Search

Background Telotristat ethyl is approved to treat carcinoid syndrome diarrhea in combination with somatostatin analogs. In TELESTAR and TELECAST phase III studies, patients with carcinoid syndrome received telotristat ethyl 250 or 500 mg 3 times per day (tid) or placebo tid in addition to somatostatin analogs. The aim of this prespecified analysis was to examine the time to reductions in bowel movements (BMs) in the TELESTAR and TELECAST studies using survival analysis methods. Methods First occurrence of sustained response was defined as the time to the first day of 2 consecutive weeks with a mean BM frequency improvement of ≥ 30% from baseline during the 12-week double-blind treatment periods. Time to first ≥ 30% worsening in BM frequency was also measured. Treatments were compared with the log-rank test; Cox regression models provided point and confidence interval estimates of the hazard ratios for each trial. Results In TELESTAR and TELECAST, majority of patients (69%) on telotristat ethyl experienced a sustained ≥ 30% improvement in BM frequency. The median time to sustained reduction of at least 30% in BM frequency was significantly faster (fewer days to onset) for telotristat ethyl compared with placebo in both TELESTAR (250 mg, HR = 2.3 [95% CI, 1.3–4.1, P = 0.004]; 500 mg, HR = 2.2 [95% CI, 1.2–3.9, P = 0.009]) and TELECAST (250 mg, HR = 3.9 [95% CI, 1.6–11.1, P = 0.003]; 500 mg, HR = 4.2 [95% CI, 1.7–11.7, P = 0.002]). In TELECAST, 42% of patients on placebo experienced sustained worsening in BM frequency compared with 20% on telotristat ethyl; no significant difference was observed in TELESTAR. Conclusion The time of onset of sustained BM frequency improvement mean and range are important when considering use of telotristat ethyl in patients with carcinoid syndrome diarrhea. Telotristat ethyl may also reduce sustained worsening in BM frequency. Trial Registration ClinicalTrials.gov Identifiers: NCT01677910, NCT02063659

461 Background: Prior studies suggest that white light cystoscopy (WLC) alone can fail to detect cases of non-muscle invasive bladder cancer (NMIBC) compared to blue light cystoscopy (BLC). We describe bladder cancer outcomes and the impact of BLC among NMIBC patients in an equal access setting. Methods: A total of 378 NMIBC patients within the Veterans Affairs system that had a CPT code for BLC from December 1, 2014 to December 31, 2020 were assessed. We determined recurrence rates and time to recurrence prior to BLC (i.e. after previous WLC if available) and following BLC. We used the Kaplan-Meier method to estimate event-free survival and Cox regression to determine the association between race and recurrence, progression, and overall survival. Results: Of 378 patients with complete data, 43 (11%) were Black and 300 (79%) White. Median follow-up was 40.7 months from bladder cancer diagnosis. There were 194 (51%) patients with either TaHG or T1 without CIS; 52 (14%) had CIS with or without TaHG or T1; and 127 (34%) had TaLG only. A total of 239 (63%) patients received BCG at any point during the study. Median time to first recurrence following BLC was longer compared to WLC alone (40 (33-NE) vs. 26 (17-39) months). The risk of recurrence was significantly lower following BLC (Hazard Ratio (HR) 0.70; 95% Confidence Interval (CI) 0.54-0.90). There was no significant difference in recurrence (Hazard Ratio (HR) 0.83; 95% Confidence Interval (CI) 0.48-1.43), progression (HR 1.46; 95% CI 0.45-4.74), and overall survival (HR 0.69; 95% CI 0.29-1.65) following BLC by Black vs. White race. Conclusions: In this study from an equal access setting in the VA, we observed significantly decreased risk of recurrence and prolonged time interval to recurrence following BLC compared to WLC alone. There was no difference in any bladder cancer outcomes by race.

Aim: Elevated serotonin in patients with neuroendocrine tumors (NETs) may impact heart failure incidence but a quantitative relationship has not been established. Materials & methods: Systematic review and meta-analysis of studies assessing 24-h urinary 5-hydroxyindoleacetic acid (u5-HIAA) and mortality in patients with NETs (2007–2017) with a primary outcome of 1-year mortality risk and 24-h u5-HIAA. Results: We identified 1715 records of which 12 studies including 755 patients (3442 person-years with 376 deaths) were eligible for meta-analysis. Mean u5-HIAA was 149.2 mg/24 h (standard deviation: 96.6) and mortality was 13.0%. The meta-regression equation showed an 11.8% (95% CI: 8.9–17.0%; I2 = 93.0%) increase in 1-year mortality for every ten-unit increase in u5-HIAA. Conclusion: Serotonin measured by its metabolite u5-HIAA is predictive of 1-year all-cause mortality in patients with NETs.

There is an unmet clinical need for adequate biomarkers to aid risk stratification and management of prostate cancer (PCa) patients. Even within the high-risk PCa category, not all patients will invariably have a poor prognosis, and improved stratification of this heterogeneous group is needed. In this context, components of the hedgehog (Hh) pathway may have promise as biomarkers, because the available evidence suggests increased Hh pathway activity may confer a poorer outcome in advanced and castrate-resistant PCa. In this study, potential associations between Hh pathway protein expression and clinicopathological factors, including time to biochemical recurrence (BCR), were investigated using a tissue microarray constructed from benign and malignant prostate samples from 75 predominantly high-risk PCa patients who underwent radical prostatectomy. Hh signaling activity was found to differ between benign and malignant prostate tissue, with a greater amount of active Hh signaling present in malignant than benign prostate epithelium. High expression of Patched 1 in malignant prostate epithelium was found to be an independent predictor of BCR in high-risk PCa patients. Glioma-associated oncogene 1 may potentially represent a clinically useful biomarker of an aggressive tumor phenotype. Evaluation of Hh signaling activity in PCa patients may be useful for risk stratification, and epithelial Patched 1 expression, in particular, may be a prognostic marker for BCR in high-risk PCa patients.

