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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 oflgl(Llgl1andLlgl2). To determine the role of the entire lgl signaling pathway in mammals we generated mice with ablation of bothLlgl1andLlgl2in skin epidermis using K14-Cre (Llgl1/2-/-cKO mice). Surprisingly, we found that ablation ofLlgl1/2genes does not impact epidermal polarity in adult mice. However, oldLlgl1/2cKO 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 generatedTrp53-/-/Llgl1/2-/-cKO andTrp53-/+/Llgl1/2-/-cKO mice. Loss ofLlgl1/2promoted squamous cell carcinoma (SCC) development inTrp53-/-cKO and caused SCC inTrp53-/+cKO mice, while no cancer was observed inTrp53-/+cKO controls. Mechanistically, we show that ablation ofLlgl1/2causes activation of aPKC and upregulation of NF-kB signaling pathway, which may be necessary for SCC inTrp53-/+/Llgl1/2-/-cKO mice. We conclude that Lgl signaling pathway functions as a tumor suppressor in mammalian skin epidermis.

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.

Transforming growth factor‐beta (TGFβ) 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 TGFβ activity by acting as a storage compartment of latent‐TGFβ, but how TGFβ 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 TGFβ signaling through the release of latent‐TGFβ from the ECM storage compartment. Mammary glands in hepsin CRISPR knockout mice showed reduced TGFβ signaling and increased epithelial branching, accompanied by increased levels of fibronectin and latent‐TGFβ1, while overexpression of hepsin in mammary tumors increased TGFβ signaling. Cell‐free and cell‐based experiments showed that hepsin is capable of direct proteolytic cleavage of fibronectin but not latent‐TGFβ and, importantly, that the ability of hepsin to activate TGFβ signaling is dependent on fibronectin. Altogether, this study demonstrates a role for hepsin as a regulator of the TGFβ pathway in the mammary gland via a novel mechanism involving proteolytic downmodulation of fibronectin., TGFβ is released from the ECM compartments of the mammary glands by hepsin mediated proteolytic cleavage of the ECM component fibronectin.

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.

A tyrosine kinase connects tension with strength of junctions between three adjacent cells

Vinculin, a mechanotransducer associated with both adherens junctions (AJ) and focal adhesions (FA) plays a central role in force transmission through these cell-cell and cell-substratum contacts. Here we describe the conditional knock out (KO) of vinculin in murine skin. Remarkably, we find that the loss of vinculin function results in the loss of bulge stem cell (BuSC) quiescence. We demonstrate that vinculin KO cells are impaired in force generation resulting in mechanically weak AJs. Mechanistically, vinculin functions by keeping α-catenin in a stretched conformation, which in turn regulates the retention of YAP1, another potent mechanotransducer and regulator of cell proliferation, to the junctions. Conditional KO of α-catenin specifically in the BuSCs further corroborates the importance of stable AJs in the maintenance of quiescence and stemness. Altogether, our data provides definitive mechanistic insights into the hitherto unexplored regulatory link between the mechanical stability of cell-junctions and the maintenance of BuSC quiescence.

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.

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.

SummaryThe significance of ERG in human prostate cancer is unclear because mouse prostate is resistant to ERG-mediated transformation. We determined that ERG activates the transcriptional program regulated by YAP1 of the Hippo signaling pathway and found that prostate-specific activation of either ERG or YAP1 in mice induces similar transcriptional changes and results in age-related prostate tumors. ERG binds to chromatin regions occupied by TEAD/YAP1 and transactivates Hippo target genes. In addition, in human luminal-type prostate cancer cells, ERG binds to the promoter of YAP1 and is necessary for YAP1 expression. These results provide direct genetic evidence of a causal role for ERG in prostate cancer and reveal a connection between ERG and the Hippo signaling pathway.

Background/aim The aim of the current study was to determine the effects of the ERG small-molecule inhibitor YK-4-279 on ERG+ prostate cancer patient-derived xenografts (PDX). Materials and methods ERG activity was blocked using YK-4-279 in three subcutaneously-implanted ERG+ (LuCaP 23.1, 86.2 and 35) and one ERG- (LuCaP 96) PDX. Treated animals tumor volume (TV), body weight (BW) and serum prostate-specific antigen (PSA) were compared to vehicle-treated control animals. Gene expression, proliferation, apoptosis, microvessel density and ERG expression were also assessed. Results Administration of YK-4-279 decreased TV (p=0.026), proliferation (p=0.0038) and PSA (p=0.022) in Severe Combined Immunodeficiency (SCID) mice bearing LuCaP 23.1 tumors. LuCaP 86.2, LuCaP 35 and LuCaP 96 showed no significant changes in TV, or PSA. Mineralocorticoid receptor (MR) and MR-direct target genes were up-regulated in treatment-resistant LuCaP 86.2 and LuCaP 35 PDX. Conclusion YK-4-279 decreased ERG+ LuCaP 23.1 tumor growth, but not LuCaP 86.2 and LuCaP 35 ERG+ tumor growth.

