Biomedical subjects

Craig N Robson

Publications and source records attributed to Craig N Robson.

17 recordsLinked to original sources

PubMed2006

NF-kappaB activation upregulates fibroblast growth factor 8 expression in prostate cancer cells.

BACKGROUND: Fibroblast growth factor 8 (FGF8) is over-expressed in prostate cancer (CaP) correlating with high-grade disease and reduced survival. The role of acetylation in transcriptional regulation of FGF8 was investigated using the histone deacetylase (HDAC) inhibitor Trichostatin A (TSA). METHODS: FGF8 transcriptional response to TSA was investigated by gene reporter assays, RT-PCR, and Western blotting. Chromatin immunoprecipitation (ChIP) assays were also performed. RESULTS: FGF8 is upregulated in response to TSA treatment along with NF-kappaB transcriptional activity. Over-expression of p65 activated FGF8 transcription. ChIP assays revealed p65 recruitment to the fgf8 promoter, containing putative NF-kappaB binding sites, post TSA stimulation. PI-3K activity is required for TSA mediated FGF8 upregulation. CONCLUSION: Using TSA treatment in prostate cancer cells, a requirement of PI-3K activity in mediating TSA function is demonstrated and a novel role for NF-kappaB in the regulation of FGF8 expression is uncovered.

Acetylation↗

PubMed2006

KGF suppresses alpha2beta1 integrin function and promotes differentiation of the transient amplifying population in human prostatic epithelium.

Prostate epithelial stem cells are self-renewing cells capable of differentiation into prostate epithelium, and are thought to contribute towards both benign and malignant conditions in the human prostate. We have previously demonstrated that prostate epithelial basal cells express high levels of integrin alpha2beta1 and this population can be subdivided into stem (alpha2beta1(hi) CD133+) and transient-amplifying population (TAP) cells (alpha2beta1(hi) CD133-). However, the molecular mechanism(s) controlling the commitment and regulation of these cells towards differentiated epithelium remains unclear. Here, we demonstrate that beta1 integrin function is required for the maintenance of basal prostatic epithelial cells and suppression of its function by either methylcellulose or, more specifically, beta1-blocking antibody (80 microg/ml) induces differentiation, with associated expression of the differentiation-specific markers prostate acid phosphatase (PAP) and cytokeratin 18 (CK18). Keratinocyte growth factor (KGF), a stromal-derived growth factor, has previously been implicated in prostate organogenesis using in vitro tissue recombination experiments. We show that treatment with KGF (10 ng/ml) potently induces epithelial differentiation with concomitant suppression of alpha2beta1 integrin expression as well as the induction of androgen receptor expression. Specifically, p38-MAPK appears to be involved and the presence of SB202190, a p38 inhibitor, significantly blocks KGF-induced differentiation. Furthermore, the expression of the high-affinity receptor tyrosine kinase to KGF (FGFR2) is predominantly detectable in alpha2beta1(hi) CD133- TAP cells when compared with stem cells (alpha2beta1(hi) CD133+), which would therefore be relatively unresponsive to the differentiating effect of KGF. Taken together, using a human primary culture model, we have demonstrated key roles for interactions between KGF and integrin-mediated function in the regulation of prostate epithelial differentiation.

Aged↗

PubMed2006

Cellular functions of TIP60.

TIP60 was originally identified as a cellular acetyltransferase protein that interacts with HIV-1 Tat. As a consequence, the role of TIP60 in transcriptional regulation has been investigated intensively. Recent data suggest that TIP60 has more divergent functions than originally thought and roles for TIP60 in many processes, such as cellular signalling, DNA damage repair, cell cycle and checkpoint control and apoptosis are emerging. TIP60 is a tightly regulated transcriptional coregulator, acting in a large multiprotein complex for a range of transcription factors including androgen receptor, Myc, STAT3, NF-kappaB, E2F1 and p53. This usually involves recruitment of TIP60 acetyltransferase activities to chromatin. Additionally, in response to DNA double strand breaks, TIP60 is recruited to DNA lesions where it participates both in the initial as well as the final stages of repair. Here, we describe how TIP60 is a multifunctional enzyme involved in multiple nuclear transactions.

