Hagit Turm

Protease-activated Receptor-1 (PAR1) Acts via a Novel Gα13-Dishevelled Axis to Stabilize β-Catenin Levels

We have previously shown a novel link between hPar-1 (human protease-activated receptor-1) and β-catenin stabilization. Although it is well recognized that Wnt signaling leads to β-catenin accumulation, the role of PAR1 in the process is unknown. We provide here evidence that PAR1 induces β-catenin stabilization independent of Wnt, Fz (Frizzled), and the co-receptor LRP5/6 (low density lipoprotein-related protein 5/6) and identify selective mediators of the PAR1-β-catenin axis. Immunohistological analyses of hPar1-transgenic (TG) mouse mammary tissues show the expression of both Gα12 and Gα13 compared with age-matched control counterparts. However, only Gα13 was found to be actively involved in PAR1-induced β-catenin stabilization. Indeed, a dominant negative form of Gα13 inhibited both PAR1-induced Matrigel invasion and Lef/Tcf (lymphoid enhancer factor/T cell factor) transcription activity. PAR1-Gα13 association is followed by the recruitment of DVL (Dishevelled), an upstream Wnt signaling protein via the DIX domain. Small interfering RNA-Dvl silencing leads to a reduction in PAR1-induced Matrigel invasion, inhibition of Lef/Tcf transcription activity, and decreased β-catenin accumulation. It is of note that PAR1 also promotes the binding of β-arrestin-2 to DVL, suggesting a role for β-arrestin-2 in PAR1-induced DVL phosphorylation dynamics. Although infection of small interfering RNA-LRP5/6 or the use of the Wnt antagonists, SFRP2 (soluble Frizzled-related protein 2) or SFRP5 potently reduced Wnt3A-mediated β-catenin accumulation, no effect was observed on PAR1-induced β-catenin stabilization. Collectively, our data show that PAR1 mediates β-catenin stabilization independent of Wnt. We propose here a novel cascade of PAR1-induced Gα13-DVL axis in cancer and β-catenin stabilization.

PAR1 plays a role in epithelial malignancies: transcriptional regulation and novel signaling pathway.

Protease‐activated receptor1 (PAR1) is the first and prototype member of an established PAR family comprising four members. The role of PAR1 in tumor biology has been established, and is characterized by a consistent direct correlation between overexpression of its levels and epithelial tumor aggressiveness. We have found that high expression of the human Par1 (hPar1) gene in epithelial tumors is controlled largely at the transcriptional level. This led us to assign Egr‐1, a transcription activator, as an inducer of hPar1, and p53, a tumor suppressor gene, as an inhibitor, both acting to achieve fine tuning of hPar1 in prostate carcinoma. High PAR1 levels maintain prosurvival signals in tumor cells while silencing or ablation of the gene induce apoptosis. Studies of our hPar1 transgenic mice, which overexpress hPar1 in the mammary glands, revealed a novel PAR1‐induced β‐catenin stabilization function. The components connecting PAR1 to β‐catenin stabilization have been determined, assigning at first Gα13 as a selective immediate component. The PAR1‐Gα13 axis recruits disheveled (DVL), an upstream signaling partner of the canonical Wnt signaling pathway. Silencing of DVL by siRNA‐DVL potently abrogates PAR1‐induced β‐catenin stabilization, demonstrating its critical role in the process. We, thus, propose that transcriptional regulation of hPar1 gene over expression in epithelia malignancies initiates a novel signaling pathway, directly connecting to β‐catenin stabilization, a core event in both tumorigenesis and developmental processes.

