Modulatory roles of microRNAs in the regulation of different signalling pathways in large bowel cancer stem cells

There are emerging data to suggest that microRNAs (miRNAs) have significant roles in regulating the function of normal cells and cancer stem cells (CSCs). This review aims to analyse the roles of miRNAs in the regulation of colon CSCs through their interaction with various signalling pathways. Studies showed a large number of miRNAs that are reported to be deregulated in colon CSCs. However, few of the studies available were able to outline the function of miRNAs in colon CSCs and uncover their signalling pathways. From those miRNAs, which are better described, miR-21 followed by miR-34, miR-200 and miR-215 are the most reported miRNAs to have roles in colon CSC regulation. In particular, miRNAs have been reported to regulate the stemness features of colon CSCs mainly via Wnt/B-catenin and Notch signalling pathways. Additionally, miRNAs have been reported to act on processes involving CSCs through cell cycle regulation genes and epithelial-mesenchymal transition. The relative paucity of data available on the significance of miRNAs in CSCs means that new studies will be of great importance to determine their roles and to identify the signalling pathways through which they operate. Such studies may in future guide further research to target these genes for more effective cancer treatment. miRNAs were shown to regulate the function of cancer stem cells in large bowel cancer by targeting a few key signalling pathways in cells.

Methionine down-regulates TLR4/MyD88/NF-kappa B signalling in osteoclast precursors to reduce bone loss during osteoporosis

Background and PurposeStudies have demonstrated that a moderate intake of amino acids is associated with development of bone health. Methionine, a sulphur-containing essential amino acid, has been largely implicated for improving cartilage formation, however its physiological significance on bone integrity and functionality have not been elucidated. We investigated whether methionine can prevent osteoporotic bone loss. Experimental ApproachThe anti-resorptive effect of methionine, (250mgkg(-1) body wt administered in drinking water for 10 weeks), was evaluated in ovariectomized (OVX) rats by monitoring changes in bone turnover, formation of osteoclasts from blood-derived mononuclear cells and changes in the synthesis of pro-osteoclastogenic cytokines. Key resultsMethionine improved bone density and significantly decreased the degree of osteoclast development from blood mononuclear cells in OVX rats, as indicated by decreased production of osteoclast markers tartarate resistant acid phosphatase b (TRAP5b) and MIP-1. siRNA-mediated knockdown of myeloid differentiation primary response 88 MyD88], a signalling molecule in the toll-like receptor (TLR) signalling cascade, abolished the synthesis of both TRAP5b and MIP-1 in developing osteoclasts. Methionine supplementation disrupted osteoclast development by inhibiting TLR-4/MyD88/NF-B pathway. Conclusions and ImplicationsTLR-4/MyD88/NF-B signalling pathway is integral for osteoclast development and this is down-regulated in osteoporotic system on methionine treatment. Methionine treatment could be beneficial for the treatment of postmenopausal osteoporosis.

