Cytoneme signaling provides essential contributions to mammalian tissue patterning.

During development, morphogens pattern tissues by instructing cell fate across long distances. Directly visualizing morphogen transport in situ has been inaccessible, so the molecular mechanisms ensuring successful morphogen delivery remain unclear. To tackle this longstanding problem, we developed a mouse model for compromised sonic hedgehog (SHH) morphogen delivery and discovered that endocytic recycling promotes SHH loading into signaling filopodia called cytonemes. We optimized methods to preserve in vivo cytonemes for advanced microscopy and show endogenous SHH localized to cytonemes in developing mouse neural tubes. Depletion of SHH from neural tube cytonemes alters neuronal cell fates and compromises neurodevelopment. Mutation of the filopodial motor myosin 10 (MYO10) reduces cytoneme length and density, which corrupts neuronal signaling activity of both SHH and WNT. Combined, these results demonstrate that cytoneme-based signal transport provides essential contributions to morphogen dispersion during mammalian tissue development and suggest MYO10 is a key regulator of cytoneme function.

Rational design, synthesis, and structure-activity relationships of 5-amino-1H-pyrazole-4-carboxylic acid derivatives as protein tyrosine phosphatase 1B inhibitors.

A series of novel amino-carboxylic based pyrazole as protein tyrosine phosphatase 1B (PTP1B) inhibitors were designed on the basis of structure-based pharmacophore model and molecular docking. Compounds containing different hydrophobic tail (1,2-diphenyl ethanone, oxdiadizole and dibenzyl amines) were synthesized and evaluated in PTP1B enzymatic assay. Structure-activity relationship based optimization resulted in identification of several potent, metabolically stable and cell permeable PTP1B inhibitors.

Modifications of flexible nonyl chain and nucleobase head group of (+)-erythro-9-(2's-hydroxy-3's-nonyl)adenine [(+)-EHNA] as adenosine deaminase inhibitors.

A series of terminal nonyl chain and nucleobase modified analogues of (+)-EHNA (III) were synthesized and evaluated for their ability to inhibit adenosine deaminase (ADA). The constrained carbon analogues of (+)-EHNA, 7a-7h, 10a-c, 12, 13, 14 and 17a-c appeared very potent with Ki values in the low nanomolar range. Thio-analogues of (+)-EHNA 24a-e wherein 5'C of nonyl chain replaced by sulfur atom found to be less potent compared to (+)-EHNA. Docking of the representative compounds into the active site of ADA was performed to understand structure-activity relationships. Compounds 7a (Ki: 1.1nM) 7b (Ki: 5.2nM) and 26a (Ki: 5.9nM) showed suitable balance of potency, microsomal stability and demonstrated better pharmacokinetic properties as compared to (+)-EHNA and therefore may have therapeutic potential for various inflammatory diseases, hypertension and cancer.

Sonic Hedgehog activates prostaglandin signaling to stabilize primary cilium length.

Sonic Hedgehog (SHH) is a driver of embryonic patterning that, when corrupted, triggers developmental disorders and cancers. SHH effector responses are organized through primary cilia (PC) that grow and retract with the cell cycle and in response to extracellular cues. Disruption of PC homeostasis corrupts SHH regulation, placing significant pressure on the pathway to maintain ciliary fitness. Mechanisms by which ciliary robustness is ensured in SHH-stimulated cells are not yet known. Herein, we reveal a crosstalk circuit induced by SHH activation of Phospholipase A2α that drives ciliary E-type prostanoid receptor 4 (EP4) signaling to ensure PC function and stabilize ciliary length. We demonstrate that blockade of SHH-EP4 crosstalk destabilizes PC cyclic AMP (cAMP) equilibrium, slows ciliary transport, reduces ciliary length, and attenuates SHH pathway induction. Accordingly, Ep4-/- mice display shortened neuroepithelial PC and altered SHH-dependent neuronal cell fate specification. Thus, SHH initiates coordination between distinct ciliary receptors to maintain PC function and length homeostasis for robust downstream signaling.

