Agelastatin A: a novel inhibitor of osteopontin-mediated adhesion, invasion, and colony formation

Effective inhibitors of osteopontin (OPN)-mediated neoplastic transformation and metastasis are still lacking. (-)-Agelastatin A is a naturally occurring oroidin alkaloid with powerful antitumor effects that, in many cases, are superior to cisplatin in vitro. In this regard, past comparative assaying of the two agents against a range of human tumor cell lines has revealed that typically (-)-agelastatin A is 1.5 to 16 times more potent than cisplatin at inhibiting cell growth, its effects being most pronounced against human bladder, skin, colon, and breast carcinomas. In this study, we have investigated the effects of (-)-agelastatin A on OPN-mediated malignant transformation using mammary epithelial cell lines. Treatment with (-)-agelastatin A inhibited OPN protein expression and enhanced expression of the cellular OPN inhibitor, Tcf-4. (-)-Agelastatin A treatment also reduced beta-catenin protein expression and reduced anchorage-independent growth, adhesion, and invasion in R37 OPN pBK-CMV and C9 cell lines. Similar effects were observed in MDA-MB-231 and MDA-MB-435s human breast cancer cell lines exposed to (-)-agelastatin A. Suppression of Tcf-4 by RNA interference (short interfering RNA) induced malignant/invasive transformation in parental benign Rama 37 cells; significantly, these events were reversed by treatment with (-)-agelastatin A. Our study reveals, for the very first time, that (-)-agelastatin A down-regulates beta-catenin expression while simultaneously up-regulating Tcf-4 and that these combined effects cause repression of OPN and inhibition of OPN-mediated malignant cell invasion, adhesion, and colony formation in vitro. We have also shown that (-)-agelastatin A inhibits cancer cell proliferation by causing cells to accumulate in the G(2) phase of cell cycle.

Modelling the MAPK signalling pathway using a two-stage identification algorithm

Signal transduction pathways describe the dynamics of cellular response to input signalling molecules at receptors on the cell membrane. The Mitogen-Activated Protein Kinase (MAPK) cascade is one of such pathways that are involved in many important cellular processes including cell growth and proliferation. This paper describes a black-box model of this pathway created using an advanced two-stage identification algorithm. Identification allows us to capture the unique features and dynamics of the pathway and also opens up the possibility of regulatory control design. In the approach described, an optimal model is obtained by performing model subset selection in two stages, where the terms are first determined by a forward selection method and then modified using a backward selection model refinement. The simulation results demonstrate that the model selected using the two-stage algorithm performs better than with the forward selection method alone.

A new polycythaemia vera-associated SOCS3 SH2 mutant (SOCS3(F136L)) cannot regulate erythropoietin responses

Recently several different JAK2 exon12 mutations have been identified in V617F negative polycythaemia vera (PV) or idiopathic erythrocytosis (IE) patients. The patients present with erythrocytosis, ligand-independent cell growth and low serum erythropoietin (EPO) levels. Within this group, a deletion of amino acids 542-543 (N542-E543del) of JAK2 is most prevalent. We have previously shown that in the presence of JAK2(V617F), suppressor of cytokine signalling 3 (SOCS3) is unable to negatively regulate EPO signalling and proliferation of V617F-expressing cells. Here we report a PV patient heterozygous for the somatic JAK2(N542-E543del) mutation and a previously unreported germline mutation within the SH2 domain of SOCS3 (F136L). Interestingly, the SOCS3(F136L) mutation was detected in a Japanese myeloproliferative disorder patient cohort at double the frequency of healthy controls. Cells expressing SOCS3(F136L) had markedly elevated EPO-induced proliferation and extended EPO-induced JAK2 phosphorylation. Additionally, compared to wild-type SOCS3, mutant SOCS3 had an extended half-life in the presence of JAK2 and JAK2(N542-E543del). Our findings suggest that this loss-of-function SOCS3 mutation may have contributed to disease onset by causing deregulated JAK2 signalling in the presence of a constitutively active JAK2(N542-E543del) mutant.