FLT3-driven redox signalling in acute myeloid leukaemia

Acute myeloid leukaemia refers to cancer of the blood and bone marrow characterised by the rapid expansion of immature blasts of the myeloid lineage. The aberrant proliferation of these blasts interferes with normal haematopoiesis, resulting in symptoms such as anaemia, poor coagulation and infections. The molecular mechanisms underpinning acute myeloid leukaemia are multi-faceted and complex, with a range of diverse genetic and cytogenetic abnormalities giving rise to the acute myeloid leukaemia phenotype. Amongst the most common causative factors are mutations of the FLT3 gene, which codes for a growth factor receptor tyrosine kinase required by developing haematopoietic cells. Disruptions to this gene can result in constitutively active FLT3, driving the de-regulated proliferation of undifferentiated precursor blasts. FLT3-targeted drugs provide the opportunity to inhibit this oncogenic receptor, but over time can give rise to resistance within the blast population. The identification of targetable components of the FLT3 signalling pathway may allow for combination therapies to be used to impede the emergence of resistance. However, the intracellular signal transduction pathway of FLT3 is relatively obscure. The objective of this study is to further elucidate this pathway, with particular focus on the redox signalling element which is thought to be involved. Signalling via reactive oxygen species is becoming increasingly recognised as a crucial aspect of physiological and pathological processes within the cell. The first part of this study examined the effects of NADPH oxidase-derived reactive oxygen species on the tyrosine phosphorylation levels of acute myeloid leukaemia cell lines. Using two-dimensional phosphotyrosine immunoblotting, a range of proteins were identified as undergoing tyrosine phosphorylation in response to NADPH oxidase activity. Ezrin, a cytoskeletal regulatory protein and substrate of Src kinase, was selected for further study. The next part of this study established that NADPH oxidase is subject to regulation by FLT3. Both wild type and oncogenic FLT3 signalling were shown to affect the expression of a key NADPH oxidase subunit, p22phox, and FLT3 was also demonstrated to drive intracellular reactive oxygen species production. The NADPH oxidase target protein, Ezrin, undergoes phosphorylation on two tyrosine residues downstream of FLT3 signalling, an effect which was shown to be p22phox-dependent and which was attributed to the redox regulation of Src. The cytoskeletal associations of Ezrin and its established role in metastasis prompted the investigation of the effects of FLT3 and NADPH oxidase activity on the migration of acute myeloid leukaemia cell lines. It was found that inhibition of either FLT3 or NADPH oxidase negatively impacted on the motility of acute myeloid leukaemia cells. The final part of this study focused on the relationship between FLT3 signalling and phosphatase activity. It was determined, using phosphatase expression profiling and real-time PCR, that several phosphatases are subject to regulation at the levels of transcription and post-translational modification downstream of oncogenic FLT3 activity. In summary, this study demonstrates that FLT3 signal transduction utilises a NADPH oxidase-dependent redox element, which affects Src kinase, and modulates leukaemic cell migration through Ezrin. Furthermore, the expression and activity of several phosphatases is tightly linked to FLT3 signalling. This work reveals novel components of the FLT3 signalling cascade and indicates a range of potential therapeutic targets.

Characterisation of the role of canonical BMP-Smad 1/5/8 signalling in the development of ventral midbrain dopaminergic neurons

Ventral midbrain (VM) dopaminergic (DA) neurons, which project to the dorsal striatum via the nigrostriatal pathway, are progressively degenerated in Parkinson’s disease (PD). The identification of the instructive factors that regulate midbrain DA neuron development, and the subsequent elucidation of the molecular bases of their effects, is vital. Such an understanding would facilitate the generation of transplantable DA neurons from stem cells and the identification of developmentally-relevant neurotrophic factors, the two most promising therapeutic approaches for PD. Two related members of the bone morphogenetic protein (BMP) family, BMP2 and growth/differentiation factor (GDF) 5, which signal via a canonical Smad 1/5/8 signalling pathway, have been shown to have neurotrophic effects on midbrain DA neurons both in vitro and in vivo, and may function to regulate VM DA neuronal development. However, the molecular (signalling pathway(s)) and cellular (direct neuronal or indirect via glial cells) mechanisms of their effects remain to be elucidated. The present thesis hypothesised that canonical Smad signalling mediates the direct effects of BMP2 and GDF5 on the development of VM DA neurons. By activating, modulating and/or inhibiting various components of the BMP-Smad signalling pathway, this research demonstrated that GDF5- and BMP2-induced neurite outgrowth from midbrain DA neurons is dependent on BMP type I receptor activation of the Smad signalling pathway. The role of glial cell-line derived neurotrophic factor (GDNF)-signalling, dynamin-dependent endocytosis and Smad interacting protein-1 (Sip1) regulation, in the neurotrophic effects of BMP2 and GDF5 were determined. Finally, the in vitro development of VM neural stem cells (NSCs) was characterised, and the ability of GDF5 and BMP2 to induce these VM NSCs towards DA neuronal differentiation was investigated. Taken together, these experiments identify GDF5 and BMP2 as novel regulators of midbrain DA neuronal induction and differentiation, and demonstrate that their effects on DA neurons are mediated by canonical BMPR-Smad signalling.