Discovery of novel and potent heterocyclic carboxylic acid derivatives as protein tyrosine phosphatase 1B inhibitors.

A series of novel heterocyclic carboxylic acid based protein tyrosine phosphatase 1B (PTP1B) inhibitors with hydrophobic tail have been synthesized and characterized. Structure-activity relationship (SAR) optimization resulted in identification of several potent, selective (over the highly homologous T-cell protein tyrosine phosphatase, TCPTP) and metabolically stable PTP1B inhibitors. Compounds 7a, 19a and 19c showed favorable cell permeability and pharmacokinetic properties in mouse with moderate to very good oral (% F=13-70) bio-availability.

Optineurin downregulation induces endoplasmic reticulum stress, chaperone-mediated autophagy, and apoptosis in pancreatic cancer cells.

Pancreatic ductal adenocarcinoma (PDAC) shows a high level of basal autophagy. Here we investigated the role of optineurin (OPTN) in PDAC cell lines, which is a prominent member of the autophagy system. To that purpose, mining of publically available databases showed that OPTN is highly expressed in PDAC and that high levels of expression are related to reduced survival. Therefore, the role of OPTN on proliferation, migration, and colony formation was investigated by transient knockdown in Miapaca, BXPC3, and Suit2-007 human PDAC cells. Furthermore, gene expression modulation in response to OPTN knockdown was assessed by microarray. The influence on cell cycle distribution and cell death signaling cascades was followed by FACS, assays for apoptosis, RT-PCR, and western blot. Finally, autophagy and ROS induction were screened by acridine orange and DCFH-DA fluorescent staining respectively. OPTN knockdown caused significant inhibition of colony formation, increased migration and no significant effect on proliferation in Miapaca, BXPC3 and Suit2-007 cells. The microarray showed modulation of 293 genes in Miapaca versus 302 in Suit2-007 cells, of which 52 genes overlapped. Activated common pathways included the ER stress response and chaperone-mediated autophagy, which was confirmed at mRNA and protein levels. Apoptosis was activated as shown by increased levels of cleaved PARP, Annexin V binding and nuclear fragmentation. OPTN knockdown caused no increased vacuole formation as assessed by acridine orange. Also, there was only marginally increased ROS production. Combination of OPTN knockdown with the autophagy inducer erufosine or LY294002, an inhibitor of autophagy, showed additive effects, which led us to hypothesize that they address different pathways. In conclusion, OPTN knockdown was related to activation of ER stress response and chaperone-mediated autophagy, which tend to confine the damage caused by OPTN knockdown and thus question its value for PDAC therapy.

Upregulation of cell cycle genes in head and neck cancer patients may be antagonized by erufosine's down regulation of cell cycle processes in OSCC cells.

The TCGA database was analyzed to identify deregulation of cell cycle genes across 24 cancer types and ensuing effects on patient survival. Pan-cancer analysis showed that head and neck squamous cell carcinoma (HNSCC) ranks amongst the top four cancers showing deregulated cell cycle genes. Also, the median gene expression of all CDKs and cyclins in HNSCC patient samples was higher than that of the global gene expression. This was verified by IHC staining of CCND1 from HNSCC patients. When evaluating the quartiles with highest and lowest expression, increased CCND1/CDK6 levels had negative implication on patient survival. In search for a drug, which may antagonize this tumor profile, the potential of the alkylphosphocholine erufosine was evaluated against cell lines of the HNSCC subtype, oral squamous cell carcinoma (OSCC) using in-vitro and in-vivo assays. Erufosine inhibited growth of OSCC cell lines concentration dependently. Initial microarray findings revealed that cyclins and CDKs were down-regulated concentration dependently upon exposure to erufosine and participated in negative enrichment of cell cycle processes. These findings, indicating a pan-cdk/cyclin inhibition by erufosine, were verified at both, mRNA and protein levels. Erufosine caused a G2/M block and inhibition of colony formation. Significant tumor growth retardation was seen upon treatment with erufosine in a xenograft model. For the decreased cyclin D1 and CDK 4/6 levels found in tumor tissue, these proteins can serve as biomarker for erufosine intervention. The findings demonstrate the potential of erufosine as cell cycle inhibitor in HNSCC treatment, alone or in combination with current therapeutic agents.

