Protease-activated receptor 2: a promising therapeutic target for women's cancers.

Cancers affecting women, such as breast, uterine, ovarian, endometrial and cervical cancers, have become increasingly prevalent. The growing incidence and death rates associated with these cancers warrant the development of innovative and alternative approaches to current treatments. This article investigates the association of women's cancers with a molecular target known as protease-activated receptor 2 (PAR2), a G-protein coupled receptor that is expressed on the surface of cancer cells. Expression levels of the PAR2 gene were curated from publicly available databases and were found to be significantly overexpressed in tissues from patients with breast, uterine, ovarian, endometrial or cervical cancer compared to normal tissues. PAR2 overexpression has been previously linked to tumor progression and, in some cases, tumor growth. Activation of PAR2 by either endogenous proteases or synthetic agonists triggers certain downstream intracellular signaling pathways that have been associated with tumor progression, cell migration and invasion, angiogenesis and apoptosis of cancer cells. While recent advances have led to the identification of several PAR2 antagonists, none has yet been developed for human use. Additionally, PAR2 inhibition has been shown also to increase the efficacy of chemotherapeutic drugs, allowing them to be potentially used at less toxic doses in combination therapies for cancer. The present work briefly summarizes the current status of PAR2 as a potential therapeutic target for treating women's cancers. Significance Statement This article highlights potential roles for PAR2 in cancers affecting women. Overexpression of the PAR2 gene in women's cancers is associated with various oncogenic processes such as tumor progression, cell migration and invasion, ultimately contributing to poorer patient prognoses. Given the increasing incidence of women's cancers, there is an urgent need to develop novel therapeutic drugs and PAR2 represents a promising target for developing new treatments.

Tuning Electrostatic and Hydrophobic Surfaces of Aromatic Rings to Enhance Membrane Association and Cell Uptake of Peptides.

Aromatic groups are key mediators of protein-membrane association at cell surfaces, contributing to hydrophobic effects and π-membrane interactions. Here we show electrostatic and hydrophobic influences of aromatic ring substituents on membrane affinity and cell uptake of helical, cyclic and cell penetrating peptides. Hydrophobicity is important, but subtle changes in electrostatic surface potential, dipoles and polarizability also enhance association with phospholipid membranes and cell uptake. A combination of fluorine and sulfur substituents on an aromatic ring induces microdipoles that enhance cell uptake of 12-residue peptide inhibitors of p53-HDM2 interaction and of cell-penetrating cyclic peptides. These aromatic motifs can be readily inserted into peptide sidechains to enhance their cell uptake.

A Potent Antagonist of Protease-Activated Receptor 2 That Inhibits Multiple Signaling Functions in Human Cancer Cells.

Protease-activated receptor 2 (PAR2) is a cell surface protein linked to G-protein dependent and independent intracellular signaling pathways that produce a wide range of physiological responses, including those related to metabolism, inflammation, pain, and cancer. Certain proteases, peptides, and nonpeptides are known to potently activate PAR2. However, no effective potent PAR2 antagonists have been reported yet despite their anticipated therapeutic potential. This study investigates antagonism of key PAR2-dependent signaling properties and functions by the imidazopyridazine compound I-191 (4-(8-(tert-butyl)-6-(4-fluorophenyl)imidazo[1,2-b]pyridazine-2-carbonyl)-3,3-dimethylpiperazin-2-one) in cancer cells. At nanomolar concentrations, I-191 inhibited PAR2 binding of and activation by structurally distinct PAR2 agonists (trypsin, peptide, nonpeptide) in a concentration-dependent manner in cells of the human colon adenocarcinoma grade II cell line (HT29). I-191 potently attenuated multiple PAR2-mediated intracellular signaling pathways leading to Ca2+ release, extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation, Ras homologue gene family, member A (RhoA) activation, and inhibition of forskolin-induced cAMP accumulation. The mechanism of action of I-191 was investigated using binding and calcium mobilization studies in HT29 cells where I-191 was shown to be noncompetitive and a negative allosteric modulator of the agonist 2f-LIGRL-NH2 The compound alone did not activate these PAR2-mediated pathways, even at high micromolar concentrations, indicating no bias in these signaling properties. I-191 also potently inhibited PAR2-mediated downstream functional responses, including expression and secretion of inflammatory cytokines and cell apoptosis and migration, in human colon adenocarcinoma grade II cell line (HT29) and human breast adenocarcinoma cells (MDA-MB-231). These findings indicate that I-191 is a potent PAR2 antagonist that inhibits multiple PAR2-induced signaling pathways and functional responses. I-191 may be a valuable tool for characterizing PAR2 functions in cancer and in other cellular, physiological, and disease settings.