A study of the regulation of Trib family members expression in normal and malignant haematopoiesis

The Tribbles family of genes consist of three members; TRIB1, TRIB2 and TRIB3. Trib1 and Trib2 have been identified as oncogenes that can induce AML in mice. However little is known about how the expressions of the Tribbles family genes are controlled in the cell during haematopoiesis or leukaemogenesis. To investigate the Tribbles genes in leukaemia a bioinformatics approach was used. TRIB2 expression was found to be elevated in T-ALL and ALL with t(1;19). TRIB1 was found not to be significantly elevated in any leukaemic subtypes. Analyses of the TRIB1 and TRIB2 gene signatures in both leukaemic and normal haematopoietic cells identified pathways and transcription factors associated with these signatures. Pathways enriched for the TRIB1 signature included TLR signalling pathways and NF-κB pathways. Transcription factors enriched for this signature include C/EBP and SRF. Enriched for the TRIB2 signature includes T cell signalling pathways and Notch signalling pathways. Transcription factors enriched for this signature include E2F and ETS. Further investigation in vitro confirmed the finding that E2F1 was as a potential regulator of TRIB2 expression. E2F1 is able to directly bind to the TRIB2 promoter region and induce TRIB2 expression. C/EBPα p42 was found to inhibit E2F1 and the p30 isoform was found to cooperate with E2F1 induced activation of the TRIB2 promoter. Indicating the potential presence of a regulatory loop involved in the regulation of the TRIB2 gene. In conclusion we have investigated the Tribbles gene signatures in both normal haematopoietic and leukaemic cells. This has led to the identification of a number of pathways and transcription factors associated with these genes. We have also identified a family of transcription factors directly responsible for the regulation of TRIB2 expression. This regulatory pathway has the potential to be targeted in the treatment of leukaemia with a high TRIB2 signature.

Investigating the role of Fas (CD95) signalling in the modification of innate immune induced inflammation

Background/Aim: It has been demonstrated that a number of pathologies occur as a result of dysregulation of the immune system. Whilst classically associated with apoptosis, the Fas (CD95) signalling pathway plays a role in inflammation. Studies have demonstrated that Fas activation augments TLR4-mediated MyD88-dependent cytokine production. Studies have also shown that the Fas adapter protein FADD is required for RIG-I-induced IFNβ production. As a similar signalling pathway exists between RIG-I, TLR3 and the MyD88- independent of TLR4, we hypothesised that Fas activation may modulate both TLR3- and TLR4-induced cytokine production. Results: Fas activation reduced poly I:C-induced IFNβ, IL-8, IL-10 and TNFα production whilst augmenting poly I:C-, poly A:U- and Sendai virus-induced IP-10 production. TLR3-, RIG-I- and MDA5-induced IP-10 luciferase activation were inhibited by the Fas adapter protein FADD using overexpression studies. Poly I:C-induced phosphorylation of p-38 and JNK MAPK were reduced by Fas activation. Overexpression of FADD induced AP-1 luciferase activation. Point mutations in the AP-1 binding site enhanced poly I:C-induced IP- 10 production. LPS-induced IL-10, IL-12, IL-8 and TNFα production were enhanced by Fas activation, whilst reducing LPS-induced IFNβ production. Absence of FADD using FADD-/- MEFs resulted in impaired IFNβ production. Overexpression studies using FADD augmented TLR4-, MyD88- and TRIF-induced IFNβ luciferase activation. Overexpression studies also suggested that enhanced TLR4-induced IFNβ production was independent of NFκB activation. Conclusion: Viral-induced IP-10 production is augmented by Fas activation by reducing the phosphorylation of p-38 and JNK MAPKs, modulating AP-1 activation. The Fas adapterprotein FADD is required for TLR4-induced IFNβ production. Studies presented here demonstrate that the Fas signalling pathway can therefore modulate the immune response. Our data demonstrates that this modulatory effect is mediated by its adapter protein FADD, tailoring the immune response by acting as a molecular switch. This ensures the appropriate immune response is mounted, thus preventing an exacerbated immune response.