The development of effective therapies inhibiting prostate cancer progression and metastasis may substantially impact prostate cancer mortality and potentially reduce the rates of invasive treatments by enhancing the safety of active surveillance strategies. Hepsin (HPN) is a cell surface serine protease amplified in a subset of human sarcomas (7.2%), as well as in ovarian (10%), lung adeno (5.4%), lung squamous cell (4.5%), adenoid cystic (5%), breast (2.6%), uterine (1.7%) and colon (1.4%) carcinomas. While HPN is not amplified in prostate cancer, it is one of the most prominently overexpressed genes in the majority of human prostate tumors and genetic experiments in mice indicate that Hepsin promotes prostate cancer metastasis, particularly metastasis to the bone marrow. We report here the development, analysis and animal trial of the small-molecule Hepsin inhibitor HepIn-13. Long-term exposure to HepIn-13 inhibited bone, liver and lung metastasis in a murine model of metastatic prostate cancer. These findings indicate that inhibition of Hepsin with small-molecule compounds could provide an effective tool for attenuation of prostate cancer progression and metastasis.

Supratentorial ependymoma can be sub-stratified into clinically relevant subtypes characterized by distinct molecular features. The subtype defined by high YAP1 activity harbored two distinct YAP1 gene fusions, YAP1-MAMLD1 and YAP1-FAM118B. In addition, YAP1 gene fusions have been detected in several other cancer types, including Epithelioid Hemangioendothelioma and Endocervical Adenocarcinoma. YAP1 is a key transcriptional co-activator and proto-oncogene that is negatively regulated by the Hippo pathway. Here, we show that both YAP1-MAMLD1 and YAP1-FAM118B, as well as additional YAP1 fusion genes found in other cancer types, are potent oncogenic drivers that cause tumor formation in the brain and the hindlimb in mice upon overexpression by somatic cell gene transfer. Using different in vitro assays, including Luciferase, RNA-, and ChIP Seq, we show that both the N-terminal YAP1 part and the C-terminal fusion partners exert activity. We can show that the YAP1 activity still relies on the binding to TEAD transcription factors, whereas the C terminal activity does not. Furthermore, the different fusion proteins have become independent from negative Hippo pathway signaling by constitutive nuclear localization and protection from degradation. In addition, by introducing point mutations and truncations to block the YAP1 and the MAMLD1 function we can show that the activity of both halves contributes to the oncogenic function of YAP1-MAMLD1. Using in vitro and in vivo assays we can show that pharmacological and genetic ablation of YAP-TEAD interaction diminishes the oncogenic potential of the fusions, indicating that this might be a potential therapeutic approach for these tumors in the future.

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.

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.

The concerted production of neurons and glia by neural stem cells (NSCs) is essential for neural circuit assembly. In the developing cerebral cortex, radial glia progenitors (RGPs) generate nearly all neocortical neurons and certain glia lineages. RGP proliferation behavior shows a high degree of non-stochasticity, thus a deterministic characteristic of neuron and glia production. However, the cellular and molecular mechanisms controlling RGP behavior and proliferation dynamics in neurogenesis and glia generation remain unknown. By using mosaic analysis with double markers (MADM)-based genetic paradigms enabling the sparse and global knockout with unprecedented single-cell resolution, we identified Lgl1 as a critical regulatory component. We uncover Lgl1-dependent tissue-wide community effects required for embryonic cortical neurogenesis and novel cell-autonomous Lgl1 functions controlling RGP-mediated glia genesis and postnatal NSC behavior. These results suggest that NSC-mediated neuron and glia production is tightly regulated through the concerted interplay of sequential Lgl1-dependent global and cell intrinsic mechanisms.

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 β4 integrin-mediated activation of SRC tyrosine kinase. SRC is 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 β4 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.

