FUS/TLS Is a Co-Activator of Androgen Receptor in Prostate Cancer Cells

Androgen receptor (AR) is a member of the nuclear receptor family of transcription factors. Upon binding to androgens, AR becomes transcriptionally active to regulate the expression of target genes that harbor androgen response elements (AREs) in their promoters and/or enhancers. AR is essential for the growth and survival of prostate cancer cells and is therefore a target for current and next-generation therapeutic modalities against prostate cancer. Pathophysiologically relevant protein-protein interaction networks involving AR are, however, poorly understood. In this study, we identified the protein FUsed/Translocated in LipoSarcoma (FUS/TLS) as an AR-interacting protein by co-immunoprecipitation of endogenous proteins in LNCaP human prostate cancer cells. The hormonal response of FUS expression in LNCaP cells was shown to resemble that of other AR co-activators. FUS displayed a strong intrinsic transactivation capacity in prostate cancer cells when tethered to basal promoters using the GAL4 system. Chromatin immunoprecipitation experiments showed that FUS was recruited to ARE III of the enhancer region of the PSA gene. Data from ectopic overexpression and “knock-down” approaches demonstrated that AR transcriptional activity was enhanced by FUS. Depletion of FUS reduced androgen-dependent proliferation of LNCaP cells. Thus, FUS is a novel co-activator of AR in prostate cancer cells

Upregulation of MAPK pathway is associated with survival in castrate-resistant prostate cancer

BACKGROUND: Recent evidence has implicated the MAP kinase (MAPK) pathway with the development of castrate-resistant prostate cancer (CRPC). We have previously reported gene amplification of critical members of this pathway with the development of castrate-resistant disease. In addition, we have shown that rising Raf-1 expression, with the development of CRPC, influences time to biochemical relapse. We therefore sought to further analyse the role of both Raf-1 and its downstream target MAPK in the molecular pathogenesis of CRPC. METHODS: Protein expression of Raf-1 and MAPK, including their activation status, was analysed using immunohistochemistry in a database of 65 paired tumour specimens obtained before and after the development of CRPC and correlated with other members of the pathway. RESULTS: Patients whose nuclear expression of MAPK rose with the development of CRPC had a significantly shorter median time to death following biochemical relapse (1.40 vs 3.00 years, P=0.0255) as well as reduced disease-specific survival when compared with those whose expression fell or remained unchanged (1.16 vs 2.62 years, P=0.0005). Significant correlations were observed between protein expression of Raf-1 and MAPK with the type 1 receptor tyrosine kinases, Her2 and epidermal growth factor receptor, as well as the transcription factor AP-1 in CRPC tumours. CONCLUSION: We conclude that the Her2/Raf-1/MAPK/AP-1 axis may promote the development of CRPC, leading to early relapse, and reduced disease-specific survival. In addition, members of the pathway may act as novel therapeutic and/or diagnostic targets for prostate cancer. British Journal of Cancer (2011) 104, 1920-1928. doi:10.1038/bjc.2011.163 www.bjcancer.com Published online 10 May 2011 (C) 2011 Cancer Research U

Identification of Kinases Regulating Prostate Cancer Cell Growth Using an RNAi Phenotypic Screen

As prostate cancer progresses to castration-resistant disease, there is an increase in signal transduction activity. Most castration-resistant prostate tumors continue to express the androgen receptor (AR) as well as androgen-responsive genes, despite the near absence of circulating androgen in these patients. The AR is regulated not only by its cognate steroid hormone, but also by interactions with a constellation of co-regulatory and signaling molecules. Thus, the elevated signaling activity that occurs during progression to castration resistance can affect prostate cancer cell growth either through the AR or independent of the AR. In order to identify signaling pathways that regulate prostate cancer cell growth, we screened a panel of shRNAs targeting 673 human kinases against LNCaP prostate cancer cells grown in the presence and absence of hormone. The screen identified multiple shRNA clones against known and novel gene targets that regulate prostate cancer cell growth. Based on the magnitude of effect on growth, we selected six kinases for further study: MAP3K11, DGKD, ICK, CIT, GALK2, and PSKH1. Knockdown of these kinases decreased cell growth in both androgen-dependent and castration-resistant prostate cancer cells. However, these kinases had different effects on basal or androgen-induced transcriptional activity of AR target genes. MAP3K11 knockdown most consistently altered transcription of AR target genes, suggesting that MAP3K11 affected its growth inhibitory effect by modulating the AR transcriptional program. Consistent with MAP3K11 acting on the AR, knockdown of MAP3K11 inhibited AR Ser 650 phosphorylation, further supporting stress kinase regulation of AR phosphorylation. This study demonstrates the applicability of lentiviral-based shRNA for conducting phenotypic screens and identifies MAP3K11, DGKD, ICK, CIT, GALK2, and PSKH1 as regulators of prostate cancer cell growth. The thorough evaluation of these kinase targets will pave the way for developing more effective treatments for castration-resistant prostate cancer