BACKGROUND: The parasympathetic nervous system (PNS), via neurotransmitter Acetylcholine (ACh), modulates fibrogenesis in animal models. However, the role of ACh in human hepatic fibrogenesis is unclear. AIMS: We aimed to determine the fibrogenic responses of human hepatic stellate cells (hHSC) to ACh and the relevance of the PNS in hepatic fibrosis in patients with non-alcoholic steatohepatitis (NASH). METHODS: Primary hHSC were analysed for synthesis of endogenous ACh and acetylcholinesterase (AChE), and gene expression of choline acetyltransferase (ChAT) and muscarinic acetylcholine receptors (mAChR). Cell proliferation and fibrogenic markers were analysed in hHSC exposed to ACh, Atropine (Atrop), Mecamylamine (Mec), methoctramine and 4-Diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP). MAChR expression was analysed in human NASH scored for fibrosis. RESULTS: We observed that hHSC synthesise ACh and AChE, and express ChAT and M1-M5 mAChR. We also show that M2 was increased during NASH progression, while both M2 and M3 were found upregulated in activated hHSC. Furthermore, endogenous ACh is required for hHSC basal growth. Exogenous ACh resulted in hHSC hyperproliferation via mAChR and PI-3 K and MEK signalling pathways, as well as increased fibrogenic markers. CONCLUSION: We show that ACh regulates hHSC activation via M2 and M3 mAChR involving the PI-3 K and MEK pathways in vitro. Finally, we provide evidence that the PNS may be involved in human NASH fibrosis.

Epithelial to mesenchymal transition (EMT) of cancer cells involves loss of epithelial polarity and adhesiveness, and gain of invasive and migratory mesenchymal behaviours. EMT occurs in prostate cancer (PCa) but it is unknown whether this is in specific areas of primary tumours. We examined whether any of eleven EMT-related proteins have altered expression or subcellular localisation within the extraprostatic extension component of locally advanced PCa compared with other localisations, and whether similar changes may occur in in vitro organotypic PCa cell cultures and in vivo PCa models. Expression profiles of three proteins (E-cadherin, Snail, and α-smooth muscle actin) were significantly different in extraprostatic extension PCa compared with intra-prostatic tumour, and 18/27 cases had an expression change of at least one of these three proteins. Of the three significantly altered EMT proteins in pT3 samples, one showed similar significantly altered expression patterns in in vitro organotypic culture models, and two in in vivo Pten-/- model samples. These results suggest that changes in EMT protein expression can be observed in the extraprostatic extension component of locally invasive PCa. The biology of some of these changes in protein expression may be studied in certain in vitro and in vivo PCa models.

Background Prostate cancer (PCa) is a heterogeneous disease with a variable natural history, genetics, and treatment outcome. The Hedgehog (Hh) signaling pathway is increasingly recognized as being potentially important for the development and progression of PCa. In this retrospective study, we compared the activation status of the Hh signaling pathway between benign and tumor tissue, and evaluated the clinical significance of Hh signaling in PCa. Methods In this tissue microarray (TMA) study, the protein expression of several Hh signaling components and Hh target proteins, along with microvessel density, were compared between benign (n = 64) and malignant (n = 170) prostate tissue, and correlated with PCa clinicopathological characteristics and biochemical recurrence (BCR). Results The Hh signaling pathway appeared to be more active in PCa than in benign prostate tissue, as demonstrated by lower expression of the negative regulators PTCH1 and GLI3 in the tumor tissue compared to benign. In addition, high epithelial GLI2 expression correlated with higher pathological Gleason score. Overall, higher epithelial GLI3 expression in the tumor was shown to be an independent marker of a favorable prognosis. Conclusion Hh signaling activation might reflect aggressive tumoral behavior, since high epithelial GLI2 expression positively correlates with a higher pathological Gleason score. Moreover, higher epithelial GLI3 expression is an independent marker of a more favorable prognosis. Electronic supplementary material The online version of this article (10.1186/s12885-017-3619-4) contains supplementary material, which is available to authorized users.

The anti-diabetes drug metformin has been shown to have anti-neoplastic effects in several tumor models through its effects on energy metabolism and protein synthesis. Recent studies show that metformin also targets Hedgehog (Hh) signaling, a developmental pathway re-activated in several tumor types, including prostate cancer (PCa). Furthermore, we and others have shown that Hh signaling is an important target for radiosensitization. Here, we evaluated the combination of metformin and the Hh inhibitor GANT61 (GLI-ANTagonist 61) with or without ionizing radiation in three PCa cell lines (PC3, DU145, 22Rv1). The effect on proliferation, radiosensitivity, apoptosis, cell cycle distribution, reactive oxygen species production, DNA repair, gene and protein expression was investigated. Furthermore, this treatment combination was also assessed in vivo. Metformin was shown to interact with Hh signaling by inhibiting the effector protein glioma-associated oncogene homolog 1 (GLI1) in PCa cells both in vitro and in vivo. The combination of metformin and GANT61 significantly inhibited PCa cell growth in vitro and enhanced the radiation response of 22Rv1 cells compared to either single agent. Nevertheless, neither the growth inhibitory effect nor the radiosensitization effect of the combination treatment observed in vitro was seen in vivo. Although the interaction between metformin and Hh signaling seems to be promising from a therapeutic point of view in vitro, more research is needed when implementing this combination strategy in vivo.