FKBPL regulates estrogen receptor signaling and determines response to endocrine therapy

The HSP90 chaperone and immunophilin FKBPL is an estrogen-responsive gene that interacts with estogen receptor a (ERa) and regulates its levels. In this study, we explored the effects of FKBPL on breast cancer proliferation. Breast cancer cells stably overexpressing FKBPL became dependent on estrogen for their growth and were dramatically more sensitive to the antiestrogens tamoxifen and fulvestrant, whereas FKBPL knockdown reverses this phenotype. FKBPL knockdown also decreased the levels of the cell cycle inhibitor p21WAF1 and increased ERa phosphorylation on Ser118 in response to 17ß-estradiol and tamoxifen. In support of the likelihood that these effects explained FKBPL-mediated cell growth inhibition and sensitivity to endocrine therapies, FKBPL expression was correlated with increased overall survival and distant metastasis-free survival in breast cancer patients. Our findings suggest that FKBPL may have prognostic value based on its impact on tumor proliferative capacity and sensitivity to endocrine therapies, which improve outcome.

Retinal pericytes control expression of nitric oxide synthase and endothelin-1 in microvascular endothelial cells

Pericytes are known to communicate with endothelial cells by direct contact and by releasing cytokines such as TGF-beta. There is also strong evidence that pericytes act as regulators of endothelial cell proliferation and differentiation. We have investigated the effect of pericyte-conditioned medium (PCM) on proliferation of human microvascular endothelial cells in vitro, together with the expression of the vasoregulatory molecules, constitutive and inducible nitric oxide synthases (ecNOS and iNOS), and endothelin-1 (ET-1). Expression was measured at the mRNA level using semiquantitative RT-PCR for all three genes and at the protein level for ecNOS and iNOS using Western blotting. Growth curves for HMECs showed that PCM inhibits proliferation, eventually leading to cell death. Exposure to PCM repressed iNOS mRNA expression fivefold after 6 h. A similar, though delayed, reduction in protein levels was observed. ecNOS mRNA was slightly induced at 6 h, though there was no significant change in ecNOS protein. By contrast, ET-1 mRNA was induced 2.3-fold after 6 h exposure to PCM. We conclude that pericytes release a soluble factor or factors that are potent inhibitors of endothelial cell growth and promote vasoconstriction by up-regulating endothelin-1 and down-regulating iNOS. (C) 2000 Academic Press.

The DUB/USP17 deubiquitinating enzymes: A gene family within a tandemly repeated sequence, is also embedded within the copy number variable Beta-defensin cluste

Background: The DUB/USP17 subfamily of deubiquitinating enzymes were originally identified as immediate early genes induced in response to cytokine stimulation in mice (DUB-1, DUB-1A, DUB-2, DUB-2A). Subsequently we have identified a number of human family members and shown that one of these (DUB-3) is also cytokine inducible. We originally showed that constitutive expression of DUB-3 can block cell proliferation and more recently we have demonstrated that this is due to its regulation of the ubiquitination and activity of the 'CAAX' box protease RCE1.

Results: Here we demonstrate that the human DUB/USP17 family members are found on both chromosome 4p16.1, within a block of tandem repeats, and on chromosome 8p23.1, embedded within the copy number variable betadefensin cluster. In addition, we show that the multiple genes observed in humans and other distantly related mammals have arisen due to the independent expansion of an ancestral sequence within each species. However, it is also apparent when sequences from humans and the more closely related chimpanzee are compared, that duplication events have taken place prior to these species separating.

Conclusions: The observation that the DUB/USP17 genes, which can influence cell growth and survival, have evolved from an unstable ancestral sequence which has undergone multiple and varied duplications in the species examined marks this as a unique family. In addition, their presence within the beta-defensin repeat raises the question whether they may contribute to the influence of this repeat on immune related conditions.