Specificity and heregulin regulation of Ebp1 (ErbB3 binding protein 1) mediated repression of androgen receptor signalling

Although ErbB receptors have been implicated in the progression of prostate cancer, little is known about proteins that may mediate their interactions with the androgen receptor (AR). Ebp1, a protein cloned via its association with the ErbB3 receptor, binds the AR and inhibits androgen-regulated transactivation of wild-type AR in COS cells. As the complement of coregulators in different cells are important for AR activity, we determined the effect of Ebp1 on AR function in prostate cancer cell lines. In addition, we examined the regulation of Ebp1 function by the ErbB3/4 ligand heregulin (HRG). In this study, we demonstrate, using several natural AR-regulated promoters, that Ebp1 repressed transcriptional activation of wild-type AR in prostate cancer cell lines. Downregulation of Ebp1 expression in LNCaP cells using siRNA resulted in activation of AR in the absence of androgen. Ebp1 associated with ErbB3 in LNCaP cells in the absence of HRG, but HRG induced the dissociation of Ebp1 from ErbB3. In contrast, HRG treatment enhanced both the association of Ebp1 with AR and also the ability of Ebp1 to repress AR transactivation. These studies suggest that Ebp1 is an AR corepressor whose biological activity can be regulated by the ErbB3 ligand, HRG

LNCaP Atlas: Gene expression associated with in vivo progression to castration-recurrent prostate cancer

<p>Abstract</p> <p>Background</p> <p>There is no cure for castration-recurrent prostate cancer (CRPC) and the mechanisms underlying this stage of the disease are unknown.</p> <p>Methods</p> <p>We analyzed the transcriptome of human LNCaP prostate cancer cells as they progress to CRPC <it>in vivo </it>using replicate LongSAGE libraries. We refer to these libraries as the LNCaP atlas and compared these gene expression profiles with current suggested models of CRPC.</p> <p>Results</p> <p>Three million tags were sequenced using <it>in vivo </it>samples at various stages of hormonal progression to reveal 96 novel genes differentially expressed in CRPC. Thirty-one genes encode proteins that are either secreted or are located at the plasma membrane, 21 genes changed levels of expression in response to androgen, and 8 genes have enriched expression in the prostate. Expression of 26, 6, 12, and 15 genes have previously been linked to prostate cancer, Gleason grade, progression, and metastasis, respectively. Expression profiles of genes in CRPC support a role for the transcriptional activity of the androgen receptor (<it>CCNH, CUEDC2, FLNA, PSMA7</it>), steroid synthesis and metabolism (<it>DHCR24, DHRS7</it>, <it>ELOVL5, HSD17B4</it>, <it>OPRK1</it>), neuroendocrine (<it>ENO2, MAOA, OPRK1, S100A10, TRPM8</it>), and proliferation (<it>GAS5</it>, <it>GNB2L1</it>, <it>MT-ND3</it>, <it>NKX3-1</it>, <it>PCGEM1</it>, <it>PTGFR</it>, <it>STEAP1</it>, <it>TMEM30A</it>), but neither supported nor discounted a role for cell survival genes.</p> <p>Conclusions</p> <p>The <it>in vivo </it>gene expression atlas for LNCaP was sequenced and support a role for the androgen receptor in CRPC.</p

Pooled analysis of WHO Surgical Safety Checklist use and mortality after emergency laparotomy