Highlights • β-Blocker propranolol worsens liver injury in model of non-alcoholic steatohepatitis. • Mechanism of hepatic injury is via activation of apoptotic pathway in hepatocytes. • β-Blockers should be avoided or used with extreme caution in patients with NASH., Prazosin an α1-adrenoceptor (AR) antagonist has been shown to reduce liver injury in a mouse model of non-alcoholic steatohepatitis (NASH) and is suggested as a potential treatment of NASH especially given its concomitant anti-fibrotic properties. The effect however, of β-AR blockade in non-cirrhotic NASH is unknown and is as such investigated here. In the presence of the β-blocker propranolol (PRL), mice fed normal chow or a half methionine and choline deficient diet, supplemented with ethionine (HMCDE), to induce NASH, showed significantly enhanced liver injury, as evidenced by higher hepatic necrosis scores and elevated serum aminotransferases (ALT). Mechanistically, we showed that murine hepatocytes express α and β adrenoceptors; that PRL directly induces hepatocyte injury and death as evidenced by increased release of lactate dehydrogenase, FASL and TNF-α from hepatocytes in the presence of PRL; and that PRL activated the apoptotic pathway in primary hepatocyte cultures, as indicated by upregulation of Fas receptor and caspase-8 proteins. The β-AR antagonist PRL therefore appears to enhance liver injury through induction of hepatocyte death via the death pathway. Further studies are now required to extrapolate these findings to humans but meanwhile, β-AR antagonists should be avoided or used with caution in patients with non-cirrhotic NASH as they may worsen liver injury.

BACKGROUND Matrix metalloproteinase 9 (MMP9) has been implicated in extracellular matrix (ECM) remodelling, angiogenesis and inflammation. However, the targets for proteolysis that lead to these physiological consequences are often undefined as is the regulation of MMP9 itself. Therefore, identification of both the potential direct and indirect targets of MMP9 is critical for further understanding the effects of its proteolytic cascades. METHODS To study these cascades on a wider scale, transgenic mouse “knock-out” models and ultra-high performance liquid chromatography mass spectroscopy (UPLC-MSE) were used to elucidate the MMP9 targets, inhibitors, and interactors found in mouse seminal vesicle fluid (SVF). RESULTS Proteomics analysis of SVF from wild type, mmp9−/− or pn1−/− mice detected differences in serine protease inhibitors (serpins), reproductive proteins, developmental regulators, and cancer proto-oncogenes, including Renin 1/2. Protease nexin 1 (PN1), an ECM-based inhibitor of urokinase, was elevated in the SVF of mmp9−/− mice. We observed that MMP9-mediated N-terminal cleavage of PN1 reduces this serpin's functional activity. Our data also suggest a feedback loop in which inhibition of PN1 is a critical step in permitting greater activity of MMP9. CONCLUSION This study extends the degradome of MMP9 and examines components relevant to seminal fluid physiology. PN1 is proposed to be a novel inhibitor of MMP9 activity and a block to collagen cleavage, a frequent antecedent to cancer cell invasion. The interaction of MMP9 with PN1 and other serpins may lead to a better understanding of seminal vesicle function and possible impacts on fertility, as well as provide novel therapeutic targets. Prostate 73: 1427–1440, 2013. © 2013 Wiley Periodicals, Inc.

// Annelies Gonnissen 1 , Sofie Isebaert 1 , Chad M McKee 2 , Ruveyda Dok 1 , Karin Haustermans 1 , Ruth J Muschel 2 1 KU Leuven, University of Leuven, Department of Oncology, Experimental Radiotherapy; University Hospitals Leuven, Radiation Oncology, Leuven, Belgium 2 University of Oxford, Department of Oncology, CRUK/MRC Oxford Institute for Radiation Oncology, Oxford, UK Correspondence to: Annelies Gonnissen, email: annelies.gonnissen@kuleuven.be Keywords: prostate cancer, Hedgehog pathway, GANT61, radiotherapy, xenograft mouse model Received: July 06, 2016 Accepted: September 28, 2016 Published: October 05, 2016 ABSTRACT Limited data exists regarding the combination of Hedgehog signaling (Hh) inhibition and radiotherapy, even though there are several indications that this might be a promising treatment strategy. In this study, we evaluated the combination of two Hh inhibitors, the SMO inhibitor GDC-0449 and the GLI inhibitor GANT61 with radiotherapy in different prostate cancer (PCa) models. In vitro , GANT61 was able to sensitize 22Rv1 PCa cells but not PC3 and DU145 PCa cells. The lack of radiosensitization in the latter cell lines was shown to be dependent on the presence of mutated p53. Introduction of WT p53 into PC3 cells resulted in radiosensization following GANT61 treatment, suggesting that the p53 transcription factor plays an important role in the GANT61-induced radiosensitization in vitro . Targeting at the level of SMO (GDC-0449) did not show cytotoxicity or synergy with radiation. Furthermore, we confirmed the radiosensitization effect of GANT61 in two in vivo xenograft PCa models. The decrease in tumor growth was associated with decreased proliferation and increased apoptosis. In conclusion, we provide evidence that GANT61 in combination with radiation treatment might represent a promising therapeutic strategy for enhancing the radiation response of PCa patients.