Acetyltransferases↗

PubMed2006

Human PIRH2 enhances androgen receptor signaling through inhibition of histone deacetylase 1 and is overexpressed in prostate cancer.

The androgen receptor (AR) is a hormone-dependent transcription factor critically involved in human prostate carcinogenesis. Optimal transcriptional control of androgen-responsive genes by AR may require complex interaction among multiple coregulatory proteins. We have previously shown that the AR coregulator TIP60 can interact with human PIRH2 (hPIRH2). In this study, we uncover important new functional role(s) for hPIRH2 in AR signaling: (i) hPIRH2 interacts with AR and enhances AR-mediated transcription with a dynamic pattern of recruitment to androgen response elements in the prostate-specific antigen (PSA) gene; (ii) hPIRH2 interacts with the AR corepressor HDAC1, leading to reduced HDAC1 protein levels and inhibition of transcriptional repression; (iii) hPIRH2 is required for optimal PSA expression; and (iv) hPIRH2 is involved in prostate cancer cell proliferation. In addition, overexpression of hPIRH2 protein was detected in 73 of 82 (89%) resected prostate cancers, with a strong correlation between increased hPIRH2 expression and aggressive disease, as signified by high Gleason sum scores and the presence of metastatic disease (P = <0.0001 and 0.0004, respectively). Collectively, our data establish hPIRH2 as a key modulator of AR function, opening a new direction for targeted therapy in aggressive human prostate cancer.

Cell Proliferation↗

PubMed2005

Epigenetic inactivation of the human sprouty2 (hSPRY2) homologue in prostate cancer.

Abnormal signalling events mediated by receptor tyrosine kinases (RTKs) contribute to human carcinogenesis. Sprouty2 (Spry2) is a key antagonistic regulator of RTK signalling and suppression of its expression or function may facilitate proliferation and angiogenesis. Using prostate cancer (CaP) as a model, we investigated the significance of Spry2 in human malignancy. We observed downregulated Spry2 expression in invasive CaP cell lines and high-grade clinical CaP (compared to benign prostatic hyperplasia (BPH) and well-differentiated tumours, P=0.041). A large CpG island is associated with hSPRY2, and extensive hypermethylation of this CpG island was observed in 76-82% of high-grade CaP, while control BPH tissues were predominantly unmethylated (P=0.0005). Furthermore, suppressed Spry2 expression correlated with methylation of the CpG region in clinical samples (P=0.004) and treatment with 5-aza-2'-deoxycytidine reactivated Spry2 expression in LNCaP and PC-3M cells. hSPRY2 maps to the long arm of chromosome 13 (13q31.1), where loss of heterozygosity (LOH) has been reported. We found no evidence of mutation; however, we demonstrated 27-40% LOH using flanking markers to hSPRY2. Hence, while biallelic epigenetic inactivation of hSPRY2 represents the main genetic event in prostate carcinogenesis, the observed 27-40% LOH presents evidence of hemizygous deletion with the remaining allele hypermethylated. We therefore propose hSPRY2 as a potential tumour suppressor locus in CaP.

Base Sequence↗

PubMed2005

Regulation of androgen receptor and histone deacetylase 1 by Mdm2-mediated ubiquitylation.