Europium-Labeled Synthetic C3a Protein as a Novel Fluorescent Probe for Human Complement C3a Receptor.

Measuring ligand affinity for a G protein-coupled receptor is often a crucial step in drug discovery. It has been traditionally determined by binding putative new ligands in competition with native ligand labeled with a radioisotope of finite lifetime. Competing instead with a lanthanide-based fluorescent ligand is more attractive due to greater longevity, stability, and safety. Here, we have chemically synthesized the 77 residue human C3a protein and conjugated its N-terminus to europium diethylenetriaminepentaacetate to produce a novel fluorescent protein (Eu-DTPA-hC3a). Time-resolved fluorescence analysis has demonstrated that Eu-DTPA-hC3a binds selectively to its cognate G protein-coupled receptor C3aR with full agonist activity and similar potency and selectivity as native C3a in inducing calcium mobilization and phosphorylation of extracellular signal-regulated kinases in HEK293 cells that stably expressed C3aR. Time-resolved fluorescence analysis for saturation and competitive binding gave a dissociation constant (Kd) of 8.7 ± 1.4 nM for Eu-DTPA-hC3a and binding affinities for hC3a (pKi of 8.6 ± 0.2 and Ki of 2.5 nM) and C3aR ligands TR16 (pKi of 6.8 ± 0.1 and Ki of 138 nM), BR103 (pKi of 6.7 ± 0.1 and Ki of 185 nM), BR111 (pKi of 6.3 ± 0.2 and Ki of 544 nM) and SB290157 (pKi of 6.3 ± 0.1 and Ki of 517 nM) via displacement of Eu-DTPA-hC3a from hC3aR. The macromolecular conjugate Eu-DTPA-hC3a is a novel nonradioactive probe suitable for studying ligand-C3aR interactions with potential value in accelerating drug development for human C3aR in physiology and disease.

Benzylamide antagonists of protease activated receptor 2 with anti-inflammatory activity.

Activation of protease activated receptor 2 (PAR2) has been implicated in inflammatory and metabolic disorders and its inhibition may yield novel therapeutics. Here, we report a series of PAR2 antagonists based on C-terminal capping of 5-isoxazolyl-L-cyclohexylalanine-L-isoleucine, with benzylamine analogues being effective new PAR2 antagonists. 5-Isoxazolyl-L-cyclohexylalanine-L-isoleucine-2-methoxybenzylamine (10) inhibited PAR2-, but not PAR1-, induced release of Ca(2+) (IC50 0.5 µM) in human colon cells, IL-6 and TNFα secretion (IC50 1-5 µM) from human kidney cells, and was anti-inflammatory in acute rat paw inflammation (ED50 5 mg/kg sc). These findings show that new benzylamide antagonists of PAR2 have anti-inflammatory activity.

Repurposing Registered Drugs as Antagonists for Protease-Activated Receptor 2.

Virtual screening of a drug database identified Carvedilol, Loratadine, Nefazodone and Astemizole as PAR2 antagonists, after ligand docking and molecular dynamics simulations using a PAR2 homology model and a putative binding mode of a known PAR2 ligand. The drugs demonstrated competitive binding and antagonism of calcium mobilization and ERK1/2 phosphorylation in CHO-hPAR2 transfected cells, while inhibiting IL-6 secretion in PAR2 expressing MDA-MB-231 breast cancer cells. This research highlights opportunities for GPCR hit-finding from FDA-approved drugs.