Prostate adenocarcinoma (CaP) patients are classified into low-, intermediate-, and high-risk groups that reflect relative survival categories. While there are accepted treatment regimens for low- and high-risk patients, intermediate-risk patients pose a clinical dilemma, as treatment outcomes are highly variable for these individuals. A better understanding of the factors that regulate the progression of CaP is required to delineate risk. For example, aberrant activation of the Hedgehog (Hh) pathway is implicated in CaP progression. Here, we identify the serine protease inhibitor protease nexin 1 (PN1) as a negative regulator of Hh signaling in prostate. Using human CaP cell lines and a mouse xenograft model of CaP, we demonstrate that PN1 regulates Hh signaling by decreasing protein levels of the Hh ligand Sonic (SHH) and its downstream effectors. Furthermore, we show that SHH expression enhanced tumor growth while overexpression of PN1 inhibited tumor growth and angiogenesis in mice. Finally, using comparative genome hybridization, we found that genetic alterations in Hh pathway genes correlated with worse clinical outcomes in intermediate-risk CaP patients, indicating the importance of this pathway in CaP.

Matrix metalloproteinase-9 (MMP-9) expression is known to enhance the invasion and metastasis of tumor cells. In previous work based on a proteomic screen, we identified the serpin protease nexin-1 (PN-1) as a potential target of MMP-9. Here, we show that PN-1 is a substrate for MMP-9 and establish a link between PN-1 degradation by MMP-9 and regulation of invasion. PN-1 levels increased in prostate carcinoma cells after downregulation of MMP-9 and in tissues of MMP-9–deficient mice, consistent with PN-1 degradation by MMP-9. We identified three MMP-9 cleavage sites in PN-1 and showed that mutations in those sites made PN-1 more resistant to MMP-9. Urokinase plasminogen activator (uPA) is inhibited by PN-1. MMP-9 augmented uPA activity in the medium of PC3-ML cells by degrading PN-1. Prostate cancer cells, overexpressing PN-1 or treated with MMP-9 shRNA, had reduced cell invasion in Matrigel. PN-1 siRNA restored uPA activity and the invasive capacity. PN-1 mutated in the serpin inhibitory domain, the reactive center loop, failed to inhibit uPA and to reduce Matrigel invasion. This study shows a novel molecular pathway in which MMP-9 regulates uPA activity and tumor cell invasion through cleavage of PN-1. Cancer Res; 70(17); 6988–98. ©2010 AACR.

Highlights: Black-Right-Pointing-Pointer Cigarette smoke may induce liver fibrosis via nicotine receptors. Black-Right-Pointing-Pointer Nicotine induces proliferation of hepatic stellate cells (HSCs). Black-Right-Pointing-Pointer Nicotine activates hepatic fibrogenic pathways. Black-Right-Pointing-Pointer Nicotine receptor antagonists attenuate HSC proliferation. Black-Right-Pointing-Pointer Nicotinic receptor antagonists may have utility as novel anti-fibrotic agents. -- Abstract: Background and aims: Cigarette smoke (CS) may cause liver fibrosis but possible involved mechanisms are unclear. Among the many chemicals in CS is nicotine - which affects cells through nicotinic acetylcholine receptors (nAChR). We studied the effects of nicotine, and involved pathways, on human primary hepatic stellate cells (hHSCs), the principal fibrogenic cells in the liver. We then determined possible disease relevance by assaying nAChR in liver samples from human non-alcoholic steatohepatitis (NASH). Methods: hHSC were isolated from healthy human livers and nAChR expression analyzed - RT-PCR and Western blotting. Nicotine induction of hHSC proliferation, upregulation of collagen1-{alpha}2 and the pro-fibrogenic cytokine transforming growth factor beta 1 (TGF-{beta}1) was determined along with involved intracellular signaling pathways. nAChR mRNA expression was finally analyzed in whole liver biopsies obtained from patients diagnosed with non-alcoholic steatohepatitis (NASH). Results: hHSCs express muscle type ({alpha}1, {beta}1, delta and epsilon) and neuronal type ({alpha}3, {alpha}6, {alpha}7, {beta}2 and {beta}4) nAChRmore » subunits at the mRNA level. Among these subunits, {alpha}3, {alpha}7, {beta}1 and {epsilon} were predominantly expressed as confirmed by Western blotting. Nicotine induced hHSC proliferation was attenuated by mecamylamine (p < 0.05). Additionally, collagen1-{alpha}2 and TGF-{beta}1 mRNA expression were significantly upregulated by nicotine and inhibited by mecamylamine. {alpha}1 and {alpha}3-nAChR mRNA expression was significantly upregulated in NASH fibrosis compared to normal livers. Conclusion: Nicotine at levels in smokers' blood is pro-fibrogenic, through actions on hHSCs expressed nAChRs. Therefore, CS, via its nicotine content, may worsen liver fibrosis. Moreover, nicotinic receptor antagonists may have utility as novel anti-fibrotic agents.« less