The androgen receptor (AR) is a member of the nuclear hormone receptor family of transcription factors and plays a critical role in regulating the expression of genes involved in androgen-dependent and -independent tumour formation. Regulation of the AR is achieved by alternate binding of either histone acetyltransferase (HAT)-containing co-activator proteins, or histone deacetylase 1 (HDAC1). Factors that control AR stability may also constitute an important regulatory mechanism, a notion that has been confirmed with the finding that the AR is a direct target for Mdm2-mediated ubiquitylation and proteolysis. Using chromatin immunoprecipitation (ChIP) and re-ChIP analyses, we show that Mdm2 associates with AR and HDAC1 at the active androgen-responsive PSA promoter in LNCaP prostate cancer cells. Furthermore, we demonstrate that Mdm2-mediated modification of AR and HDAC1 catalyses protein destabilization and attenuates AR sactivity, suggesting that ubiquitylation of the AR and HDAC1 may constitute an additional mechanism for regulating AR function. We also show that HDAC1 and Mdm2 function co-operatively to reduce AR-mediated transcription that is attenuated by the HAT activity of the AR co-activator Tip60, suggesting interplay between acetylation status and receptor ubiquitylation in AR regulation. In all, our data indicates a novel role for Mdm2 in regulating components of the AR transcriptosome.

Androgens↗

PubMed2005

Inhibition of ubiquitination and stabilization of human ubiquitin E3 ligase PIRH2 by measles virus phosphoprotein.

Using a C-terminal domain (PCT) of the measles virus (MV) phosphoprotein (P protein) as bait in a yeast two-hybrid screen, a cDNA identical to the recently described human p53-induced-RING-H2 (hPIRH2) cDNA was isolated. A glutathione S-transferase-hPIRH2 fusion protein expressed in bacteria was able to pull down P protein when mixed with an extract from P-expressing HeLa cells in vitro, and myc-tagged hPIRH2 could be reciprocally co-immunoprecipitated with MV P protein from human cells. Additionally, immunoprecipitation experiments demonstrated that hPIRH2-myc, MV P, and nucleocapsid (N) proteins form a ternary complex. The hPIRH2 binding site was mapped to the C-terminal X domain region of the P protein by using a yeast two-hybrid assay. The PCT binding site was mapped on hPIRH2 by using a novel yeast two-hybrid tagged PCR approach and by co-immunoprecipitation of hPIRH2 cysteine mutants and mouse/human PIRH2 chimeras. The hPIRH2 C terminus could mediate the interaction with MV P which was favored by the RING-H2 motif. When coexpressed with an enhanced green fluorescent protein-tagged hPIRH2 protein, MV P alone or in a complex with MV N was able to redistribute hPIRH2 to outside the nucleus, within intracellular aggregates. Finally, MV P efficiently stabilized hPIRH2-myc expression and prevented its ubiquitination in vivo but had no effect on the stability or ubiquitination of an alternative ubiquitin E3 ligase, Mdm2. Thus, MV P protein is the first protein from a pathogen that is able to specifically interact with and stabilize the ubiquitin E3 ligase hPIRH2 by preventing its ubiquitination.

Animals↗

PubMed2005

A human T-cell lymphotropic virus type 1 enhancer of Myc transforming potential stabilizes Myc-TIP60 transcriptional interactions.

The human T-cell lymphotropic virus type 1 (HTLV-1) infects and transforms CD4+ lymphocytes and causes adult T-cell leukemia/lymphoma (ATLL), an aggressive lymphoproliferative disease that is often fatal. Here, we demonstrate that the HTLV-1 pX splice-variant p30II markedly enhances the transforming potential of Myc and transcriptionally activates the human cyclin D2 promoter, dependent upon its conserved Myc-responsive E-box enhancer elements, which are associated with increased S-phase entry and multinucleation. Enhancement of c-Myc transforming activity by HTLV-1 p30II is dependent upon the transcriptional coactivators, transforming transcriptional activator protein/p434 and TIP60, and it requires TIP60 histone acetyltransferase (HAT) activity and correlates with the stabilization of HTLV-1 p30II/Myc-TIP60 chromatin-remodeling complexes. The p30II oncoprotein colocalizes and coimmunoprecipitates with Myc-TIP60 complexes in cultured HTLV-1-infected ATLL patient lymphocytes. Amino acid residues 99 to 154 within HTLV-1 p30II interact with the TIP60 HAT, and p30II transcriptionally activates numerous cellular genes in a TIP60-dependent or TIP60-independent manner, as determined by microarray gene expression analyses. Importantly, these results suggest that p30II functions as a novel retroviral modulator of Myc-TIP60-transforming interactions that may contribute to adult T-cell leukemogenesis.