Three Homology Models of PAR2 Derived from Different Templates: Application to Antagonist Discovery.

Protease activated receptor 2 (PAR2) is an unusual G-protein coupled receptor (GPCR) involved in inflammation and metabolism. It is activated through cleavage of its N-terminus by proteases. The new N-terminus functions as a tethered ligand that folds back and intramolecularly activates PAR2, initiating multiple downstream signaling pathways. The only compounds reported to date to inhibit PAR2 activation are of moderate potency. Three structural models for PAR2 have been constructed based on sequence homology with known crystal structures for bovine rhodopsin, human ORL-1 (also called nociceptin/orphanin FQ receptor), and human PAR1. The three PAR2 model structures were compared and used to predict potential interactions with ligands. Virtual screening for ligands using the Chembridge database, and either ORL-1 or PAR1 derived PAR2 models led to identification of eight new small molecule PAR2 antagonists (IC50 10-100 µM). Notably, the most potent compound 1 (IC50 11 µM) was derived from the less homologous template protein, human ORL-1. The results suggest that virtual screening against multiple homology models of the same GPCR can produce structurally diverse antagonists and that this may be desirable even when some models have less sequence homology with the target protein.

Inflammatory responses induced by lipopolysaccharide are amplified in primary human monocytes but suppressed in macrophages by complement protein C5a.

Monocytes and macrophages are important innate immune cells equipped with danger-sensing receptors, including complement and Toll-like receptors. Complement protein C5a, acting via C5aR, is shown in this study to differentially modulate LPS-induced inflammatory responses in primary human monocytes versus macrophages. Whereas C5a enhanced secretion of LPS-induced IL-6 and TNF from primary human monocytes, C5a inhibited these responses while increasing IL-10 secretion in donor-matched human monocyte-derived macrophages differentiated by GM-CSF or M-CSF. Gαi/c-Raf/MEK/ERK signaling induced by C5a was amplified in macrophages but not in monocytes by LPS. Accordingly, the Gαi inhibitor pertussis toxin and MEK inhibitor U0126 blocked C5a inhibition of LPS-induced IL-6 and TNF production from macrophages. This synergy was independent of IL-10, PI3K, p38, JNK, and the differentiating agent. Furthermore, C5a did not inhibit IL-6 production from macrophages induced by other TLR agonists that are selective for Toll/IL-1R domain-containing adapter inducing IFN-ß (polyinosinic-polycytidylic acid) or MyD88 (imiquimod), demonstrating selectivity for C5a regulation of LPS responses. Finally, suppression of proinflammatory cytokines IL-6 and TNF in macrophages did not compromise antimicrobial activity; instead, C5a enhanced clearance of the Gram-negative bacterial pathogen Salmonella enterica serovar Typhimurium from macrophages. C5aR is thus a regulatory switch that modulates TLR4 signaling via the Gαi/c-Raf/MEK/ERK signaling axis in human macrophages but not monocytes. The differential effects of C5a are consistent with amplifying monocyte proinflammatory responses to systemic danger signals, but attenuating macrophage cytokine responses (without compromising microbicidal activity), thereby restraining inflammatory responses to localized infections.

Stereoelectronic effects dictate molecular conformation and biological function of heterocyclic amides.

Heterocycles adjacent to amides can have important influences on molecular conformation due to stereoelectronic effects exerted by the heteroatom. This was shown for imidazole- and thiazole-amides by comparing low energy conformations (ab initio MP2 and DFT calculations), charge distribution, dipole moments, and known crystal structures which support a general principle. Switching a heteroatom from nitrogen to sulfur altered the amide conformation, producing different three-dimensional electrostatic surfaces. Differences were attributed to different dipole and orbital alignments and spectacularly translated into opposing agonist vs antagonist functions in modulating a G-protein coupled receptor for inflammatory protein complement C3a on human macrophages. Influences of the heteroatom were confirmed by locking the amide conformation using fused bicyclic rings. These findings show that stereoelectronic effects of heterocycles modulate molecular conformation and can impart strikingly different biological properties.