Induction of ER and mitochondrial stress by the alkylphosphocholine erufosine in oral squamous cell carcinoma cells.

Endoplasmic reticulum (ER) plays an essential role in cell function and survival. Accumulation of unfolded or misfolded proteins in the lumen of the ER activates the unfolded protein response (UPR), resulting in ER stress and subsequent apoptosis. The alkylphosphocholine erufosine is a known Akt-mTOR inhibitor in oral squamous cell carcinoma (OSCC). In the present study, we evaluate erufosine's role to induce ER and mitochondrial stress leading to autophagy, apoptosis, and ROS induction. The cellular toxicity of erufosine was determined in two OSCC cell lines and gene expression and enrichment analyses were performed. A positive enrichment of ER stress upon erufosine exposure was observed, which was verified at protein levels for the ER stress sensors and their downstream mediators. Knockdown and pharmacological inhibition of the ER stress sensors PERK and XBP1 revealed their involvement into erufosine's cellular effects, including proliferation, apoptosis, and autophagy induction. Autophagy was confirmed by increased acidic vacuoles and LC3-B levels. Upon erufosine exposure, calcium influx into the cytoplasm of the two OSCC cell lines was seen. Apoptosis was confirmed by nuclear staining, Annexin-V, and immunoblotting of caspases. The induction of mitochondrial stress upon erufosine exposure was predicted by gene set enrichment analysis (GSEA) and shown by erufosine's effect on mitochondrial membrane potential, ATP, and ROS production in OSCC cells. These data show that ER and mitochondrial targeting by erufosine represents a new facet of its mechanism of action as well as a promising new framework in the treatment of head and neck cancers.

Erufosine increases RhoB expression in oral squamous carcinoma cells independent of its tumor suppressive mode of action - a short report.

PURPOSE: Recently, we found that erufosine (erucylphospho-N,N,N trimethylpropylammonium) can induce up-regulation of RhoB expression in oral squamous carcinoma (OSCC) cells, thereby hinting at a tumor suppressive role. Therefore, we aimed to evaluate the role of RhoB in the tumor suppressive mode of action of erufosine on OSCC cells. METHODS: Anti-proliferative effects of erufosine were determined in HN-5 and FaDu OSCC-derived cells using a MTT assay. RhoB up-regulation was detected using microarray and qRT-PCR-based expression assays at IC25, IC50 and IC75 concentrations of erufosine. The results obtained were verified by Western blotting. In addition, siRNA-mediated RhoB knockdown was carried out and combined with erufosine treatment, after which cell cycle, colony formation and migration assays were performed to evaluate its combined effects. RESULTS: We found that after erufosine treatment of HN-5 and FaDu cells for 24, 48 and 72 h the IC50 values ranged from 43 to 37 µM and 27- to 15 µM, respectively. Microarray and qRT-PCR-based expression analyses revealed RhoB up-regulation up to 9-fold and 20-fold, respectively. Using Western blotting, an increase in RhoB protein expression was observed, as well as a decrease in pAkt (Ser473 and Thr308) expression and an increase in PARP cleavage. Combined siRNA-mediated RhoB knockdown and erufosine treatment resulted in slightly reduced RhoB and pAkt levels compared to erufosine treatment alone. Subsequent cell cycle analyses revealed an increased apoptotic induction, but a reduced G2 cell cycle arrest, of the combination. At the functional level, synergistic effects were observed using cell migration and colony formation assays. CONCLUSIONS: Our data show that erufosine can cause up-regulation of RhoB expression in OSCC cells. Combining erufosine treatment with siRNA-mediated RhoB knockdown did, however, not reveal a role of RhoB in its tumor suppressive mode of action.