Acetyltransferases↗

PubMed2004

Adenovirus-mediated expression of a soluble fibroblast growth factor receptor inhibits in vitro growth of prostate DU145 cells.

BACKGROUND: Fibroblast growth factor (FGF) family plays a key role in prostate cancer. The soluble FGF receptor (sFGFR) has been studied with regards to inhibiting cancer growth and was shown to have a dominant negative effect on cellular signaling and function. Using replication deficient adenovirus-mediated gene transfer, we tested if sFGFR expression may have a suppressive effect on in vitro growth of prostate cancer cells. METHODS: Western analysis was used to verify expression of sFGFR1 and to examine the effect of sFGFR1 on MAP kinase phosphorylation. The effect on proliferation and invasiveness of DU145 cells was examined using the WST-1 and Matrigel Invasion assay, respectively. RESULTS: Activation of MAP kinase (pERK1 and 2) by exogenous FGF1, 2, and 7 was suppressed to baseline levels by sFGFR, which was not seen with EGF. Proliferation and invasion of DU145 cells were significantly suppressed by sFGFR. CONCLUSIONS: A replication deficient adenoviral vector system reproducibly expresses sFGFR in prostate cells. Suppression of in vitro growth in DU145 cells by sFGFR provides the basis of a novel therapeutic approach.

Adenoviridae↗

PubMed2004

CD133, a novel marker for human prostatic epithelial stem cells.

Stem cells are clonogenic cells with self-renewal and differentiation properties, which may represent a major target for genetic damage leading to prostate cancer and benign prostatic hyperplasia. Stem cells remain poorly characterised because of the absence of specific molecular markers that permit us to distinguish them from their progeny, the transit amplifying cells, which have a more restricted proliferative potential. Human CD133 antigen, also known as AC133, was recently identified as a haematopoietic stem cell marker. Here we show that a small population (approximately 1%) of human prostate basal cells express the cell surface marker CD133 and are restricted to the alpha(2)beta(1)(hi) population, previously shown to be a marker of stem cells in prostate epithelia. alpha(2)beta(1)(hi)/CD133(+) cells exhibit two important attributes of epithelial stem cells: they possess a high in vitro proliferative potential and can reconstitute prostatic-like acini in immunocompromised male nude mice.

AC133 Antigen↗

PubMed2004

Upregulation and nuclear recruitment of HDAC1 in hormone refractory prostate cancer.

BACKGROUND: Histone deacetylase 1 (HDAC1) is a co-repressor involved in differentiation and proliferation control. It is upregulated in malignant compared to benign tissue, and targets a number of transcription factors including p53. METHODS: By immunohistochemistry, HDAC1 protein expression was investigated in human prostate specimens and the CWR22 mouse xenograft model. Flow cytometry and deconvolution immunofluorescence were also performed. RESULTS: HDAC1 was upregulated in pre-malignant and malignant lesions, with the highest increase in expression in hormone refractory (HR) cancer. Using the CWR22 xenograft model we showed androgen dependent regulation of HDAC1. HDAC1 overexpression led to a significant increase in proliferation and a shift towards the undifferentiated cytokeratin (CK) profile in a PC3M derivative clone constitutively expressing HDAC1. CONCLUSION: This study underlines the importance of HDAC1 in cell proliferation and the development of prostate cancer (CaP) and proposes a mechanism for HDAC1 nuclear recruitment. HDAC1 may constitute a crucial therapeutic target particularly in the most lethal phase of androgen independence.

Androgens↗

PubMed2004

Putative involvement of the histone acetyltransferase Tip60 in ribosomal gene transcription.