Background & aims Obesity induced, non-alcoholic fatty liver disease (NAFLD), is now the major cause in affluent countries, of the spectrum of steatosis-to-cirrhosis. Obesity and NAFLD rates in reproductive age women, and adolescents, are rising worldwide. Our hypothesis was that maternal obesity and lactation transmit to the offspring a pre-disposition to dysmetabolism, obesity and NAFLD. Methods Female mice were fed standard or obesogenic chow, before, throughout pregnancy, and during lactation. The critical developmental period was studied by cross-fostering offspring of lean and obese dams. Offspring were then weaned onto standard chow and studied at 3months. Read-outs included markers of metabolic dysfunction, biochemical and histological indicators of NAFLD, induction of liver fibrogenesis, and activation of pro-fibrotic pathways. Mechanisms involved in programming a dysmetabolic and NAFLD phenotype were investigated by assaying breast milk components. Results Offspring of obese dams had a dysmetabolic, insulin resistant and NAFLD phenotype compared to offspring of lean dams. Offspring of lean dams that were suckled by obese dams showed an exaggerated dysmetabolic and NAFLD phenotype, with increased body weight, as well as increased levels of insulin, leptin, aspartate transaminase, interleukin-6, tumour necrosis factor-alpha, liver triglycerides, steatosis, hepatic fibrogenesis, renal norepinephrine, and liver alpha1-D plus beta1-adrenoceptors, indicative of sympathetic nervous system activation. Obese dams also had raised breast milk leptin levels compared to lean dams. Conclusions Maternal obesity programs development of a dysmetabolic and NAFLD phenotype, which is critically dependent on the early postnatal period and possibly involving alteration of hypothalamic appetite nuclei signalling by maternal breast milk and neonatal adipose tissue derived, leptin.

Background and aims The prevalence of pancreatic adenocarcinoma (PAC) parallels rising rates of obesity and dysmetabolism, a possible link being non-alcoholic fatty pancreas disease (NAFPD). We have recently shown that maternal obesity programmes the development of a dysmetabolic and fatty liver (non-alcoholic fatty liver disease, NAFLD) phenotype in adult offspring. Since the pancreas and liver originate from the same embryonic bud, it is plausible that maternal obesity may similarly programme the development of NAFPD. Our objective was to determine the effect of maternal obesity on development of NAFPD in offspring and ascertain contributions of the intra/extra-uterine periods. Methods Female C57BL/6J mice were fed either a standard chow (3% fat, 7% sugar) or a hypercalorific diet (16% fat, 33% sugar) for six weeks prior to mating and throughout pregnancy and lactation. Female offspring were cross-fostered for suckling to dams on the same or opposite diet to yield four groups: offspring of lean suckled by lean dams (n = 6), offspring of obese suckled by obese dams (n = 6), offspring of lean suckled by obese dams (n = 5) and offspring of obese suckled by lean dams (n = 6). All offspring were weaned onto a standard chow diet at 21 days and sacrificed at 3 months post-partum for tissue collection. Results Offspring subjected to an adverse suckling environment showed significant increases in body weight, pancreatic triglyceride content, TGF-β, collagen gene expression and SBP at rest along with an enhanced restraint stress response, indicating a dysmetabolic and NAFPD phenotype. Conclusions Developmental programming is involved in the pathogenesis of NAFPD and appears to be largely dependent on an adverse extra-uterine environment.

395 Background: Telotristat ethyl (TE) is approved to treat inadequately controlled carcinoid syndrome (CS) diarrhea in combination with somatostatin analog (SSA) therapy. Understanding the time to onset of sustained BM frequency improvement is important when considering the use of TE in patients with CS diarrhea. We present data on time to sustained improvement and worsening in BM frequency during the double-blind treatment (DBT) period of TELECAST, a phase 3, randomized, placebo-controlled, double-blind study in patients with CS that required additional treatment, primarily due to gastrointestinal symptoms. Methods: Stable dose SSA was not required. Patients were treated with TE 250 mg 3 times per day (tid), TE 500 mg tid, or placebo tid. Sustained response was defined as the time from the first double-blind dose date to the first day of 2 consecutive weeks with BM frequency change of at least 30% from baseline. The time to the first sustained response in the TELECAST study was examined among treatment groups and analyzed using Cox regression. Results: There were 26 patients on placebo and 25 patients on either 250 mg or 500 mg telotristat groups. Sustained improvement in BM frequency over the 12-week DBT period was achieved in 6, 16, and 16 patients on placebo, TE 250 mg, and TE 500 mg treatment groups. Median time to sustained ≥ 30% improvement was 4-5 weeks with TE at both dosing levels, with no median reached on placebo. First occurrence of sustained improvements in BM frequency occurred within 4-68 days. Time to sustained worsening was delayed on the 500-mg dose of TE (no percentile reached) and is statistically significant (p = 0.021, HR: 0.25) Conclusions: Treatment with telotristat ethyl may provide sustained BM frequency reduction within 4-5 weeks (median) of initiating therapy. No median was reached on placebo due to fewer patients with sustained improvement. Additionally, TE 500 mg tid may also offer additional clinical benefit by reducing sustained BM worsening. Clinical trial information: NCT02063659.