Background The World Health Organization (WHO) Surgical Safety Checklist has fostered safe practice for 10 years, yet its place in emergency surgery has not been assessed on a global scale. The aim of this study was to evaluate reported checklist use in emergency settings and examine the relationship with perioperative mortality in patients who had emergency laparotomy. Methods In two multinational cohort studies, adults undergoing emergency laparotomy were compared with those having elective gastrointestinal surgery. Relationships between reported checklist use and mortality were determined using multivariable logistic regression and bootstrapped simulation. Results Of 12 296 patients included from 76 countries, 4843 underwent emergency laparotomy. After adjusting for patient and disease factors, checklist use before emergency laparotomy was more common in countries with a high Human Development Index (HDI) (2455 of 2741, 89.6 per cent) compared with that in countries with a middle (753 of 1242, 60.6 per cent; odds ratio (OR) 0.17, 95 per cent c.i. 0.14 to 0.21, P <0001) or low (363 of 860, 422 per cent; OR 008, 007 to 010, P <0.001) HDI. Checklist use was less common in elective surgery than for emergency laparotomy in high-HDI countries (risk difference -94 (95 per cent c.i. -11.9 to -6.9) per cent; P <0001), but the relationship was reversed in low-HDI countries (+121 (+7.0 to +173) per cent; P <0001). In multivariable models, checklist use was associated with a lower 30-day perioperative mortality (OR 0.60, 0.50 to 073; P <0.001). The greatest absolute benefit was seen for emergency surgery in low- and middle-HDI countries. Conclusion Checklist use in emergency laparotomy was associated with a significantly lower perioperative mortality rate. Checklist use in low-HDI countries was half that in high-HDI countries.Peer reviewe

Global variation in anastomosis and end colostomy formation following left-sided colorectal resection

Background End colostomy rates following colorectal resection vary across institutions in high-income settings, being influenced by patient, disease, surgeon and system factors. This study aimed to assess global variation in end colostomy rates after left-sided colorectal resection. Methods This study comprised an analysis of GlobalSurg-1 and -2 international, prospective, observational cohort studies (2014, 2016), including consecutive adult patients undergoing elective or emergency left-sided colorectal resection within discrete 2-week windows. Countries were grouped into high-, middle- and low-income tertiles according to the United Nations Human Development Index (HDI). Factors associated with colostomy formation versus primary anastomosis were explored using a multilevel, multivariable logistic regression model. Results In total, 1635 patients from 242 hospitals in 57 countries undergoing left-sided colorectal resection were included: 113 (6·9 per cent) from low-HDI, 254 (15·5 per cent) from middle-HDI and 1268 (77·6 per cent) from high-HDI countries. There was a higher proportion of patients with perforated disease (57·5, 40·9 and 35·4 per cent; P < 0·001) and subsequent use of end colostomy (52·2, 24·8 and 18·9 per cent; P < 0·001) in low- compared with middle- and high-HDI settings. The association with colostomy use in low-HDI settings persisted (odds ratio (OR) 3·20, 95 per cent c.i. 1·35 to 7·57; P = 0·008) after risk adjustment for malignant disease (OR 2·34, 1·65 to 3·32; P < 0·001), emergency surgery (OR 4·08, 2·73 to 6·10; P < 0·001), time to operation at least 48 h (OR 1·99, 1·28 to 3·09; P = 0·002) and disease perforation (OR 4·00, 2·81 to 5·69; P < 0·001). Conclusion Global differences existed in the proportion of patients receiving end stomas after left-sided colorectal resection based on income, which went beyond case mix alone

Enzalutamide and blocking androgen receptor in advanced prostate cancer: lessons learnt from the history of drug development of antiandrogens

Yusuke Ito, Marianne D Sadar Genome Sciences Centre, BC Cancer, Vancouver, BC, Canada Abstract: Enzalutamide is a nonsteroidal antiandrogen for the treatment of metastatic castration-resistant prostate cancer (mCRPC) both before and after chemotherapy. Enzalutamide is more effective than its predecessor bicalutamide, which was analyzed in head-to-head studies of patients with CRPC. This family of nonsteroidal antiandrogens is now comprised of four drugs approved by the US Food and Drug Administration with two investigational drugs in clinical trials. Antiandrogens have been employed clinically for more than five decades to provide a rich resource of information. Steady-state concentration minimums (Cmin or trough) in the range of ~1&ndash;13 &mu;g/mL are measured in patients at therapeutic doses. Interestingly, enzalutamide which is considered to have strong affinity for the androgen receptor (AR) requires Cmin levels &gt;10&nbsp;&mu;g/mL. The sequence of antiandrogens and the clinical order of application in regard to other drugs that target the androgen axis remain of high interest. One novel first-in-class drug, called ralaniten, which binds to a unique region in the N-terminus domain of both the full-length and the truncated constitutively active splice variants of the AR, is currently in clinical trials for patients who previously received abiraterone, enzalutamide, or both. This highlights the trend to develop drugs with novel mechanisms of action and potentially differing mechanisms of resistance compared with antiandrogens. Better and more complete inhibition of the transcriptional activity of the AR appears to continue to provide improvements in the clinical management of mCRPC. Keywords: prostate cancer, enzalutamide, antiandrogens, Cmin, trough, pharmacology, cross-resistance, clinical trials, PSA response, ralaniten, EPI-002&nbsp