The Impact of Blue Light Cystoscopy Use Among Nonmuscle Invasive Bladder Cancer Patients in an Equal Access Setting: Implications on Recurrence and Time to Recurrence.

INTRODUCTION: Prior studies suggest that white light cystoscopy (WLC) alone can fail to detect cases of non-muscle invasive bladder cancer (NMIBC) vs. blue light cystoscopy (BLC). We describe bladder cancer outcomes and the impact of BLC among NMIBC patients in an equal access setting. MATERIALS AND METHODS: We assessed 378 NMIBC patients within the Veterans Affairs system that had a CPT code for BLC from December 1, 2014 to December 31, 2020. We determined recurrence rates and time to recurrence prior to BLC (ie, after previous WLC if available) and following BLC. We used the Kaplan-Meier method to estimate event-free survival and Cox regression to determine association between BLC and recurrence, progression, and overall survival; and further, whether these outcomes differed by race. 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. Median time to first recurrence following BLC was longer vs. WLC alone (40 [33-NE] vs. 26 [17-39] months). Recurrence risk 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 (HR 0.69; 95% CI, 0.39-1.20), progression (HR 1.13; 95% CI, 0.32-3.96), and overall survival (HR 0.74; 95% CI, 0.31-1.77) following BLC by Black vs. White race. CONCLUSION: In this study from an equal access setting in the VA, we observed significantly decreased recurrence risk and prolonged time interval to recurrence following BLC vs. WLC alone. There was no difference in bladder cancer outcomes by race.

Time to Sustained Improvement in Bowel Movement Frequency with Telotristat Ethyl: Analyses of Phase III Studies in Carcinoid Syndrome.

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.

Serotonin levels and 1-year mortality in patients with neuroendocrine tumors: a systematic review and meta-analysis.

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.

Patched 1 Expression Correlates with Biochemical Relapse in High-Risk Prostate Cancer Patients.

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.

Tissue microarray analysis indicates hedgehog signaling as a potential prognostic factor in intermediate-risk prostate cancer.

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.

The Effect of Metformin and GANT61 Combinations on the Radiosensitivity of Prostate Cancer Cells.

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.

The hedgehog inhibitor GANT61 sensitizes prostate cancer cells to ionizing radiation both in vitro and in vivo.

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.

Altered expression of epithelial-to-mesenchymal transition proteins in extraprostatic prostate cancer.

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.

Acetylcholine induces fibrogenic effects via M2/M3 acetylcholine receptors in non-alcoholic steatohepatitis and in primary human hepatic stellate cells.

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 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. RESULTS: 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. CONCLUSION: 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.

Amphiregulin activates human hepatic stellate cells and is upregulated in non alcoholic steatohepatitis.

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.

Protease nexin 1 induces apoptosis of prostate tumor cells through inhibition of X-chromosome-linked inhibitor of apoptosis protein.

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.

Sympathetic nervous system catecholamines and neuropeptide Y neurotransmitters are upregulated in human NAFLD and modulate the fibrogenic function of hepatic stellate cells.

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/ß-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-ß 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/ß-adrenoceptors and NPY receptors, upregulated in human cirrhotic NAFLD. Adrenoceptor and NPY antagonists may be novel anti-fibrotic agents in human NAFLD.

Propranolol, a ß-adrenoceptor antagonist, worsens liver injury in a model of non-alcoholic steatohepatitis.

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 ß-AR blockade in non-cirrhotic NASH is unknown and is as such investigated here. In the presence of the ß-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 ß 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 ß-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, ß-AR antagonists should be avoided or used with caution in patients with non-cirrhotic NASH as they may worsen liver injury.

Proteomic analysis reveals a proteolytic feedback loop in murine seminal fluid.

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-MS(E) ) 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.

Protease nexin 1 inhibits hedgehog signaling in prostate adenocarcinoma.

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.

Nicotine induces fibrogenic changes in human liver via nicotinic acetylcholine receptors expressed on hepatic stellate cells.

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-α2 and the pro-fibrogenic cytokine transforming growth factor beta 1 (TGF-ß1) 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 (α1, ß1, delta and epsilon) and neuronal type (α3, α6, α7, ß2 and ß4) nAChR subunits at the mRNA level. Among these subunits, α3, α7, ß1 and ε were predominantly expressed as confirmed by Western blotting. Nicotine induced hHSC proliferation was attenuated by mecamylamine (p<0.05). Additionally, collagen1-α2 and TGF-ß1 mRNA expression were significantly upregulated by nicotine and inhibited by mecamylamine. α1 and α3-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.

Matrix metalloproteinase-9 regulates tumor cell invasion through cleavage of protease nexin-1.

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.

Maternal obesity during pregnancy and lactation programs the development of offspring non-alcoholic fatty liver disease in mice.

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.