Tip60 is a histone acetyltransferase (HAT) implicated in a wide range of cellular functions, including mRNA synthesis and DNA repair. In the present report we propose a model based on which Tip60 is actively involved in ribosomal gene transcription through acetylation of UBF, a ribosomal specific transcription factor, as well as through its direct recruitment to the human ribosomal gene promoter, as shown by chromatin immunoprecipitation experiments. Electron microscopy studies revealed that Tip60 resides in sites of active rDNA transcription within the nucleolus, while it co-localizes with UBF as shown by confocal microscopy. In addition, in vivo transcription assays demonstrated that the nucleolar fraction of Tip60 localizes to sites of newly synthesized rRNA. Finally, functional assays established that Tip60 complexes with, and targets UBF for acetylation. The present study underlines the importance of acetylation in rDNA transcription and directly implicates Tip60 in the process of ribosomal gene transcription.

Acetyltransferases↗

PubMed2003

Control of human PIRH2 protein stability: involvement of TIP60 and the proteosome.

Murine PIRH2 (mPIRH2) was recently identified as a RING finger-containing ubiquitin-protein isopeptide ligase that interacts with both p53 and the human androgen receptor. mpirh2 is a p53-responsive gene that is up-regulated by UV, and mPIRH2 protein has the capacity to polyubiquitylate p53, perhaps leading to p53 destruction. mpirh2 therefore has properties similar to those of the oncogene mdm2. Here, we have identified human PIRH2 (hPIRH2) as a TIP60-interacting protein. To investigate its regulation, we characterized hPIRH2 in parallel with hPIRH2 variants possessing mutations of conserved RING finger residues. We observed that wild-type hPIRH2 is an unstable protein with a short half-life and is a target for RING domain-dependent proteasomal degradation. Accordingly, we found that hPIRH2 was ubiquitylated in cells. The TIP60-hPIRH2 association appeared to regulate hPIRH2 stability; coexpression of TIP60 enhanced hPIRH2 protein stability and altered hPIRH2 subcellular localization. These results suggest that hPIRH2 activities can be controlled, at the post-translational level, in multiple ways.

Acetyltransferases↗

PubMed2003

The scaffolding protein RACK1 interacts with androgen receptor and promotes cross-talk through a protein kinase C signaling pathway.

The androgen receptor (AR), a member of the nuclear hormone receptor superfamily, functions as a ligand-dependent transcription factor that regulates genes involved in cell proliferation and differentiation. Using a C-terminal region of the human AR in a yeast two-hybrid screen, we have identified RACK1 (receptor for activated C kinase-1) as an AR-interacting protein. In this report we found that RACK1, which was previously shown to be a protein kinase C (PKC)-anchoring protein that determines the localization of activated PKCbetaII isoform, facilitates ligand-independent AR nuclear translocation upon PKC activation by indolactam V. We also observed RACK1 to suppress ligand-dependent and -independent AR transactivation through PKC activation. In chromatin immunoprecipitation assays, we demonstrate a decrease in AR recruitment to the AR-responsive prostate-specific antigen (PSA) promoter following stimulation of PKC. Furthermore, prolonged exposure to indolactam V, a PKC activator, caused a reduction in PSA mRNA expression in prostate cancer LNCaP cells. Finally, we found PKC activation to have a repressive effect on AR and PSA protein expression in androgen-treated LNCaP cells. Our data suggest that RACK1 may function as a scaffold for the association and modification of AR by PKC enabling translocation of AR to the nucleus but rendering AR unable to activate transcription of its target genes.

Active Transport, Cell Nucleus↗

PubMed2003

Expression of Tip60, an androgen receptor coactivator, and its role in prostate cancer development.