AR-driven transcriptome away from genes associated with prostate epithelial differentiation towards genes associated with cell survival and mitosis. Previously we showed that Gli proteins bind to the enigmatic N-terminal tau5 transactivation domain of AR and co-activate full-length and truncated ARs. When exogenously overexpressed in androgen growth-dependent LNCaP cells, Gli proteins can elicit androgen growth-independence (AI). Here we show that interference with Gli-AR binding in AI LNCaP variants suppresses the alternate AR transcriptome, restores the AR differentiation transcriptome and suppresses AI growth. METHODS: Androgen growth-independent, enzalutamide(ENZ-) resistant LNCaP cell variants, LNCaP-AI and LN95, were treated with Gli inhibitors (GANT61 and HPI-1) or transfected with Gli3 siRNA or transfected with a small decoy peptide from the Gli2 AR-binding domain (Gli-DP) to disrupt intracellular Gli-AR interactions. RNAs were extracted and expressions of differentiation genes (DGs), PSA and KLK2 or mitotic-regulatory genes (MRGs), UBE2C, CDK1 and CDC20 were measured by real-time qRT-PCR and compared to control-treated (vehicle, non-targeting siRNA or empty vector transfected) cells. Cell growth after Gli manipulation(s) was measured on the Incuyte-Zoom instrument that quantifies realtime cell growth and was compared to control cells. Increasing doses of Enzalutamide (ENZ) were titrated into Gli-DP transfected cells to test whether it cooperates with Gli-AR binding disruption in suppressing AI growth. RESULTS: Suppression of global Gli activity, Gli3-specific knockdown and exogenous Gli-DP expression significantly suppressed expressions of MRGs but increased expressions of DGs in both AI cell types. These actions also significantly slowed growth of the cells, with Gli inhibitors and Gli3 knockdown showing complete growth suppression whereas the Gli-DP achieved half-suppression. ENZ treatments further increased growth suppression of the Gli-DP expressing cells. CONCLUSIONS: Our results identify the potential for Gli-AR binding interference as a means to control AI/CRPC growth. This interference further cooperates with ENZ to provide very robust growth suppression showing that combined targeting of AR N-terminal and C-terminal domains holds the key for more effective treatments for CRPC.

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.

Mechanistic studies of deregulated ERG in prostate cancer and other cancers continue to enhance its role in cancer biology and its utility as a biomarker and therapeutic target. Here, we show that ERG, through its physical interaction with androgen receptor, induces AR aggregation and endoplasmic reticulum stress in the prostate glands of ERG transgenic mice. Histomorphological alterations and the expression of ER stress sensors Atf6, Ire1α, Perk, their downstream effectors Grp78/BiP and eIF2α in ERG transgenic mouse prostate glands indicate the presence of chronic ER stress. Transient activation of apoptotic cell death during early age correlated well with the differential regulation of ER stress sensors, in particular Perk. Epithelial cells derived from ERG transgenic mouse prostates have increased prostasphere formation with resistance to radiation induced cell death. Continued activation of cell survival factors, Atf6 and Ire1α during chronic ER stress due to presence of ERG in prostate epithelium induces survival pathways and provides a selection pressure in the continuum of ERG dependent neoplastic process. These novel insights will enhance the understanding of the mechanistic functions of ERG in prostate tumor biology and towards development of early targeted therapeutic strategies for prostate cancer.

Introduction: AR-mediated activation of ETS Related Gene (ERG) represents one of the most common and validated prostate cancer driver genes. Recently, we have shown novel morphologic phenotypes of endoplasmic reticulum (ER) stress in prostate glands of ARR2PB-ERG transgenic mouse. Since AR regulates ERG expression through TMPRSS2 promoter in human prostate cancer, we continue to investigate the post-translational interactions between ERG and AR leading to ER stress and subsequently to cell survival mechanisms. Understanding these mechanisms will potentially have major therapeutic implications. Methods: Light and electron microscopy were used to examine the morphologic and subcellular differences. AR aggregations, Co-IP and Proximal Ligation Assay for protein-protein interactions were studied in LNCaP, HEK293 cells. N-terminal and C-deletions of AR were utilized to identify specific AR domain interactions with ERG. Luminal cell surface markers on the isolated mouse prostate glands and spontaneously immortalized mouse prostate epithelial cells from ERG transgenic mouse (MoE1) were analyzed by FACS analysis. Results: Coexpression of ERG and AR showed significant aggregation of AR in filter assays. Co-IP experiments and PLA assays revealed that significant interactions occur through N-terminal domain of AR with ERG. Epithelial cells of ERG-Tg mouse prostates showed ~70% increase in CD49f (low) and Sca-1 (med) population with increased sphere formation capability and resistance to radiation-induced cell death. Both epithelial cells grown into spheres and established MoE1 cells displayed increased CD49f (low) and significant increase in the EpCAM negative population. Conclusions: Overall, our experiments demonstrate the mechanistic link that the physical interactions between ERG and AR lead to ER stress in prostate epithelium through AR misfolding/aggregation. Our observations of ERG-induced AR aggregation as one of the initial events that lead to ER stress and to cell survival indicate a critical function for ERG in the etiology of prostate cancer initiation and progression. Citation Format: Taduru L. Sreenath, Shiela S. Macalindong, Natallia Mikhalkevich, Shashwat Sharad, Ahmed Mohamed, Denise Young, Borbiev Talaibek, Charles Xavier, Rishita Gupta, Muhammad Jamal, Kevin Babcock, Shyh-Han Tan Tan, Marja T. Nevalainen, Albert Dobi, Gyorgy Petrovics, Isabell A. Sesterhenn, Inger L. Rosner, Charles J. Bieberich, Peter Nelson, Valeri Vasioukhin, Shiv Srivastava. ETS-related gene mediated androgen receptor aggregation and endoplasmic reticulum stress in prostate cancer development [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A010.