Role of Nitric Oxide-Signalling in Hypoxia-Induced Immune Escape in Cancer

A key step in malignant progression is the acquired ability of tumour cells to escape immune-mediated lysis. A potential mechanism by which tumour cells avoid immune destruction involves the shedding of MHC Class I Chain-Related Protein A (MICA), a Natural Killer (NK) cell-activating ligand, from the tumour cell membrane. Hypoxia has been shown to cause increased MICA shedding; however, this hypoxia-induced effect can be attenuated by pharmacological activation of the cyclic guanosine monophosphate (cGMP)-dependent nitric oxide (NO)-signalling pathway in cancer cells. The primary objective of the present study was to determine whether treatment of tumour-bearing nude mice with the NO-mimetic glyceryl trinitrate (GTN) attenuates in vivo tumour growth and if so, whether this effect is dependent on the presence of an intact NK cell compartment. Results indicated that continuous transdermal administration of GTN (1.8 µg/h) can significantly attenuate the growth of transplanted human DU-145 prostate tumours but that this effect of GTN is lost in mice whose NK-cells have been depleted. Tumours and serum from the mice in this study were analysed to determine whether GTN treatment had any effect on the expression levels of proteins integral to the proposed MICA shedding mechanism; however, the results of these studies were inconclusive. As phosphodiesterase (PDE) inhibition represents a potential method to enhance NO-signalling, experiments were performed to determine whether treatment with the PDE5/6 inhibitor zaprinast could also attenuate hypoxia-induced MICA shedding and decrease in vivo growth of DU-145 tumours. Results demonstrated that treatment with zaprinast (10 mg/kg) significantly attenuates MICA shedding in DU-145 cancer cells and significantly decreases in vivo tumour growth. Taken together, the results of these experiments indicate that GTN attenuates tumour growth by sensitising tumour cells to innate immunity, likely by increasing membrane-associated tumour cell MICA levels through the reactivation of NO-signalling, and that zaprinast decreases tumour growth likely through a similar mechanism. These findings are important because they indicate that agents capable of reactivating NO-signalling, such as NO-mimetics and PDE inhibitors, can potentially be used as immunosensitisers in the treatment and/or prevention of cancer.

Association Analysis of Canonical Wnt Signalling Genes in Diabetic Nephropathy.

Several studies have provided compelling evidence implicating the Wnt signalling pathway in the pathogenesis of diabetic nephropathy. Gene expression profiles associated with renal fibrosis have been attenuated through Wnt pathway modulation in model systems implicating Wnt pathway members as potential therapeutic targets for the treatment of diabetic nephropathy. We assessed tag and potentially functional single nucleotide polymorphisms (SNPs; n = 31) in four key Wnt pathway genes (CTNNB1, AXIN2, LRP5 and LRP6) for association with diabetic nephropathy using a case-control design.

Suppressors of cytokine signalling (SOCS): putative modulators of cytokine bioactivity in health and disease

Cytokines are important modulators of homeostatic processes such as development, haematopoiesis and host defence, A recently identified family of proteins, the supressors of cytokine signalling (SOCS) act as negative regulators of the key cytokine-activated signalling pathway, the Janus kinase/signal transducers and activators of transcription (JAK/STAT) cascade, In the current review, the discovery, structural features, regulation of expression, mechanisms of JAK/STAT inhibition and putative role in health and disease of the SOCS family are discussed.

Klebsiella pneumoniae targets an EGF receptor-dependent pathway to subvert inflammation