Prostate cancer (CaP) is initially androgen sensitive and responsive to hormone ablation therapy. However, cancer growth recurs despite androgen deprivation in the majority of cases of advanced disease. The molecular basis of this progression still remains unknown. The significance of androgen receptor (AR) coactivator proteins in this androgen-dependent malignancy is only beginning to emerge. In the present study, we examined the role of Tat interactive protein, 60 kDa (Tip60), an AR coactivator, in CaP progression. In hormone refractory CaP biopsies, we observed a nuclear accumulation of Tip60 expression in contrast to a more diffuse distribution pattern observed in benign prostate hyperplasia and primary CaP. Furthermore, in both the prostate xenograft model CWR22 and the LNCaP CaP cell line, we observed that androgen withdrawal promoted upregulation of Tip60 as well as nuclear accumulation. In contrast, androgen exposure resulted in decreased Tip60 expression that was more closely linked to a cytoplasmic presence. Chromatin immunoprecipitation analysis revealed Tip60's recruitment to the PSA gene promoter in both androgen-dependent and -independent cell lines. Thus, in vitro and in vivo data support a possible role for Tip60 in the molecular pathway leading to the development of androgen-independent CaP following long-term androgen deprivation therapy.

Acetyltransferases↗

PubMed2002

Regulation of FGF8 expression by the androgen receptor in human prostate cancer.

Fibroblast growth factor 8 (FGF8) has been shown to play a key role in prostate carcinogenesis. It was initially cloned as an androgen induced protein in mouse mammary cancer SC3 cells. In this study, we examined if FGF8 was also regulated by the androgen receptor in human prostate cancer. FGF8b protein expression in resected clinical prostate cancer correlated closely with expression of the androgen receptor (AR). In the androgen sensitive CWR22 prostate xenograft, we observed up-regulation of FGF8b immunoreactivity in testosterone supplemented mice while castration markedly reduced its signal. Furthermore, FGF8b protein expression in AR positive LNCaP cells was similarly enhanced by androgens. The proximal promoter of the human FGF8 gene was cloned into a luciferase reporter construct (FGF8.luc). FGF8.luc activity in AR positive LNCaP and SC3 cells was increased 2.5-fold by androgens. In AR negative DU145 cells, maximal induction of FGF8.luc required both co-transfection of the AR and the presence of androgens. The anti-androgen bicalutamide completely abolished AR mediated FGF8.luc induction. Deletion constructs from FGF8.luc have further defined an active promoter region and an androgen responsive region. Nucleotide analysis of this androgen responsive region has revealed putative androgen response elements. Finally, using ChIP assays we confirmed in vivo interaction between the AR and the androgen responsive region of the FGF8 promoter. Taken together these data provide first evidence that expression of the mitogen FGF8 in prostate cancer is, at least in part, regulated by the androgen receptor at the transcriptional level.

Androgen Antagonists↗

PubMed2002

Tip60 and histone deacetylase 1 regulate androgen receptor activity through changes to the acetylation status of the receptor.

The androgen receptor (AR), a member of the nuclear hormone receptor superfamily, is thought to play an important role in the development of prostate cancer. The AR is a hormone-dependent transcription factor that activates expression of numerous androgen-responsive genes. Histone acetyltransferase-containing proteins have been shown to increase activity of several transcription factors, including nuclear hormone receptors, by eliciting histone acetylation, which facilitates promoter access to the transcriptional machinery. Conversely, histone deacetylases (HDACs) have been identified which reduce levels of histone acetylation and are associated with transcriptional repression by various transcription factors. We have previously shown that Tip60 (Tat-interactive protein, 60 kDa) is a bona fide co-activator protein for the AR. Here we show that Tip60 directly acetylates the AR, which we demonstrate is a requisite for Tip60-mediated transcription. To define a mechanism for repression of AR function, we demonstrate that AR activity is specifically down-regulated by the histone deacetylase activity of HDAC1. Furthermore, using both mammalian two-hybrid and immunoprecipitation experiments, we show that AR and HDAC1 interact, suggestive of a direct role for down-regulation of AR activity by HDAC1. In chromatin immunoprecipitation assays, we provide evidence that AR, Tip60, and HDAC1 form a trimeric complex upon the endogenous AR-responsive PSA promoter, suggesting that acetylation and deacetylation of the AR is an important mechanism for regulating transcriptional activity.

Acetylation↗