C5aR and C3aR antagonists each inhibit diet-induced obesity, metabolic dysfunction, and adipocyte and macrophage signaling.

Mammalian survival depends on metabolizing nutrients, storing energy, and combating infection. Complement activation in blood triggers energy-depleting immune responses to fight infections. Here we identify surprising energy-conserving roles for complement proteins C5a and C3a and their receptors, C5aR and C3aR, roles that are contraindicated in complement biology. Rats fed a high-carbohydrate high-fat diet developed obesity, visceral adiposity, adipose inflammation, glucose/insulin intolerance, and cardiovascular dysfunction that correlated with increased plasma C3a, adipose C5aR, and C3aR. These in vivo changes were dramatically attenuated by receptor-selective antagonists of either C5aR (5 mg/kg/d p.o.) or C3aR (30 mg/kg/d p.o.), which both reduced proinflammatory adipokines and altered expression of inflammatory genes in adipose tissue. In vitro C5a and C3a (100 nM) exhibited novel insulin-like effects on 3T3-L1 adipocytes, promoting energy conservation by increasing glucose and fatty acid uptake while inhibiting cAMP signaling and lipolysis, and induced PGE(2) release from macrophages, effects all blocked by each respective antagonist (10 µM). These studies reveal important new links between complement signaling and metabolism, highlight new complement functions on adipocytes and in adipose tissue, demonstrate how aberrant immune responses may exacerbate obesity and metabolic dysfunction, and show that targeting C3aR or C5aR with antagonists is a new strategy for treating metabolic dysfunction.

Diet-induced obesity, adipose inflammation, and metabolic dysfunction correlating with PAR2 expression are attenuated by PAR2 antagonism.

Excessive uptake of fatty acids and glucose by adipose tissue triggers adipocyte dysfunction and infiltration of immune cells. Altered metabolic homeostasis in adipose tissue promotes insulin resistance, type 2 diabetes, hypertension, and cardiovascular disease. Inflammatory and metabolic processes are mediated by certain proteolytic enzymes that share a common cellular target, protease-activated receptor 2 (PAR2). This study showed that human and rat obesity correlated in vivo with increased expression of PAR2 in adipose tissue, primarily in stromal vascular cells (SVCs) including macrophages. PAR2 was expressed more than other PARs on human macrophages and was increased by dietary fatty acids (palmitic, stearic, and myristic). A novel PAR2 antagonist, GB88 (5-isoxazoyl-Cha-Ile-spiroindene-1,4-piperidine), given orally at 10 mg/kg/d (wk 8-16) reduced body weight by ∼10% in obese rats fed a high-carbohydrate high-fat (HCHF) diet for 16 wk, and strongly attenuated adiposity, adipose tissue inflammation, infiltrated macrophages and mast cells, insulin resistance, and cardiac fibrosis and remodeling; while reversing liver and pancreatic dysfunction and normalizing secretion of PAR2-directed glucose-stimulated insulin secretion in MIN6 ß cells. In summary, PAR2 is a new biomarker for obesity, and its expression is stimulated by dietary fatty acids; PAR2 is a substantial contributor to inflammatory and metabolic dysfunction; and a PAR2 antagonist inhibits diet-induced obesity and inflammatory, metabolic, and cardiovascular dysfunction.

Protease-activated receptor 2 attenuates doxorubicin-induced apoptosis in colon cancer cells.