A GREM1 gene variant associates with diabetic nephropathy

Gremlin, a cell growth and differentiation factor, promotes the development of diabetic nephropathy in animal models, but whether GREM1 gene variants associate with diabetic nephropathy is unknown. We comprehensively screened the 5' upstream region (including the predicted promoter), all exons, intron-exon boundaries, complete untranslated regions, and the 3' region downstream of the GREM1 gene. We identified 31 unique variants, including 24 with a minor allele frequency exceeding 5%, and 9 haplotype-tagging single nucleotide polymorphisms (htSNPs). We selected one additional variant that we predicted to alter transcription factor binding. We genotyped 709 individuals with type 1 diabetes of whom 267 had nephropathy (cases) and 442 had no evidence of kidney disease (controls). Three individual SNPs significantly associated with nephropathy at the 5% level, and two remained significant after adjustment for multiple testing. Subsequently, we genotyped a replicate population comprising 597 cases and 502 controls: this population supported an association with one of the SNPs (rs1129456; P = 0.0003). Combined analysis, adjusted for recruitment center (n = 8), suggested that the T allele conferred greater odds of nephropathy (OR 1.69; 95% CI 1.36 to 2.11). In summary, the GREM1 variant rs1129456 associates with diabetic nephropathy, perhaps explaining some of the genetic susceptibility to this condition.

AKT1 Is Associated with Schizophrenia Across Multiple Symptom Dimensions in the Irish Study of High Density Schizophrenia Families

Background: The phosphatidylinositol 3-kinase (PI3K)-AKT signal transduction pathway is critical to cell growth and survival. In vitro functional studies indicate that the candidate schizophrenia susceptibility gene DTNBP1 influences AKT signaling to promote neuronal viability. The AKT1 gene has also been implicated in schizophrenia by association studies and decreased protein expression in the brains of schizophrenic patients. 
 Methods: The association of DTNBP1 in the Irish Study of High Density Schizophrenia Families (ISHDSF) prompted our investigation of AKT1 for association with disease in this sample. Eight single nucleotide polymorphisms spanning AKT1 were analyzed for association with schizophrenia across four definitions of affection and according to Operational Criteria Checklist of Psychotic Illness (OPCRIT) symptom scales. We examined expression of AKT1 messenger RNA from postmortem brain tissue of schizophrenic, bipolar, and control individuals. 
 Results: No single marker showed significant association, but the risk haplotype previously found over-transmitted to Caucasian schizophrenic patients was significantly under-transmitted in the ISHDSF (.01 < p < .05), across all OPCRIT symptom dimensions. Exploratory haplotype analysis confirmed association with schizophrenia toward the 5’ end of AKT1 (.008 < p < .049, uncorrected). We found significantly decreased RNA levels in prefrontal cortex of schizophrenic individuals, consistent with reduced AKT1 protein levels reported in schizophrenic brain. 
 Conclusions: The replication of association of AKT1 gene variants in a further Caucasian family sample adds support for involvement of AKT signaling in schizophrenia, perhaps encompassing a broader clinical phenotype that includes mood dysregulation. We show that AKT signaling might be compromised in schizophrenic and bipolar patients via reduced RNA expression of specific AKT isoforms.

Proteasome inhibitors in cancer therapy

The ubiquitin proteasome pathway plays a critical role in regulating many processes in the cell which are important for tumour cell growth and survival. Inhibition of proteasome function has emerged as a powerful strategy for anti-cancer therapy. Clinical validation of the proteasome as a therapeutic target was achieved with bortezomib and has prompted the development of a second generation of proteasome inhibitors with improved pharmacological properties. This review summarises the main mechanisms of action of proteasome inhibitors in cancer, the development of proteasome inhibitors as therapeutic agents and the properties and progress of next generation proteasome inhibitors in the clinic.

