role for the actin cytoskeleton in establishing cellular organization. During epidermal sheet formation, a polarized network of nascent intercellular junctions and radial actin cables assemble in the apical plane of the monolayer. These actin fibers anchor to a central actin-myosin network, creating a tension-based plane of cytoskeleton across the apical surface of the sheet. Movement of the sheet surface relative to its base expands the zone of intercellular overlap, catalyzing new sites for nascent intercellular junctions. This polarized cytoskeleton is dependent upon alpha-catenin, Rho, and Rock, and its regulation may be important for wound healing and/or stratification, where coordinated tissue movements are involved.