Design, Synthesis of Novel, Potent, Selective, Orally Bioavailable Adenosine A2A Receptor Antagonists and Their Biological Evaluation.

Our initial structure-activity relationship studies on 7-methoxy-4-morpholino-benzothiazole derivatives featured by aryloxy-2-methylpropanamide moieties at the 2-position led to identification of compound 25 as a potent and selective A2A adenosine receptor (A2AAdoR) antagonist with reasonable ADME and pharmacokinetic properties. However, poor intrinsic solubility and low to moderate oral bioavailability made this series unsuitable for further development. Further optimization using structure-based drug design approach resulted in discovery of potent and selective adenosine A2A receptor antagonists bearing substituted 1-methylcyclohexyl-carboxamide groups at position 2 of the benzothiazole scaffold and endowed with better solubility and oral bioavailability. Compounds 41 and 49 demonstrated a number of positive attributes with respect to in vitro ADME properties. Both compounds displayed good pharmacokinetic properties with 63% and 61% oral bioavailability, respectively, in rat. Further, compound 49 displayed oral efficacy in 6-OHDA lesioned rat model of Parkinson diseases.

CCR5 blockage by maraviroc induces cytotoxic and apoptotic effects in colorectal cancer cells.

Alterations in the expression of C-C chemokine receptor type 5 (CCR5 or CD195) have been correlated with disease progression in different cancers. Recently, a few investigations have reported the blockage of this receptor by an antagonist (maraviroc) and its antineoplastic effects on tumor cell growth. However, little is known about the mechanistic reasons behind these antineoplastic effects of CCR5 blockage by maraviroc. In this study, we blocked the CCR5 receptor by maraviroc in SW480 and SW620 colorectal cancer cells to study the resulting changes in biological properties and related pathways. This blockage induced significantly reduced proliferation and a profound arrest in G1 phase of the cell cycle. Concomitantly, maraviroc caused significant signs of apoptosis at morphological level. Significant modulation of multiple apoptosis-relevant genes was also noticed at mRNA levels. In addition, we found remarkable increases in cleaved caspases at protein level. These modulations led us to propose a signaling pathway for the observed apoptotic effects. In conclusion, blocking the CCR5 by maraviroc induces significant cytotoxic and apoptotic effects in colorectal cancer cells. Thus, maraviroc can be considered a model compound, which may foster the development of further CCR5 antagonists to be used for the treatment of colorectal cancer.

Metastasis-related processes show various degrees of activation in different stages of pancreatic cancer rat liver metastasis.

BACKGROUND: Most pancreatic ductal adenocarcinoma (PDAC) patients who undergo tumor resection will develop postoperative liver metastasis within the first 2 years. Our hypothesis was that, during liver colonization, the temporal modulation of processes related to metastasis will change in a specific manner and that information on these changes might be used for new therapeutic approaches. MATERIAL AND METHODS: PDAC rat ASML cells were inoculated into the liver of BDX rats and re-isolated after different time periods of liver colonization (early, intermediate, advanced, and terminal). The total RNA of these samples was used to evaluate the expression profiles of more than 23,000 genes by chip array analysis. RESULTS: Depending on the time span following re-isolation, 7-15% of all known genes were deregulated. These genes were assigned to metastasis-related processes during the 4 stages of colonization. Except for apoptosis, all other processes were not activated in the early and middle colonization stages. In the terminal phase of liver colonization, cell proliferation, cell homing, cell movement, and vasculogenesis were significantly activated. CONCLUSION: We hypothesize that targeting the relatively few deregulated genes in the early stage of liver colonization could ultimately improve the survival of PDAC patients.