Drug resistance represents a major problem in cancer treatment. Doxorubicin (adriamycin) is an injectable DNA intercalating drug that halts cancer cell growth by inhibiting topoisomerase 2, but its long-term effectiveness is compromised by onset of resistance. This study demonstrates that expression of the PAR2 gene in human colon adenocarcinoma tissue samples was the highest among 32 different cancer types (n = 10,989), and higher in colon adenocarcinoma tissues (n = 331) than normal colon tissues (n = 308), revealing an association between PAR2 expression and human colon cancer. HT29 cells are a human colorectal adenocarcinoma cell line that is sensitive to the chemotherapeutic drug doxorubicin and also expresses PAR2. We find that PAR2 activation in HT29 cells, either by an endogenous protease agonist (trypsin) or an exogenous peptide agonist (2f-LIGRL-NH2), significantly reduces doxorubicin-induced cell death, reactive oxygen species production, caspase 3/7 activity and cleavage of caspase-8 and caspase-3. Moreover, PAR2-mediated MEK1/2-ERK1/2 pathway induced by 2f-LIGRL-NH2 leads to upregulated anti-apoptotic MCL-1 and Bcl-xL proteins that promote cellular survival. These findings suggest that activation of PAR2 compromises efficacy of doxorubicin in colon cancer. Further support for this conclusion came from experiments with human colon cancer HT29 cells, either with the PAR2 gene deleted or in the presence of a pharmacological antagonist of PAR2, which showed full restoration of all doxorubicin-mediated effects. Together, these findings reveal a strong link between PAR2 activation and signalling in human colon cancer cells and increased survival against doxorubicin-induced cell death. They support PAR2 antagonism as a possible new strategy for enhancing doxorubicin therapy.

Ras-Related Protein Rab5a Regulates Complement C5a Receptor Trafficking, Chemotaxis, and Chemokine Secretion in Human Macrophages.

Complement activation and Rab GTPase trafficking are commonly observed in inflammatory responses. Recruitment of innate immune cells to sites of infection or injury and secretion of inflammatory chemokines are promoted by complement component 5a (C5a) that activates the cell surface protein C5a receptor1 (C5aR1). Persistent activation can lead to a myriad of inflammatory and autoimmune diseases. Here, we demonstrate that the mechanism of C5a induced chemotaxis of human monocyte-derived macrophages (HMDMs) and their secretion of inflammatory chemokines are controlled by Rab5a. We find that C5a activation of the G protein coupled receptor C5aR1 expressed on the surface of HMDMs, recruits ß-arrestin2 via Rab5a trafficking, then activates downstream phosphatidylinositol 3-kinase (PI3K)/Akt signaling that culminates in chemotaxis and secretion of pro-inflammatory chemokines from HMDMs. High-resolution lattice light-sheet microscopy on live cells showed that C5a activates C5aR1-GFP internalization and colocalization with Rab5a-tdTomato but not with dominant negative mutant Rab5a-S34N-tdTomato in HEK293 cells. We found that Rab5a is significantly upregulated in differentiated HMDMs and internalization of C5aR1 is dependent on Rab5a. Interestingly, while knockdown of Rab5a inhibited C5aR1-mediated Akt phosphorylation, it did not affect C5aR1-mediated ERK1/2 phosphorylation or intracellular calcium mobilization in HMDMs. Functional analysis using transwell migration and µ-slide chemotaxis assays indicated that Rab5a regulates C5a-induced chemotaxis of HMDMs. Further, C5aR1 was found to mediate interaction of Rab5a with ß-arrestin2 but not with G proteins in HMDMs. Furthermore, C5a-induced secretion of pro-inflammatory chemokines (CCL2, CCL3) from HMDMs was attenuated by Rab5a or ß-arrestin2 knockdown or by pharmacological inhibition with a C5aR1 antagonist or a PI3K inhibitor. These findings reveal a C5a-C5aR1-ß-arrestin2-Rab5a-PI3K signaling pathway that regulates chemotaxis and pro-inflammatory chemokine secretion in HMDMs and suggests new ways of selectively modulating C5a-induced inflammatory outputs.

Venom composition and bioactive RF-amide peptide toxins of the saddleback caterpillar, Acharia stimulea (Lepidoptera: Limacodidae).