Estrogen Receptor-beta Affects the Prognosis of Human Malignant Mesothelioma

Malignant pleural mesothelioma is an asbestos-related neoplasm with poor prognosis, refractory to current therapies, the incidence of which is expected to increase in the next decades. Female gender was identified as a positive prognostic factor among other clinical and biological prognostic markers for malignant mesothelioma, yet a role of estrogen receptors (ERs) has not been studied. Our goal was to investigate ERs expression in malignant mesothelioma and to assess whether their expression correlates with prognosis. Immunohistochemical analysis revealed intense nuclear ER beta staining in normal pleura that was reduced in tumor tissues. Conversely, neither tumors nor normal pleura stained positive for ER alpha. Multivariate analysis of 78 malignant mesothelioma patients with pathologic stage, histologic type, therapy, sex, and age at diagnosis indicated that FRO expression is an independent prognostic factor of better survival. Moreover, studies in vitro confirmed that treatment with 17 beta-estradiol led to an ER beta-mediated inhibition of malignant mesothelioma cell proliferation as well as p21(CIP1) and p27(KIP1) up-regulation. Consistently cell growth was suppressed by ER beta overexpression, causing a G(2)-M-phase cell cycle arrest, paralleled by cyclin B1 and survivin down-regulation. Our data support the notion that ER beta acting as a tumor suppressor is of high potential relevance to prediction of disease progression and to therapeutic response of malignant mesothelioma patients. [Cancer Res 2009;69(11):4598-604]

Long-term bone marrow culture profiles in patients with myelodysplastic syndromes are not explicable by defective apoptosis

It has been suggested that increased intramedullary apoptosis may explain the paradox between peripheral blood cytopenias and the hyper- or normo-cellular bone marrow observed in the myelodysplastic syndromes (MDS). We wished to see if culture performance could be related to the presence of apoptotic cells in a group of patients with MDS (12 patients) and other patients with peripheral blood cytopenias (six patients) which caused diagnostic difficulty. There was no correlation between LTBMC or adherent cell growth and the presence of apoptotic cells in the original marrow sample. A variable degree of apoptosis was observed in both groups of patients. LTBMC profiles correlated well with diagnosis but were unrelated to the extent of intramedullary apoptosis. This suggests that apoptosis is a much more ubiquitous process in disease than previously thought. (C) 1998 Elsevier Science Ltd. All rights reserved.

PsT-1: A new Tryptophyllin peptide from the skin secretion of Waxy Monkey Leaf Frog, Phyllomedusa sauvagei

The Waxy Monkey Leaf Frog, Phyllomedusa sauvagei, has been extensively-studied for many years, and a broad spectrum of bioactive peptides has been found in its skin secretions. Here we report the discovery of a novel tryptophyllin (TPH) peptide, named PsT-1, from this frog species. Skin secretions from specimens of P. sauvagei were collected by mild electrical stimulation. Peptides were identified and characterized by transcriptome cloning, and the structure was confirmed by MALDI-TOF mass spectrometry and automated Edman degradation. This novel peptide was encoded by a single precursor of 61 amino acid residues, whose primary structure was deduced from cloned skin cDNA. Analysis of different amphibian tryptophyllins revealed that PsT-1 exhibited a high degree of primary structural similarity to its homologues, PdT-1 and PdT-2, from the Mexican giant leaf frog, Pachymedusa dacnicolor. A synthetic replicate of PsT-1 was found to inhibit bradykinin-induced vasorelaxation of phenylephrine pre-constricted rat tail artery smooth muscle. It was also found that PsT-1 had an anti-proliferative effect on three different human prostate cancer cell lines (LNCaP/PC3/DU145), by use of an MTT assay coupled with direct cell counting as measures of cell growth. These data indicate that PsT-1 is a likely bradykinin receptor antagonist and its biological effects are probably mediated through bradykinin receptors. As a BK antagonist, PST-1, with antagonistic effects on BK in artery smooth muscle, inhibition of proliferation in prostate cancer cells and lack of undesirable side effects, may have potential in cardiovascular, inflammatory and anticancer therapy.