Oncogenic activation of the ETS Related Gene (ERG) in humans was originally identified in subsets of Ewing sarcomas, myeloid leukemias and, recently, in the majority of prostate cancers. Expression of human ERG protein and consequently its functions in normal and disease states needs to be better understood in light of its suggested role in cell differentiation and proliferation. Here, we analyzed temporal and spatial expression of the Erg (mouse protein) by immunohistochemical analysis during mouse embryonic and adult organogenesis using a highly specific ERG monoclonal antibody (ERG MAb). This study establishes widespread immunolocalization of Erg protein in endothelial cells and restricted expression in precartilage and hematopoietic tissues. Intriguingly, Erg is not expressed in any epithelial tissue including prostate epithelium, or in infiltrating lymphocytes that are occasionally seen in the prostate environment, a common site of tumors with ERG rearrangements and unscheduled ERG expression. These findings will further aid in investigations of Erg functions in normal and disease conditions.

Oncogenic activation of the ETS Related Gene (ERG) in humans was originally identified in subsets of Ewing sarcomas, myeloid leukemias and, recently, in the majority of prostate cancers. Expression of human ERG protein and consequently its functions in normal and disease states needs to be better understood in light of its suggested role in cell differentiation and proliferation. Here, we analyzed temporal and spatial expression of the Erg (mouse protein) by immunohistochemical analysis during mouse embryonic and adult organogenesis using a highly specific ERG monoclonal antibody (ERG MAb). This study establishes widespread immunolocalization of Erg protein in endothelial cells and restricted expression in precartilage and hematopoietic tissues. Intriguingly, Erg is not expressed in any epithelial tissue including prostate epithelium, or in infiltrating lymphocytes that are occasionally seen in the prostate environment, a common site of tumors with ERG rearrangements and unscheduled ERG expression. These findings will further aid in investigations of Erg functions in normal and disease conditions.

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. Prostate 70: 591–600, 2010. © 2009 Wiley-Liss, Inc.

Hepsin is a type II transmembrane serine protease overexpressed in the majority of human prostate cancers. We recently demonstrated that hepsin promotes prostate cancer progression and metastasis and thus represents a potential therapeutic target. Here we report the identification of novel small-molecule inhibitors of hepsin catalytic activity. We utilized purified human hepsin for high-throughput screening of established drug and chemical diversity libraries and identified sixteen inhibitory compounds with IC50 values against hepsin ranging from 0.23-2.31 μM and relative selectivity of up to 86-fold or greater. Two compounds are orally administered drugs established for human use. Four compounds attenuated hepsin-dependent pericellular serine protease activity in a dose dependent manner with limited or no cytotoxicity to a range of cell types. These compounds may be used as leads to develop even more potent and specific inhibitors of hepsin to prevent prostate cancer progression and metastasis. [Mol Cancer Ther 2008;7(10):3343–51]

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 unique and evolutionary 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 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 identify a novel protein 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.