Limacodidae is a family of lepidopteran insects comprising >1500 species. More than half of these species produce pain-inducing defensive venoms in the larval stage, but little is known about their venom toxins. Recently, we characterised proteinaceous toxins from the Australian limacodid caterpillar Doratifera vulnerans, but it is unknown if the venom of this species is typical of other Limacodidae. Here, we use single animal transcriptomics and venom proteomics to investigate the venom of an iconic limacodid, the North American saddleback caterpillar Acharia stimulea. We identified 65 venom polypeptides, grouped into 31 different families. Neurohormones, knottins, and homologues of the immune signaller Diedel make up the majority of A.stimulea venom, indicating strong similarities to D. vulnerans venom, despite the large geographic separation of these caterpillars. One notable difference is the presence of RF-amide peptide toxins in A. stimulea venom. Synthetic versions of one of these RF-amide toxins potently activated the human neuropeptide FF1 receptor, displayed insecticidal activity when injected into Drosophila melanogaster, and moderately inhibited larval development of the parasitic nematode Haemonchus contortus. This study provides insights into the evolution and activity of venom toxins in Limacodidae, and provides a platform for future structure-function characterisation of A.stimulea peptide toxins.

Landscaping macrocyclic peptides: stapling hDM2-binding peptides for helicity, protein affinity, proteolytic stability and cell uptake.

Cyclic peptides that modulate protein-protein interactions can be valuable therapeutic candidates if they can be delivered intact to their target proteins in cells. Here we systematically compare the effects of different helix-inducing cyclization constraints on the capacity of a macrocyclic peptide component to confer α-helicity, protein-binding affinity, resistance to degradative proteases and cell uptake to a 12-residue peptide fragment of tumor suppressor protein p53. We varied the helix-inducing constraint (hydrocarbon, lactam, aliphatic or aromatic thioether, etc.) and the position of the cyclization linker (i to i + 4 or i to i + 7 bridges) in order to sculpt the macrocyclic size, stabilize its structure, and promote cell uptake. We find that rigidifying the macrocycle leads to higher alpha helicity, target affinity and proteolytic stability to different extents, whereas cell uptake of compounds shown here is mostly driven by hydrophobicity and aromaticity of the macrocycle.

Helical structure in cyclic peptides: effect of N-methyl amides versus esters.

An alpha helical turn can be reproduced in a cyclic pentapeptide if the first and fifth amino acid sidechains are correctly joined. Here structural studies (CD, NMR, in silico) reveal why N-methylation at positions not involved in hydrogen bonds disrupts helicity whereas ester bonds can maintain helicity and promote greater cell uptake.

The histone deacetylase Hdac7 supports LPS-inducible glycolysis and Il-1ß production in murine macrophages via distinct mechanisms.

TLRs reprogram macrophage metabolism, enhancing glycolysis and promoting flux through the tricarboxylic acid cycle to enable histone acetylation and inflammatory gene expression. The histone deacetylase (HDAC) family of lysine deacetylases regulates both TLR-inducible glycolysis and inflammatory responses. Here, we show that the TLR4 agonist LPS, as well as agonists of other TLRs, rapidly increase enzymatic activity of the class IIa HDAC family (HDAC4, 5, 7, 9) in both primary human and murine macrophages. This response was abrogated in murine macrophages deficient in histone deacetylase 7 (Hdac7), highlighting a selective role for this specific lysine deacetylase during immediate macrophage activation. With the exception of the TLR3 agonist polyI:C, TLR-inducible activation of Hdac7 enzymatic activity required the MyD88 adaptor protein. The rapid glycolysis response, as assessed by extracellular acidification rate, was attenuated in Hdac7-deficient mouse macrophages responding to submaximal LPS concentrations. Surprisingly however, reconstitution of these cells with either wild-type or an enzyme-dead mutant of Hdac7 enhanced LPS-inducible glycolysis, whereas only the former promoted production of the inflammatory mediators Il-1ß and Ccl2. Thus, Hdac7 enzymatic activity is required for TLR-inducible production of specific inflammatory mediators, whereas it acts in an enzyme-independent fashion to reprogram metabolism in macrophages responding to submaximal LPS concentrations. Hdac7 is thus a bifurcation point for regulated metabolism and inflammatory responses in macrophages. Taken together with existing literature, our findings support a model in which submaximal and maximal activation of macrophages via TLR4 instruct glycolysis through distinct mechanisms, leading to divergent biological responses.

Modifying a Hydroxyl Patch in Glucagon-like Peptide 1 Produces Biased Agonists with Unique Signaling Profiles.