Etk/Bmx Regulates Proteinase-Activated-Receptor1 (PAR1) in Breast Cancer Invasion: Signaling Partners, Hierarchy and Physiological Significance

Background While protease-activated-receptor 1 (PAR1) plays a central role in tumor progression, little is known about the cell signaling involved. Methodology/Principal Findings We show here the impact of PAR1 cellular activities using both an orthotopic mouse mammary xenograft and a colorectal-liver metastasis model in vivo, with biochemical analyses in vitro. Large and highly vascularized tumors were generated by cells over-expressing wt hPar1, Y397Z hPar1, with persistent signaling, or Y381A hPar1 mutant constructs. In contrast, cells over-expressing the truncated form of hPar1, which lacks the cytoplasmic tail, developed small or no tumors, similar to cells expressing empty vector or control untreated cells. Antibody array membranes revealed essential hPar1 partners including Etk/Bmx and Shc. PAR1 activation induces Etk/Bmx and Shc binding to the receptor C-tail to form a complex. Y/A mutations in the PAR1 C-tail did not prevent Shc-PAR1 association, but enhanced the number of liver metastases compared with the already increased metastases obtained with wt hPar1. We found that Etk/Bmx first binds via the PH domain to a region of seven residues, located between C378-S384 in PAR1 C-tail, enabling subsequent Shc association. Importantly, expression of the hPar1-7A mutant form (substituted A, residues 378-384), which is incapable of binding Etk/Bmx, resulted in inhibition of invasion through Matrigel-coated membranes. Similarly, knocking down Etk/Bmx inhibited PAR1-induced MDA-MB-435 cell migration. In addition, intact spheroid morphogenesis of MCF10A cells is markedly disrupted by the ectopic expression of wt hPar1. In contrast, the forced expression of the hPar1-7A mutant results in normal ball-shaped spheroids. Thus, by preventing binding of Etk/Bmx to PAR1 -C-tail, hPar1 oncogenic properties are abrogated. Conclusions/Significance This is the first demonstration that a cytoplasmic portion of the PAR1 C-tail functions as a scaffold site. We identify here essential signaling partners, determine the hierarchy of binding and provide a platform for therapeutic vehicles via definition of the critical PAR1 -associating region in the breast cancer signaling niche.

A new in vivo model of pantothenate kinase-associated neurodegeneration reveals a surprising role for transcriptional regulation in pathogenesis

Pantothenate Kinase-Associated Neurodegeneration (PKAN) is a neurodegenerative disorder with a poorly understood molecular mechanism. It is caused by mutations in Pantothenate Kinase, the first enzyme in the Coenzyme A (CoA) biosynthetic pathway. Here, we developed a Drosophila model of PKAN (tim-fbl flies) that allows us to continuously monitor the modeled disease in the brain. In tim-fbl flies, downregulation of fumble, the Drosophila PanK homologue in the cells containing a circadian clock results in characteristic features of PKAN such as developmental lethality, hypersensitivity to oxidative stress, and diminished life span. Despite quasi-normal circadian transcriptional rhythms, tim-fbl flies display brain-specific aberrant circadian locomotor rhythms, and a unique transcriptional signature. Comparison with expression data from flies exposed to paraquat demonstrates that, as previously suggested, pathways others than oxidative stress are affected by PANK downregulation. Surprisingly we found a significant decrease in the expression of key components of the photoreceptor recycling pathways, which could lead to retinal degeneration, a hallmark of PKAN. Importantly, these defects are not accompanied by changes in structural components in eye genes suggesting that changes in gene expression in the eye precede and may cause the retinal degeneration. Indeed tim-fbl flies have diminished response to light transitions, and their altered day/night patterns of activity demonstrates defects in light perception. This suggest that retinal lesions are not solely due to oxidative stress and demonstrates a role for the transcriptional response to CoA deficiency underlying the defects observed in dPanK deficient flies. Moreover, in the present study we developed a new fly model that can be applied to other diseases and that allows the assessment of neurodegeneration in the brains of living flies.

Salient experiences are represented by unique transcriptional signatures in the mouse brain

It is well established that inducible transcription is essential for the consolidation of salient experiences into long-term memory. However, whether inducible transcription relays information about the identity and affective attributes of the experience being encoded, has not been explored. To this end, we analyzed transcription induced by a variety of rewarding and aversive experiences, across multiple brain regions. Our results describe the existence of robust transcriptional signatures uniquely representing distinct experiences, enabling near-perfect decoding of recent experiences. Furthermore, experiences with shared attributes display commonalities in their transcriptional signatures, exemplified in the representation of valence, habituation and reinforcement. This study introduces the concept of a neural transcriptional code, which represents the encoding of experiences in the mouse brain. This code is comprised of distinct transcriptional signatures that correlate to attributes of the experiences that are being committed to long-term memory.