Despite aggressive standard and novel targeted therapies, malignant gliomas recur invariably and develop therapy resistance. Our previous studies have shown that oligodendrocyte progenitor cells (OPCs) undergo asymmetric cell division whereby they generate self-renewing and differentiating cells in a single division by unequally segregating cell fate determinants. We found that premalignant OPC, the origin of glioma in genetically engineered mouse models, exhibit disrupted asymmetric divisions, together with other hallmarks of cancer, including hyper-proliferation (Sugiarto et al., 2011, Cancer Cell 20:328-40). Yet, it is not clear whether disruption of asymmetric division of OPCs is a contributing factor or a consequence of neoplastic transformation. Lethal giant larvae 1 (Lgl1), a gene that was initially identified as a tumor suppressor in Drosophila, has been implicated in the asymmetric localization of cell fate determinants in neural progenitor cells (Klezovitch et al., 2004, Genes Dev 18:559-571). We investigated the effects of Lgl1 depletion on distinct hallmarks of glioma. We find that in murine OPC carrying conditional null alleles of Lgl1, depletion of Lgl1 resulted in reduced rates of asymmetric cell division and other defects previously associated with glioma precursors. Our data suggest that loss of asymmetric divisions contribute to neoplastic transformation. Asymmetric cell divisions of stem and progenitor cells are a point of disruption to which novel therapies can be targeted. Underlying mechanisms for the phenotypes of Lgl1 knockout and their relevance for malignant glioma will be discussed.

The intracellular epithelial protein tyrosine kinase 6 (PTK6) was cloned from cultured human melanocytes (24) and human breast cancer cells (it is also called Brk for breast tumor kinase) (31). PTK6 was also identified in a screen for tyrosine kinases expressed in the mouse small intestine (it is also called Sik for Src-related intestinal kinase) (40, 45). While PTK6 is expressed in many breast carcinoma cell lines and in a high percentage of primary breast tumors that have been examined, it has not been detected in normal human breast tissue (3, 31) or in the mouse mammary gland (26). In normal tissues PTK6 is expressed in differentiated nondividing cells, with high levels in linings of the gastrointestinal tract (26, 45). PTK6 is expressed in skin (45), and overexpression of PTK6 in mouse keratinocytes resulted in increased expression of the differentiation marker filaggrin during calcium-induced differentiation, suggesting a positive role in differentiation (46). PTK6 was also recently implicated in the regulation of human keratinocyte differentiation (48). Higher levels of PTK6 were detected in normal human oral epithelial cells than in oral epithelial cancer cells (34). In the normal prostate, PTK6 is present in the nuclei of epithelial cells, but it is relocalized to the cytoplasm in prostate tumors (10). PTK6 belongs to a novel subfamily of intracellular tyrosine kinases that is distinct from the Src family and includes Srms, FRK (Rak/Gtk/Iyk/Bsk), and Src42A/Dsrc41 (reviewed in reference 39). These kinases share a low degree of sequence homology with known kinases, including one another, but the exon structure of the genes encoding these kinases is highly conserved and distinct. Lack of myristoylation distinguishes most PTK6 family kinases from the Src family, and these proteins are not specifically targeted to the membrane. In fact, both PTK6 (10, 11, 15) and FRK (9) appear to have nuclear functions. The first substrates identified for PTK6 were the novel adaptor protein BKS/STAP-2 (32), which has been implicated in the regulation of STAT3 (30), and the KH domain-containing RNA binding protein Sam68 (11). We also identified the Sam68-like mammalian proteins SLM-1 and SLM-2 as substrates of PTK6 (15). PTK6 phosphorylates Sam68, SLM-1, and SLM-2 in the nucleus and inhibits their RNA binding activities (11, 15). PTK6 was also found to phosphorylate the cytoskeletal protein paxillin following epidermal growth factor stimulation, which promoted activation of Rac1, cell migration, and invasion in vitro (8). Recently the serine threonine kinase Akt/PKB was identified as a substrate of PTK6, and PTK6-mediated tyrosine phosphorylation led to inhibition of Akt activity in unstimulated cells (50). Overexpression of active PTK6 blocked the phosphorylation of the Forkhead transcription factor FoxO1 (FKHR), a downstream Akt target. These results suggested that PTK6 may function as a signaling molecule whose kinase activity normally limits the activity of Akt in unstimulated cells (50). The dramatic induction of the PTK6 tyrosine kinase in a significant percentage of human breast tumors suggests a role for this tyrosine kinase in the etiology of breast cancer (17). However, expression patterns of PTK6 in normal epithelia are not consistent with a role in promoting growth. Ectopic expression of mouse PTK6 in Rat1a cells did not enhance growth but led to increased apoptosis following serum deprivation and UV exposure (16). To explore functions of PTK6, we developed and characterized a PTK6-deficient mouse model, focusing on the small intestine, where PTK6 is expressed at highest levels in adult animals.

During development, cells monitor and adjust their rates of accumulation to produce organs of predetermined size. We show here that central nervous system–specific deletion of the essential adherens junction gene, α E-catenin, causes abnormal activation of the hedgehog pathway, resulting in shortening of the cell cycle, decreased apoptosis, and cortical hyperplasia. We propose that αE-catenin connects cell-density–dependent adherens junctions with the developmental hedgehog pathway and that this connection may provide a negative feedback loop controlling the size of developing cerebral cortex.