TAZ Expression as a Prognostic Indicator in Colorectal Cancer

The Hippo pathway restricts the activity of transcriptional coactivators TAZ (WWTR1) and YAP. TAZ and YAP are reported to be overexpressed in various cancers, however, their prognostic significance in colorectal cancers remains unstudied. The expression levels of TAZ and YAP, and their downstream transcriptional targets, AXL and CTGF, were extracted from two independent colon cancer patient datasets available in the Gene Expression Omnibus database, totaling 522 patients. We found that mRNA expressions of both TAZ and YAP were positively correlated with those of AXL and CTGF (p<0.05). High level mRNA expression of TAZ, AXL or CTGF significantly correlated with shorter survival. Importantly, patients co-overexpressing all 3 genes had a significantly shorter survival time, and combinatorial expression of these 3 genes was an independent predictor for survival. The downstream target genes for TAZ-AXL-CTGF overexpression were identified by Java application MyStats. Interestingly, genes that are associated with colon cancer progression (ANTXR1, EFEMP2, SULF1, TAGLN, VCAN, ZEB1 and ZEB2) were upregulated in patients co-overexpressing TAZ-AXL-CTGF. This TAZ-AXL-CTGF gene expression signature (GES) was then applied to Connectivity Map to identify small molecules that could potentially be utilized to reverse this GES. Of the top 20 small molecules identified by connectivity map, amiloride (a potassium sparing diuretic,) and tretinoin (all-trans retinoic acid) have shown therapeutic promise in inhibition of colon cancer cell growth. Using MyStats, we found that low level expression of either ANO1 or SQLE were associated with a better prognosis in patients who co-overexpressed TAZ-AXL-CTGF, and that ANO1 was an independent predictor of survival together with TAZ-AXL-CTGF. Finally, we confirmed that TAZ regulates Axl, and plays an important role in clonogenicity and non-adherent growth in vitro and tumor formation in vivo. These data suggest that TAZ could be a therapeutic target for the treatment of colon cancer.

Epigenetics in Vascular Disease - Therapeutic Potential of New Agents

Vascular diseases, including atherosclerosis, angioplasty-induced restenosis, vessel graft arteriosclerosis and hypertension-related stenosis, remain the most prevalent cause of death in the developed world. The aetiology of vascular diseases is multifactorial with both genetic and environmental factors. Recently, some of the most promising research identifies the epigenetic modification of the genome to play a major role in the disease development, linking the environmental insults with gene regulation. In this process, modification of DNA by methylation, and histone modification by acetylation, methylation, phosphorylation and/or SUMOylation are reported. Importantly, recent studies demonstrated that histone deacetylase (HDAC) enzymes are crucial in endothelial integrity, smooth muscle proliferation and in the formation of arteriosclerosis in animal models. The study of HDACs has shown remarkable specificity of HDAC family members in vascular cell growth/death that influences the disease process. Interestingly, the effects of HDACs on arteriosclerosis development in animal models have been observed after HDAC inhibition using specific inhibitors. This provides a new approach for the treatment of vascular disease using the agents that influence the epigenetic process in vascular cells. This review updates the rapid advances in epigenetics of vascular diseases focusing on the role of HDAC family in atherosclerosis. It will also discuss the underlying mechanisms of histone acetylation in vascular cells and highlight the therapeutic potential of such agents.

Androgens and estrogens stimulate ribosome biogenesis in prostate and breast cancer cells in receptor dependent manner

Ribosome biogenesis is a fundamental cellular process intimately linked to cell growth and proliferation, which is upregulated in most of cancers especially in aggressive cancers. In breast and prostate cancers steroid hormone receptor signalling is the principal stimulus for cancer growth and progression. Here we investigated the link between estrogen and androgen receptor signalling and the initial stage of ribosome biogenesis - transcription of rRNA genes. We have discovered that estrogen or androgen treatment can positively regulate rRNA synthesis in breast and prostate cancer cells respectively and that this effect is receptor dependent. This novel and interesting finding suggests a previously unidentified link between steroid hormone receptor signalling pathways and the regulation of ribosome biogenesis.