Comprehensive Analysis of Transcription Dynamics from Brain Samples Following Behavioral Experience

The encoding of experiences in the brain and the consolidation of long-term memories depend on gene transcription. Identifying the function of specific genes in encoding experience is one of the main objectives of molecular neuroscience. Furthermore, the functional association of defined genes with specific behaviors has implications for understanding the basis of neuropsychiatric disorders. Induction of robust transcription programs has been observed in the brains of mice following various behavioral manipulations. While some genetic elements are utilized recurrently following different behavioral manipulations and in different brain nuclei, transcriptional programs are overall unique to the inducing stimuli and the structure in which they are studied(1,2). In this publication, a protocol is described for robust and comprehensive transcriptional profiling from brain nuclei of mice in response to behavioral manipulation. The protocol is demonstrated in the context of analysis of gene expression dynamics in the nucleus accumbens following acute cocaine experience. Subsequent to a defined in vivo experience, the target neural tissue is dissected; followed by RNA purification, reverse transcription and utilization of microfluidic arrays for comprehensive qPCR analysis of multiple target genes. This protocol is geared towards comprehensive analysis (addressing 50-500 genes) of limiting quantities of starting material, such as small brain samples or even single cells. The protocol is most advantageous for parallel analysis of multiple samples (e.g. single cells, dynamic analysis following pharmaceutical, viral or behavioral perturbations). However, the protocol could also serve for the characterization and quality assurance of samples prior to whole-genome studies by microarrays or RNAseq, as well as validation of data obtained from whole-genome studies.

DVL as a scaffold protein capturing classical GPCRs

The classical G-protein-coupled receptors (GPCRs) are characterized by their ability to interact with heterotrimeric G proteins upon activation and by structural features such as seven transmembrane spanning domains. Frizzleds (Fzs) are comparable seven transmembrane receptors (7 TMRs) that are activated via Wnts and play a critical role in embryogenesis, tissue hemostasis and oncogenicity. It remains controversial, however, whether they may be considered GPCRs. Hence, the ten members of Fzs constitute a distinct atypical family of seven-transmembrane receptors. Canonical Wnt/β-catenin signaling leads to the core process of β-catenin stabilization and, ultimately, to the translocation of β-catenin to the nucleus where it acts as a co-transcription factor and induces Wnt target gene transcription. We have documented that activation by proteinase-activated receptor1(PAR1), a classical 7TMR, recruits dishevelled (DVL), an upstream Wnt signaling protein, to mediate β-catenin stabilization. DVL is selectively bound to activated Gα13 subunit, coupled to PAR1 following activation. Formation of the PAR1-induced DVL-Gα13 axis is carried out independently of Wnt, Fz and the co-receptor LRP5/6 (low density lipoprotein - related protein 5/6) since neither siRNA-LRP5/6 co-receptors nor the presence of SFRPs; secreted Fz receptor proteins (Wnt antagonists) affect PAR1-induced β-catenin stabilization. Similarly, PAR1 induced placenta cytotrophoblast physiological invasion process was not affected by inhibiting Wnt, but was abrogated by siRNA-DVL. We propose that DVL serves as a central mediator protein that links classical GPCRs to β-catenin stabilization in both pathological (tumor) and physiological (placenta) invasion processes.

Low-density lipoprotein receptor-related protein 6 is a novel coreceptor of protease-activated receptor-2 in the dynamics of cancer-associated β-catenin stabilization