The existing models of cancer progression assume that a linear sequence of genetic and epigenetic events occurs during this process. In this representation every new event (either loss of a tumor-suppressor, or activation of a proto-oncogene) makes cells even more malignant. The result is a "super" cell that can form metastases at the distant sites. Metastatic cells are believed to carry all genetic and epigenetic characteristics that are necessary for metastasis formation. Recently, we have shown that cell-surface protease hepsin causes disorganization of the basement membrane and promotes prostate cancer progression and metastasis. In human prostate cancer hepsin is upregulated in the precancerous lesions and this upregulation is maintained in the primary tumors. Remarkably and completely unexpected for a metastasis-promoting gene, hepsin is expressed at low levels in metastatic lesions and the message is completely absent in metastasis-derived prostate cancer cell lines. These results demonstrate that genes that play an important role in metastatic process may exercise their role only at the specific fragments of cancer progression pathway (for example, during initial invasion and tissue disorganization in the primary organ) and may have no role in metastatic lesions. Future treatment of cancer patients may rely heavily on monitoring of tumor progression, as treatment efficient in attenuation of initial tumor progression may be inefficient or even adverse at the advance stages of disease.

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.

We have generated an epidermis-specific desmoplakin (DP) mouse knockout, and show that epidermal integrity requires DP; mechanical stresses to DP-null skin cause intercellular separations. The number of epidermal desmosomes in DP-null skin is similar to wild type (WT), but they lack keratin filaments, which compromise their function. DP-null keratinocytes have few desmosomes in vitro, and are unable to undergo actin reorganization and membrane sealing during epithelial sheet formation. Adherens junctions are also reduced. In vitro, DP transgene expression rescues these defects. DP is therefore required for assembly of functional desmosomes, maintaining cytoskeletal architecture and reinforcing membrane attachments essential for stable intercellular adhesion.

When surface epithelium was conditionally targeted for ablation of alpha-catenin, hair follicle development was blocked and epidermal morphogenesis was dramatically affected, with defects in adherens junction formation, intercellular adhesion, and epithelial polarity. Differentiation occurred, but epidermis displayed hyperproliferation, suprabasal mitoses, and multinucleated cells. In vitro, alpha-catenin null keratinocytes were poorly contact inhibited and grew rapidly. These differences were not dependent upon intercellular adhesion and were in marked contrast to keratinocytes conditionally null for another essential intercellular adhesion protein, desmoplakin (DP). KO keratinocytes exhibited sustained activation of the Ras-MAPK cascade due to aberrations in growth factor responses. Thus, remarkably, features of precancerous lesions often attributed to defects in cell cycle regulatory genes can be generated by compromising the function of alpha-catenin.

Sik (mouse Src-related intestinal kinase) and its orthologue BRK (human breast tumor kinase) are intracellular tyrosine kinases that are distantly related to the Src family and have a similar structure, but they lack the myristoylation signal. Here we demonstrate that Sik and BRK associate with the RNA binding protein Sam68 (Src associated during mitosis, 68 kDa). We found that Sik interacts with Sam68 through its SH3 and SH2 domains and that the proline-rich P3 region of Sam68 is required for Sik and BRK SH3 binding. In the transformed HT29 adenocarcinoma cell cell line, endogenous BRK and Sam68 colocalize in Sam68-SLM nuclear bodies (SNBs), while transfected Sik and Sam68 are localized diffusely in the nucleoplasm of nontransformed NMuMG mammary epithelial cells. Transfected Sik phosphorylates Sam68 in SNBs in HT29 cells and in the nucleoplasm of NMuMG cells. In functional studies, expression of Sik abolished the ability of Sam68 to bind RNA and act as a cellular Rev homologue. While Sam68 is a substrate for Src family kinases during mitosis, Sik/BRK is the first identified tyrosine kinase that can phosphorylate Sam68 and regulate its activity within the nucleus, where it resides during most of the cell cycle.

We have found that epithelial cells engage in a process of cadherin-mediated intercellular adhesion that utilizes calcium and actin polymerization in unexpected ways. Calcium stimulates filopodia, which penetrate and embed into neighboring cells. E-cadherin complexes cluster at filopodia tips, generating a two-rowed zipper of embedded puncta. Opposing cell surfaces are clamped by desmosomes, while vinculin, zyxin, VASP, and Mena are recruited to adhesion zippers by a mechanism that requires alpha-catenin. Actin reorganizes and polymerizes to merge puncta into a single row and seal cell borders. In keratinocytes either null for alpha-catenin or blocked in VASP/Mena function, filopodia embed, but actin reorganization/polymerization is prevented, and membranes cannot seal. Taken together, a dynamic mechanism for intercellular adhesion is unveiled involving calcium-activated filopodia penetration and VASP/Mena-dependent actin reorganization/polymerization.