Glucagon-like peptide-1 (GLP-1) lowers blood glucose by inducing insulin but also has other poorly understood properties. Here, we show that hydroxy amino acids (Thr11, Ser14, Ser17, Ser18) in GLP-1(7-36) act in concert to direct cell signaling. Mutating any single residue to alanine removes one hydroxyl group, thereby reducing receptor affinity and cAMP 10-fold, with Ala11 or Ala14 also reducing ß-arrestin-2 10-fold, while Ala17 or Ala18 also increases ERK1/2 phosphorylation 5-fold. Multiple alanine mutations more profoundly bias signaling, differentially silencing or restoring one or more signaling properties. Mutating three serines silences only ERK1/2, the first example of such bias. Mutating all four residues silences ß-arrestin-2, ERK1/2, and Ca2+ maintains the ligand and receptor at the membrane but still potently stimulates cAMP and insulin secretion in cells and mice. These novel findings indicate that hydrogen bonding cooperatively controls cell signaling and highlight an important regulatory hydroxyl patch in hormones that activate class B G protein-coupled receptors.

Temporal perturbation of histone deacetylase activity reveals a requirement for HDAC1-3 in mesendoderm cell differentiation.

Histone deacetylases (HDACs) are a class of enzymes that control chromatin state and influence cell fate. We evaluated the chromatin accessibility and transcriptome dynamics of zinc-containing HDACs during cell differentiation in vitro coupled with chemical perturbation to identify the role of HDACs in mesendoderm cell fate specification. Single-cell RNA sequencing analyses of HDAC expression during human pluripotent stem cell (hPSC) differentiation in vitro and mouse gastrulation in vivo reveal a unique association of HDAC1 and -3 with mesendoderm gene programs during exit from pluripotency. Functional perturbation with small molecules reveals that inhibition of HDAC1 and -3, but not HDAC2, induces mesoderm while impeding endoderm and early cardiac progenitor specification. These data identify unique biological functions of the structurally homologous enzymes HDAC1-3 in influencing hPSC differentiation from pluripotency toward mesendodermal and cardiac progenitor populations.

PAR2 induces ovarian cancer cell motility by merging three signalling pathways to transactivate EGFR.

BACKGROUND AND PURPOSE: Specific cellular functions mediated by GPCRs are often associated with signalling through a particular G protein or ß-arrestin. Here, we examine signalling through a GPCR, protease-activated receptor 2 (PAR2), in a high-grade serous ovarian cancer cell line (OV90). EXPERIMENTAL APPROACH: Human ovarian cancer tissues (n = 1,200) and nine human ovarian cancer cell lines were assessed for PAR2 expression. PAR2 signalling mechanisms leading to cell migration and invasion were dissected using cellular assays, western blots, CRISPR-Cas9 gene knockouts, pharmacological inhibitors of PAR2 and downstream signalling proteins in OV90 cancer cells. KEY RESULTS: PAR2 was significantly overexpressed in clinical ovarian cancer tissues and in OV90 ovarian cancer cells. PAR2 agonists, an endogenous protease (trypsin) and a synthetic peptide (2f-LIGRL-NH2 ), induced migration and invasion of OV90 ovarian cancer cells through activating a combination of Gαq/11 , Gα12/13 and ß-arrestin1/2, but not Gαs or Gαi . This novel cooperative rather than parallel signalling resulted in downstream serial activation of Src kinases, then transactivation of epidermal growth factor receptor (EGFR), followed by downstream MEK-ERK1/2-FOS/MYC/STAT3-COX2 signalling. Either a PAR2 antagonist (I-191), CRISPR-Cas9 gene knockouts (PAR2 or Gα proteins or ß-arrestin1/2), or inhibitors of each downstream protein attenuated human ovarian cancer cell motility. CONCLUSION AND IMPLICATIONS: This study highlights a novel shared signalling cascade, requiring each of Gαq/11 , Gα12/13 and ß-arrestin1/2 for PAR2-induced ovarian cancer cell migration and invasion. This mechanism controlling a cellular function is unusual in not being linked to a specific individual G protein or ß-arrestin-mediated signalling pathway.