INTRODUCTION AND OBJECTIVES: Cellular senescence occurs in response to sub-lethal stress after androgen deprivation therapy, radiation and chemotherapy whereby cells exit the cell cycle and develop distinct morphologic and biochemical characteristics. Senescence is a therapeutic approach, however potentially deleterious consequences make elimination of these cells important. The objective of this study was to determine whether the increased gluconeogenesis induced during senescence could be exploited to synergistically eliminate PCa cells using metformin (Met), an oral biguanide hyperglycemic approved for type II diabetes. METHODS: High throughput screening of small molecule collections identified two novel robust senescence inducing agents, AZQ (diaziquone) a quinone and AZD5438 a novel CDK inhibitor. Senescence was confirmed using beta-galactosidase staining, cell arrest and increased size by flow cytometry. mTOR a serine-threonine protein kinase activity was evaluated using western. PCa cells were seeded on 96-well plates, low(L) and high(H) AZQ (0.1 and 0.25 microM) or AZD 5438 (0.1 and 0.4 microM) concentrations administered and after 3 days of senescence induction Met (1 and 5mM) added. Cells were collected 24 and 72h later and stained with Hoechts to evaluate the cell number. CalcuSyn software (Biosoft, Cambridge, UK) modeled synergism. RESULTS: AZQ and AZD robustly increased beta-galactosidase staining, cell size and p27 and p16 induction in Du145, PC3, PPC1 cell lines. mTOR signaling through mTOR complex 1 significantly increased in AZQ-induced senescent cells. Used alone, AZD(L) and AZQ(L) demonstrate a 30% reduction in cell number and Met(L) alone 18%. Met after senescence induction (72h) decreased cell number 70% and 55% (AZD and AZQ respectively). CalcuSyn calculations revealed moderate synergistic effects with combinations of either AZD(H) or AZQ (L and H) and Met(L and H) at 24hr. A strong synergistic decrease in cell viability was demonstrated at 72h with AZQ(H) and Met(L) (CI1⁄4 0.267). CONCLUSIONS: Senescent cells targeted by metformin may offer an alternative treatment strategy for removing castrate-resistant PCa cells. Increased gluconeogenesis during senescence represents a novel ‘Achilles heel’ for these dormant/arrested cancer cells. mTOR activity is activated during the conversion of cells to senescence, and Met inhibition of this pathway is a putative mechanism. Our results suggest that combination treatment with a senescence-inducing drug and metformin may be a feasible targeted therapeutic approach.

Protease nexin 1 (PN1), a member of serine protease inhibitors (SERPINs) family, is known for its ability to bind and inhibit a wide range of proteases. Recently, we have found that PN1 is able to induce prostate cancer cell undergoing apoptosis through a distinct mechanism of engaging uPA-uPAR complexes and the regulation of two independent downstream signaling cascades, leading to the repression of X-linked inhibitor of apoptosis protein (XIAP). In this process, PN1 expression reduces the expression of NF-κB signalling component p65 and thereby lessens xiap transcription. Alternately, PN1 activity can prevent the stability of XIAP by reducing XIAP phosphorylation at serine 87 via a blockade of AKT signaling. A combination of exogenous PN1 and TRAIL leads to a substantial growth lag in prostate cancer xenografts, indicating the potential of PN1 as a promising target for improving prostate cancer therapy. Furthermore, human prostate tissue arrays show inverse levels of PN1 and XIAP in tumor and normal prostate. Hence, the PN1-uPA regulatory axis may serve as an inducer of tumor cell apoptosis by modulating survival pathways, and therefore delay the tumor growth of prostate cancer.

Amphiregulin (AR) involvement in liver fibrogenesis and hepatic stellate cells (HSC) regulation is under study. Non-alcoholic fatty liver disease (NAFLD) and its more severe form non-alcoholic steatohepatitis (NASH) may progress to cirrhosis and hepatocellular cancer (HCC). Our aim was to investigate ex vivo the effect of AR on human primary HSC (hHSC) and verify in vivo the relevance of AR in NAFLD fibrogenesis. hHSC isolated from healthy liver segments were analyzed for expression of AR and its activator, TNF-α converting enzyme (TACE). AR induction of hHSC proliferation and matrix production was estimated in the presence of antagonists. AR involvement in fibrogenesis was also assessed in a mouse model of NASH and in humans with NASH. hHSC time dependently expressed AR and TACE. AR increased hHSC proliferation through several mitogenic signaling pathways such as EGFR, PI3K and p38. AR also induced marked upregulation of hHSC fibrogenic markers and reduced hHSC death. AR expression was enhanced in the HSC of a murine model of NASH and of severe human NASH. In conclusion, AR induces hHSC fibrogenic activity via multiple mitogenic signaling pathways and is upregulated in murine and human NASH, suggesting that AR antagonists may be clinically useful anti-fibrotics in NAFLD.