Desmosomes first assemble in the E3.5 mouse trophectoderm, concomitant with establishment of epithelial polarity and appearance of a blastocoel cavity. Throughout development, they increase in size and number and are especially abundant in epidermis and heart muscle. Desmosomes mediate cell–cell adhesion through desmosomal cadherins, which differ from classical cadherins in their attachments to intermediate filaments (IFs), rather than actin filaments. Of the proteins implicated in making this IF connection, only desmoplakin (DP) is both exclusive to and ubiquitous among desmosomes. To explore its function and importance to tissue integrity, we ablated the desmoplakin gene. Homozygous −/− mutant embryos proceeded through implantation, but did not survive beyond E6.5. Mutant embryos proceeded through implantation, but did not survive beyond E6.5. Surprisingly, analysis of these embryos revealed a critical role for desmoplakin not only in anchoring IFs to desmosomes, but also in desmosome assembly and/or stabilization. This finding not only unveiled a new function for desmoplakin, but also provided the first opportunity to explore desmosome function during embryogenesis. While a blastocoel cavity formed and epithelial cell polarity was at least partially established in the DP (−/−) embryos, the paucity of desmosomal cell–cell junctions severely affected the modeling of tissue architecture and shaping of the early embryo.

A critical aspect of gut morphogenesis is initiation of a leftward tilt, and failure to do so leads to gut malrotation and volvulus. The direction of tilt is specified by asymmetric cell behaviors within the dorsal mesentery (DM), which suspends the gut tube, and is downstream of Pitx2, the key transcription factor responsible for the transfer of left-right (L-R) information from early gastrulation to morphogenesis. Although Pitx2 is a master regulator of L-R organ development, its cellular targets that drive asymmetric morphogenesis are not known. Using laser microdissection and targeted gene misexpression in the chicken DM, we show that Pitx2-specific effectors mediate Wnt signaling to activate the formin Daam2, a key Wnt effector and itself a Pitx2 target, linking actin dynamics to cadherin-based junctions to ultimately generate asymmetric cell behaviors. Our work highlights how integration of two conserved cascades may be the ultimate force through which Pitx2 sculpts L-R organs.

Introduction: High levels of the ERG expression due to gene fusions (predominantly TMPRSS2-ERG) is frequently detected in prostate cancer (CaP) in Western countries. Better understanding of the roles of the ERG oncogenic functions in CaP initiation and/or progression will enhance its utility as the therapeutic target. Transgenic mice generated to understand role of ERG in the CaP development have shown focal proliferative and dysplastic PIN lesions and adenocarcinoma in older mice. The present study was aimed towards understanding the molecular mechanism underlying the role of ERG in early stages of oncogenic transformation of prostate epithelium. Methods: Morphological analyses of mouse prostate glands were performed by H&E staining and by electron microscopy. Luminal cell surface markers were evaluated by FACS analysis. Luminal cells were assessed for their potential to proliferate and form spheres by prostate sphere formation assay. Effects of ERG on ER stress and UPR marker proteins were analyzed in transgenic mice prostates and ERG expressing cell culture models. Representative ER stress pathway in ERG-positive human prostate cancer was analyzed by CPDR-Affymetrix GeneChip data from well and poorly differentiated tumors. Results: Histological examination of ERG-Tg mouse prostates revealed increased luminal cell death due to apoptosis. Subsequently, TEM analysis revealed significant morphological differences such as increased numbers of lysosomes, autophagosomes, concentric membrane bodies with ribosomes, and lipid droplets in the cytoplasm of transgenic secretory luminal epithelial cells. Epithelial cells of ERG-Tg mouse prostates showed increase in CD49f (low) and Sca-1 (med) population with increased sphere formation capability and resistance to induced cell death. ERG-Tg mouse prostate tissues and LNCaP-ERG transfectants showed increased expression of ER stress sensors and UPR proteins. Further, in human prostate tumors, a strong correlation was also observed between expression of the ERG and P4HB/PDI, an ER stress response protein. Conclusions: A critical of function of ERG in early prostate tumorigenesis may involve ER stress resulting into the activation of UPR, autophagy and cell survival through clonal selection. These observation also define potential new therapeutic targets in CaP-ERG network. Funding: This research in part was supported by the National Cancer Institute R01CA162383 (S. S.) and USU-CPDR funds. Citation Format: Taduru L. Sreenath, Shiela Macalindong, Natallia Mikhalkevich, Shashwat Sharad, Parameet Kumar, Denise Young, Rishita Gupta, Shilpa Katta, Ahmed Mohamed, Shyh-Han Tan, Albert Dobi, Gyorgy Petrovics, Isabell A. Sesterhenn, Charles J. Bieberich, Peter Nelson, David G. McLeod, Valeri Vasioukhin, Shiv Srivastava. ERG oncogenic activation leads to the endoplasmic reticulum stress and cell survival mechanisms. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4175.