Protease-activated receptor-2 (PAR2) plays a central role in cancer; however, the molecular machinery of PAR2-instigated tumors remains to be elucidated. We show that PAR2 is a potent inducer of β-catenin stabilization, a core process in cancer biology, leading to its transcriptional activity. Novel association of low-density lipoprotein-related protein 6 (LRP6), a known coreceptor of Frizzleds (Fz), with PAR2 takes place following PAR2 activation. The association between PAR2 and LRP6 was demonstrated employing co-immunoprecipitation, bioluminescence resonance energy transfer (BRET), and confocal microscopy analysis. The association was further supported by ZDOCK protein-protein server. PAR2-LRP6 interaction promotes rapid phosphorylation of LRP6, which results in the recruitment of Axin. Confocal microscopy of PAR2-driven mammary gland tumors in vivo, as well as in vitro confirms the association between PAR2 and LRP6. Indeed, shRNA silencing of LRP6 potently inhibits PAR2-induced β-catenin stabilization, demonstrating its critical role in the induced path. We have previously shown a novel link between protease-activated receptor-1 (PAR1) and β-catenin stabilization, both in a transgenic (tg) mouse model with overexpression of human PAR1 (hPar1) in the mammary glands, and in cancer epithelial cell lines. Unlike in PAR1-Gα13 axis, both Gα12 and Gα13 are equally involved in PAR2-induced β-catenin stabilization. Disheveled (DVL) is translocated to the cell nucleus through the DVL-PDZ domain. Collectively, our data demonstrate a novel PAR2-LRP6-Axin interaction as a key axis of PAR2-induced β-catenin stabilization in cancer. This newly described axis enhances our understanding of cancer biology, and opens new avenues for future development of anti-cancer therapies.

PAR Genes: Molecular Probes to Pathological Assessment in Breast Cancer Progression

Taking the issue of tumor categorization a step forward and establish molecular imprints to accompany histopathological assessment is a challenging task. This is important since often patients with similar clinical and pathological tumors may respond differently to a given treatment. Protease-activated receptor-1 (PAR1), a G protein-coupled receptor (GPCR), is the first member of the mammalian PAR family consisting of four genes. PAR1 and PAR2 play a central role in breast cancer. The release of N-terminal peptides during activation and the exposure of a cryptic internal ligand in PARs, endow these receptors with the opportunity to serve as a “mirror-image” index reflecting the level of cell surface PAR1&2-in body fluids. It is possible to use the levels of PAR-released peptide in patients and accordingly determine the choice of treatment. We have both identified PAR1 C-tail as a scaffold site for the immobilization of signaling partners, and the critical minimal binding site. This binding region may be used for future therapeutic modalities in breast cancer, since abrogation of the binding inhibits PAR1 induced breast cancer. Altogether, both PAR1 and PAR2 may serve as molecular probes for breast cancer diagnosis and valuable targets for therapy.

The Role of Thrombin and its Receptors in Epithelial Malignancies: Lessons from a Transgenic Mouse Model and Transcriptional Regulation

The otherwise well-orchestrated epithelial sheets are disrupted when they acquire the ability to overexpress the prototype mammalian thrombin receptor, human protease-activated receptor-1 (hPar1). This is exhibited by down-regulation of cell–cell contacts and alterations in cell–matrix interactions. The notion that hPar1 is one of a series of genes that is part of a malignant program stems from studies indicating that hPar1 expression directly correlates with tumor metastasis and the time-limited physiological invasion of the placenta to the uterus decidua. Our transgenic mouse model of tissue-targeted hPar1 overexpression in the mammary glands exhibits a phenotype of hyperplasia, characterized by a dense network of ductal side branching and accelerated proliferation. The transgenic mammary glands exhibit increased levels of wnt-4 and -7b, and the striking stabilization of β-catenin. This novel association between hPar1 and nuclear β-catenin may provide a key determinant in the molecular pathway of hPar1 oncogenicity. While studying the properties of hPar1 in tumor biology we demonstrated its role as a survival factor that protects cells from undergoing apoptosis. Withdrawal of the hPar1 gene leads to selective apoptosis especially in young sprouting blood vessels, whereas mature vessels remain unaffected. We also provide evidence showing that hPar1 gene overexpression in tumors stems from enhanced transcriptional activity. This is evaluated on the basis of elicited run-on transcription rate in highly metastatic vs. low metastatic cells (on a background of equal stability rates). Indeed, we have shown that the transcription factor Egr-1 induced hPar1 gene overexpression in prostate cancer. In addition, the tumor suppressor gene p53 also acts on hPar1 as one of its target genes, regulating its level of expression in the context of a given tumor. It still remains to determine the profile of individual fingerprints and specific motifs that bind to a panel of transcription factors, as well as tumor suppressor genes which are critically involved in altering hPar1 transcription levels according to the type of tumor.