The parasympathetic nervous system (PNS), via neurotransmitter acetylcholine (ACh), modulates fibrogenesis in animal models. However, the role of ACh in human hepatic fibrogenesis is unclear.We aimed to determine the fibrogenic responses of human hepatic stellate cells (hHSC) to ACh and the relevance of the PNS in hepatic fibrosis in patients with non-alcoholic steatohepatitis (NASH).Primary hHSC were analyzed for synthesis of endogenous ACh and acetylcholinesterase and gene expression of choline acetyltransferase and muscarinic ACh receptors (mAChR). Cell proliferation and fibrogenic markers were analyzed in hHSC exposed to ACh, atropine, mecamylamine, methoctramine, and 4-diphenylacetoxy-N-methylpiperidine methiodide. mAChR expression was analyzed in human NASH scored for fibrosis.We observed that hHSC synthesize ACh and acetylcholinesterase and express choline acetyltransferase and M1-M5 mAChR. We also show that M2 was increased during NASH progression, while both M2 and M3 were found upregulated in activated hHSC. Furthermore, endogenous ACh is required for hHSC basal growth. Exogenous ACh resulted in hHSC hyperproliferation via mAChR and phosphoinositide 3-kinase and Mitogen-activated protein kinase kinase (MEK) signaling pathways, as well as increased fibrogenic markers.We show that ACh regulates hHSC activation via M2 and M3 mAChR involving the phosphoinositide 3-kinase and MEK pathways in vitro. Finally, we provide evidence that the PNS may be involved in human NASH fibrosis.

Previously we reported that testicular germ cells undergo FAS-mediated apoptosis after exposure of mice to the Sertoli cell toxicant mono-(2-ethylhexyl) phthalate (MEHP) and that this process is partially dependent on the TRP53 protein (p53). Recent reports have suggested that TRP53 may influence the ubiquitinylation and consequent proteosomal degradation of a negative regulator of FAS, CFLAR (L) (c-FLIP [L]), in human colon cancer cells. To further characterize the relationship between CFLAR and TRP53, we used the transformed germ cell line GC-2spd (ts), which harbors a temperature-sensitive Trp53 mutation that allows for TRP53 activation at 328C. We report here that GC-2 cells expressed a 10-fold increase in basal cell membrane FAS levels and an increased sensitivity to FAS agonistic antibody (JO2)-triggered apoptosis only when they were maintained at the permissive TRP53 temperature. After JO2 exposure, CFLAR (L) protein levels were enhanced only at the nonpermissive TRP53 temperature (378C) while real-time PCR results indicated an absence of Cflar (L) mRNA changes in GC-2 cells regardless of the temperature. Furthermore, transfection of GC-2 cells at 378C with siRNA against Cflar resulted in reduction of CFLAR (L) protein levels and increased sensitivity to JO2-mediated apoptosis. The CFLAR (L) protein was also more strongly ubiquitinylated in response to JO2 treatment at the permissive TRP53 temperature. Taken together, these data suggest that the TRP53 protein influences the sensitivity of GC-2 cells to undergo FAS-mediated apoptosis by modulating the expression of FAS on their cell membranes and subsequently influencing the degradation of the antiapoptotic protein CFLAR (L). apoptosis, CFLAR (c-FLIP), FAS, GC-2 cells, signal transduction, testis, toxicology, TRP53, ubiquitinylation

// Chad M. McKee 2, * , Yunchuan Ding 1, * , Jianfeng Zhou 1 , Chunrui Li 1 , Liang Huang 1 , Xiangke Xin 1 , Jing He 1 , Joshua E. Allen 3 , Wafik S. El-Deiry 3 , Yunhong Cao 2 , Ruth J. Muschel 2 , Danmei Xu 1 1 Department of Internal Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Hubei, China 2 Gray Institute of Radiation Oncology and Biology, Medical Science Division, University of Oxford, Oxford, United Kingdom 3 Penn State Hershey Cancer Institute, Penn State University, Hershey, PA, USA * These authors have contributed equally to this work Correspondence to: Ruth J. Muschel, e-mail: ruth.muschel@oncology.ox.ac.uk Danmei Xu, e-mail: xudanmei99@yahoo.co.uk Keywords: PN1, XIAP, prostate cancer, NF-κB, AKT Received: November 18, 2014 Accepted: December 11, 2014 Published: February 17, 2015 ABSTRACT Protease nexin 1 (PN1) is an endogenous serine protease inhibitor (SERPIN), expressed at high levels in the prostate, and capable of inhibiting the proliferation of prostate cancer cells. We previously showed that PN1-uPA complexes inhibited Sonic Hedgehog (SHH) signalling through engagement of the LRP receptor. Here, we describe an alternative anti-proliferative mechanism through which PN1 expression leads to apoptosis. In prostate cancer cells, increased expression of PN1 led to substantial reduction of XIAP levels and apoptosis mediated through the uPAR, but not the LRP receptor. The alterations in XIAP were effected in two ways 1) via alteration in the NF-κB pathway, a pathway known to signal XIAP transcription and 2) by promoting XIAP instability. The AKT pathway is known to phosphorylate XIAP at serine 87 leading to protein stability and PN1 expression is shown to interfere with this process. As a result of both mechanisms, programmed cell death is substantially increased. Consistent with these observations, reduced PN1 protein correlated with elevated p65/XIAP expression and with higher Gleason scores in human prostate tissue arrays. Thus, PN1 expression appears to differentially down-regulate distinct oncogenic pathways depending upon the cell surface receptor engaged by its complexes and demonstrates a novel molecular mechanism by which the protein can promote tumor cell apoptosis.