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.

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.

Oncogene mutations are frequently found in several tumour types and, among these, point mutations of the ras gene are particularly significant. A predominance of N-ras mutations has been found in the bone marrow DNA of patients with myelodysplastic syndrome (MDS) or acute myelogenous leukaemia (AML). On the other hand, increased levels of plasma DNA have previously been observed in patients suffering from various malignant diseases. In the present work we have investigated, by polymerase chain reaction (PCR), point mutations of the N-ras gene in the DNA of plasma, blood cells and bone marrow of 10 patients suffering from AML or MDS. The different ras mutations detected in five cases were always present in the plasma DNA while sometimes absent in the DNA of peripheral blood cells or bone marrow. This indicates that a bone marrow biopsy or aspiration does not necessarily contain all the malignant clones involved in the disease. Plasma could thus prove to be an easily accessible and useful material for detection and monitoring of myeloid disorders.

Cell polarity plays a critical role in the development of all metazoans; however, the mechanisms of cell polarity and the specific role of cell polarity pathways in mammalian organisms are still poorly understood. Lethal giant larvae (Lgl) is an apical-basal polarity gene identified in Drosophila, where it functions as a tumor suppressor controlling self-renewal and differentiation of progenitor cells. There are two orthologs of Lgl in mammalian genomes: Llgl1 and Llgl2. While mammalian Lgls are assumed to be tumor suppressor genes, little is known about their function in vivo. Here we report the functional analysis of murine Llgl2. We generated Llgl2(-/-) mice and found that Llgl2 functions as a polarity protein required for proper branching morphogenesis during placental development. Llgl2(-/-) pups are born as runts but quickly catch up in size and grow into normal-size adults. Surprisingly, no prominent phenotypes or spontaneous tumors were observed in adult Llgl2(-/-) mice. Analyses of placental trophoblasts reveal a critical role for Llgl2 in cell polarization and polarized cell invasion. We conclude that mammalian Llgl2 is required for proper polarized invasion of trophoblasts and efficient branching morphogenesis during placental development, but, unlike its Drosophila ortholog, it does not function as a canonical tumor suppressor gene.

The Hippo pathway regulates contact inhibition of cell proliferation and, ultimately, organ size in diverse multicellular organisms. Inactivation of the Hippo pathway promotes nuclear localization of the transcriptional coactivator Yap1, a Hippo pathway effector, and can cause cancer. Here, we show that deletion of αE (α epithelial) catenin in the hair follicle stem cell compartment resulted in the development of skin squamous cell carcinoma in mice. Tumor formation was accelerated by simultaneous deletion of αE-catenin and the tumor suppressor-encoding gene p53. A small interfering RNA screen revealed a functional connection between αE-catenin and Yap1. By interacting with Yap1, αE-catenin promoted its cytoplasmic localization, and Yap1 showed constitutive nuclear localization in αE-catenin-null cells. We also found an inverse correlation between αE-catenin abundance and Yap1 activation in human squamous cell carcinoma tumors. These findings identify αE-catenin as a tumor suppressor that inhibits Yap1 activity and sequesters it in the cytoplasm.

This Podcast features conversations with authors of articles related to Hippo signaling that appear in Science and Science Signaling . James Martin discusses his group’s finding that Hippo signaling regulates the size of the mouse heart by antagonizing Wnt signaling through a mechanism that involves cooperation between the transcription factors Yap and β-catenin, as described in a Research Report published in the 22 April 2011 issue of Science . Valeri Vasioukhin and Mark Silvis discuss their findings related to Hippo signaling in tumorigenesis, as reported in the 24 May 2011 issue of Science Signaling . They report that the adhesion protein αE-catenin acts as a tumor suppressor by regulating the localization and activity of the transcriptional coactivator Yap, which is also regulated by Hippo signaling.