The NF-kB transcriptional factor plays a key role governing the activation of immune responses. Klebsiella pneumoniae is an important cause of community-acquired and nosocomial pneumonia. Evidence indicates that K. pneumoniae infections are characterized by lacking an early in?ammatory response. Recently, we have demonstrated that Klebsiella antagonizes the activation of NF-kB via the deubiquitinase CYLD. In this work, by applying a high-throughput siRNA gain-of-function screen interrogating the human kinome, we identi?ed 17 kinases that when targeted by siRNA restored IL-1b-dependent NF-kB translocation in infected cells. Further characterization revealed that K. pneumoniae activates an EGF receptor (EGFR)- phosphatidylinositol 3-OH kinase (PI3K)–AKT–PAK4–ERK–GSK3b signalling pathway to attenuate the cytokine-dependent nuclear translocation of NF-kB. Our data also revealed that CYLD is a downstream effector of K. pneumoniae-induced EGFR–
PI3K–AKT–PAK4–ERK–GSK3b signalling pathway. Our efforts to identify the bacterial factor(s) responsible for EGFR activation demonstrate that a capsule (CPS) mutant did not activate EGFR hence
suggesting that CPS could mediate the activation of EGFR. Supporting this notion, puri?ed CPS did activate EGFR as well as the EGFR-dependent PI3K–AKT–PAK4–ERK–GSK3b signalling pathway. CPS-mediated EGFR activation was dependent on a TLR4–MyD88–c-SRC-dependent pathway. Several promising drugs have been developed to antagonize this cascade. We propose that agents targeting this signalling pathway might provide selective alternatives for the management of K. pneumoniae pneumonias.

Haplotype association analysis of genes within the WNT signalling pathways in diabetic nephropathy

Background: Renal interstitial fibrosis and glomerular sclerosis are hallmarks of diabetic nephropathy (DN) and several studies have implicated members of the WNT pathways in these pathological processes. This study comprehensively examined common genetic variation within the WNT pathway for association with DN.

Methods: Genes within the WNT pathways were selected on the basis of nominal significance and consistent direction of effect in the GENIE meta-analysis dataset. Common SNPs and common haplotypes were examined within the selected WNT pathway genes in a white population with type 1 diabetes, discordant for DN (cases: n = 718; controls: n = 749). SNPs were genotyped using Sequenom or Taqman assays. Association analyses were performed using PLINK, to compare allele and haplotype frequencies in cases and controls. Correction for multiple testing was performed by either permutation testing or using false discovery rate.

Results: A logistic regression model including collection centre, duration of diabetes, and average HbA1c as covariates highlighted three SNPs in GSK3B (rs17810235, rs17471, rs334543), two in DAAM1 (rs1253192, rs1252906) and one in NFAT5 (rs17297207) as being significantly (P< 0.05) associated with DN, however these SNPs did not remain significant after correction for multiple testing. Logistic regression of haplotypes, with ESRD as the outcome, and pairwise interaction analyses did not yield any significant results after correction for multiple testing.

Conclusions: These results indicate that both common SNPs and common haplotypes of WNT pathway genes are not strongly associated with DN. However, this does not completely exclude these or the WNT pathways from association with DN, as unidentified rare genetic or copy number variants could still contribute towards the genetic architecture of DN.© 2013 Kavanagh et al.; licensee BioMed Central Ltd.

Analysis of single nucleotide polymorphisms implicate mTOR signalling in the development of new-onset diabetes after transplantation

Introduction
Despite excellent first year outcomes in kidney transplantation, there remain significant long-term complications related to new-onset diabetes after transplantation (NODAT). The purpose of this study was to validate the findings of previous investigations of candidate gene variants in patients undergoing a protocolised, contemporary immunosuppression regimen, using detailed serial biochemical testing to identify NODAT development.

Methods
One hundred twelve live and deceased donor renal transplant recipients were prospectively followed-up for NODAT onset, biochemical testing at days 7, 90, and 365 after transplantation. Sixty-eight patients were included after exclusion for non-white ethnicity and pre-transplant diabetes. Literature review to identify candidate gene variants was undertaken as described previously.

Results
Over 25% of patients developed NODAT. In an adjusted model for age, sex, BMI, and BMI change over 12 months, five out of the studied 37 single nucleotide polymorphisms (SNPs) were significantly associated with NODAT: rs16936667:PRDM14 OR 10.57;95% CI 1.8–63.0;p = 0.01, rs1801282:PPARG OR 8.5; 95% CI 1.4–52.7; p = 0.02, rs8192678:PPARGC1A OR 0.26; 95% CI 0.08–0.91; p = 0.03, rs2144908:HNF4A OR 7.0; 95% CI 1.1–45.0;p = 0.04 and rs2340721:ATF6 OR 0.21; 95%CI 0.04–1.0; p = 0.05.