Background Sympathetic nervous system (SNS) signalling regulates murine hepatic fibrogenesis through effects on hepatic stellate cells (HSC), and obesity-related hypertension with SNS activation accelerates progression of non-alcoholic fatty liver disease (NAFLD), the commonest cause of chronic liver disease. NAFLD may lead to cirrhosis. The effects of the SNS neurotransmitters norepinephrine (NE), epinephrine (EPI) and neuropeptide Y (NPY) on human primary HSC (hHSC) function and in NAFLD pathogenesis are poorly understood. Aims to determine the mechanistic effects of NE/EPI/NPY on phenotypic changes in cultured hHSC, and to study SNS signalling in human NAFLD livers. Methods Freshly isolated hHSC were assessed for expression of cathecholamine/neuropeptide Y receptors and for the synthesis of NE/EPI. The effects of NE/EPI/NPY and adrenoceptor antagonists prazosin (PRZ)/propranolol (PRL) on hHSC fibrogenic functions and the involved kinases and interleukin pathways were examined. Human livers with proven NAFLD were then assessed for upregulation of SNS signalling components. Results Activated hHSC express functional α/β-adrenoceptors and NPY receptors, which are upregulated in the livers of patients with cirrhotic NAFLD. hHSC in culture synthesize and release NE/EPI, required for their optimal basal growth and survival. Exogenous NE/EPI and NPY dose-dependently induced hHSC proliferation, mediated via p38 MAP, PI3K and MEK signalling. NE and EPI but not NPY increased expression of collagen-1α2 via TGF-β without involvement of the pro-fibrogenic cytokines leptin, IL-4 and IL-13 or the anti-fibrotic cytokine IL-10. Conclusions hHSC synthesize and require cathecholamines for optimal survival and fibrogenic functionality. Activated hHSC express directly fibrogenic α/β-adrenoceptors and NPY receptors, upregulated in human cirrhotic NAFLD. Adrenoceptor and NPY antagonists may be novel anti-fibrotic agents in human NAFLD.

Hydrocarbon radical stabilization energy (RSE) estimates based on the differences in R-H vs CH(3)-H bond dissociation energies have inherent advantages over RSEs based on R-CH(3) vs CH(3)-CH(3), as well as R-R vs CH(3)-CH(3) comparisons, since the R-CH(3) and R-R reference systems are prone to unbalanced contaminating intramolecular interactions involving the R groups. When the effects of steric crowding, branching, protobranching, conjugation, and hyperconjugation are taken into account, R-CH(3) and R-R based RSE values are nearly identical to R-H RSEs. Corrections for electronegativity differences between H and R are not needed to achieve agreement.

The ability of the TRAIL/DR5 signaling pathway to induce apoptosis has generally been limited to tumor cells. Here we report that in primary testis explants, addition of TRAIL (0.5 mug/ml) caused a three-fold increase in germ cell apoptosis. Furthermore, exposure of C57BL/6 mice to the testicular toxicant, mono-(2-ethylhexyl) phthalate (MEHP), caused an increased p53 stability and elevated DR5 mRNA levels coincident with increases in the levels of apoptosis in spermatocytes. To further assess the mechanisms responsible for the sensitivity of germ cells to undergo TRAIL/DR5-mediated apoptosis, we used the germ cell lines GC-1spg and GC-2spd(ts) (a temperature sensitive spermatocyte-like cell line that allows for p53 nuclear localization at 32 degrees C but not 37 degrees C). Addition of TRAIL and the anti-DR5 monoclonal antibody, MD5-1, triggered a robust synergistic increase of apoptosis in p53 permissive GC-2 cells (32 degrees C) but not in GC-1 cells. In addition, DR5 levels on the plasma membrane of permissive cells were considerably enhanced concomitant with p53 expression and after MD5-1 treatment. These data represent the first indication that testicular germ cells, specifically spermatocytes, can undergo TRAIL-mediated apoptosis and the clinically relevant observation that pretreatment with a DR5 monoclonal antibody can greatly sensitize their apoptotic response to TRAIL.

Prostate adenocarcinoma is the most frequent cancer and is the second leading cause of cancer death of men in the Western world. Aberrant activation of the Hedgehog (Hh) pathway is implicated in the growth and progression of prostate cancer. We show that the Hh pathway is inhibited by the serine protease inhibitor, protease nexin 1 (PN1), an extracellular matrix (ECM) protein highly expressed in normal prostate. We demonstrate here that PN1 regulates Hh signalling by reduction of the Hh ligand, Sonic (SHH), and its downstream effectors in tissue culture and in vivo. Conversely, matrix metalloproteinase 9 (MMP9) enhances Hh signalling through degradation of PN1. MRI imaging was used to determine variation in tumour formation in an orthotopic prostate tumour model using PN1 and MMP9 knock-out mice. In PN1-/- animals, SHH was increased and tumor growth was stimulated. In contrast, PN1 levels were elevated in MMP9-/- mice while Hh signalling was reduced with concomitant decreased tumor growth. Finally, genetic alterations in Hh pathway genes identified by comparative genomic hybridization (CGH) from intermediate risk prostate cancer patients correlated with a worse clinical outcome (HR=2.67, p=0.00067), indicating the importance of this pathway in human cancer. Our data identify PN1 as a novel regulator of SHH and its downstream targets allowing PN1 levels to influence prostate tumor growth. Regulation of the PN1-Hh pathway could constitute a significant therapeutic advance in treating cancers that exhibit high expression of Hh markers. These data also provide a mechanism through which MMP-9 can regulate hedgehog pathway signalling in tissues rich in PN1. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2463. doi:1538-7445.AM2012-2463