Conclusion
This study represents a replication study of candidate SNPs associated with developing NODAT and implicates mTOR as the central regulator via altered insulin sensitivity, pancreatic β cell, and mitochondrial survival and dysfunction as evidenced by the five SNPs.

General significance
1) Highlights the importance of careful biochemical phenotyping with oral glucose tolerance tests to diagnose NODAT in reducing time to diagnosis and missed cases.
2)This alters potential genotype:phenotype association.
3)The replication study generates the hypothesis that mTOR signalling pathway may be involved in NODAT development.

p63 drives invasion in keratinocytes expressing HPV16 E6/E7 genes through regulation of Src-FAK signalling

Using microarray information from oro-pharyngeal data sets and results from primary human foreskin keratinocytes (HFK) expressing Human Papilloma Virus (HPV)-16 E6/E7 proteins, we show that p63 expression regulates signalling molecules which initiate cell migration such as Src and focal adhesion kinase (FAK) and induce invasion in 3D-organotypic rafts; a phenotype that can be reversed by depletion of p63. Knockdown of Src or FAK in the invasive cells restored focal adhesion protein paxillin at cell periphery and impaired the cell migration. In addition, specific inhibition of FAK (PF573228) or Src (dasatinib) activities mitigated invasion and attenuated the expression/activity of matrix metalloproteinase 14 (MMP14), a pivotal MMP in the MMP activation cascade. Expression of constitutively active Src in non-invasive HFK expressing E6/E7 proteins upregulated the activity of c-Jun and MMP14, and induced invasion in rafts. Depletion of Src, FAK or AKT in the invasive cells normalised the expression/activity of c-Jun and MMP14, thus implicating the Src-FAK/AKT/AP-1 signalling in MMP14-mediated extra-cellular matrix remodelling. Up-regulation of Src, AP-1, MMP14 and p63 expression was confirmed in oro-pharyngeal cancer. Since p63 transcriptionally regulated expression of many of the genes in this signalling pathway, it suggests that it has a central role in cancer progression.

Ligand binding and signalling pathways of PTH receptors in sea bream (Sparus auratus) enterocytes

Whole animal studies have indicated that Ca2+ uptake by the gastrointestinal tract is regulated by the action of parathyroid hormone-related peptide (PTHrP) in teleost fish. We have characterised PTH receptors (PTHR) in piscine enterocytes and established, by using aminoterminal PTHrP peptides, the amino acid residues important for receptor activation and for stabilising the ligand/receptor complex. Ligand binding of 125I-(1–35tyr) PTHrP to the membrane fraction of isolated sea bream enterocytes revealed the existence of a single saturable high-affinity receptor (KD=2.59 nM; Bmax=71 fmol/mg protein). Reverse transcription/polymerase chain reaction with specific primers for sea bream PTH1R and PTH3R confirmed the mRNA expression of only the later receptor. Fugu (1–34) PTHrP increased cAMP levels in enterocytes but had no effect on total inositol phosphate accumulation. The aminoterminal peptides (2–34)PTHrP, (3–34)PTHrP and (7–34) PTHrP bound efficiently to the receptor but were severely defective in stimulating cAMP in enterocyte cells indicating that the first six residues of piscine (1–34)PTHrP, although not important for receptor binding, are essential for activation of the adenylate cyclase/phosphokinase A (AC-PKA)-receptor-coupled intracellular signalling pathway. Therefore, PTHrP in teleosts acts on the gastrointestinal tract through PTH3R and the AC-PKA intracellular signalling pathway and might regulate Ca2+ uptake at this site. Ligand-receptor binding and activity throughout the vertebrates appears to be allocated to the same amino acid residues of the amino-terminal domain of the PTHrP molecule.

Investigating the role of Annexin A2 in Epidermal Growth Factor (EGF) induced signalling in cancer

Dissertação de mestrado, Qualidade em Análises, Faculdade de Ciências e Tecnologia, Universidade do Algarve; Universitat de Barcelona; Gdansk University of Technology, Universidad de Cádiz, Universitas Bergensis; 2015