Stanniocalcin-1 Regulates Extracellular ATP-Induced Calcium Waves in Human Epithelial Cancer Cells by Stimulating ATP Release from Bystander Cells

Background: The epithelial cell response to stress involves the transmission of signals between contiguous cells that can be visualized as a calcium wave. In some cell types, this wave is dependent on the release of extracellular trinucleotides from injured cells. In particular, extracellular ATP has been reported to be critical for the epithelial cell response to stress and has recently been shown to be upregulated in tumors in vivo. Methodology/Principal Findings: Here, we identify stanniocalcin-1 (STC1), a secreted pleiotrophic protein, as a critical mediator of calcium wave propagation in monolayers of pulmonary (A549) and prostate (PC3) epithelial cells. Addition of STC1 enhanced and blocking STC1 decreased the distance traveled by an extracellular ATP-dependent calcium wave. The same effects were observed when calcium was stimulated by the addition of exogenous ATP. We uncover a positive feedback loop in which STC1 promotes the release of ATP from cells in vitro and in vivo. Conclusions/Significance: The results indicated that STC1 plays an important role in the early response to mechanical injury by epithelial cells by modulating signaling of extracellular ATP. This is the first report to describe STC1 as a modulator o

Stanniocalcin 2 alters PERK signalling and reduces cellular injury during cerulein induced pancreatitis in mice

BACKGROUND: Stanniocalcin 2 (STC2) is a secreted protein activated by (PKR)-like Endoplasmic Reticulum Kinase (PERK) signalling under conditions of ER stress in vitro. Over-expression of STC2 in mice leads to a growth-restricted phenotype; however, the physiological function for STC2 has remained elusive. Given the relationship of STC2 to PERK signalling, the objective of this study was to examine the role of STC2 in PERK signalling in vivo. RESULTS: Since PERK signalling has both physiological and pathological roles in the pancreas, STC2 expression was assessed in mouse pancreata before and after induction of injury using a cerulein-induced pancreatitis (CIP) model. Increased Stc2 expression was identified within four hours of initiating pancreatic injury and correlated to increased activation of PERK signalling. To determine the effect of STC2 over-expression on PERK, mice systemically expressing human STC2 (STC2Tg) were examined. STC2Tg pancreatic tissue exhibited normal pancreatic morphology, but altered activation of PERK signalling, including increases in Activating Transcription Factor (ATF) 4 accumulation and autophagy. Upon induction of pancreatic injury, STC2Tg mice exhibited limited increases in circulating amylase levels and increased maintenance of cellular junctions. CONCLUSIONS: This study links STC2 to the pathological activation of PERK in vivo, and suggests involvement of STC2 in responding to pancreatic acinar cell injury

TGFβ signaling regulates Epithelial-mesenchymal plasticity in ovarian cancer ascites-derived spheroids

Epithelial-mesenchymal transition (EMT) serves as a key mechanism driving tumor cell migration, invasion, and metastasis in many carcinomas. Transforming growth factor-beta (TGFβ) signaling is implicated in several steps during cancer pathogenesis and acts as a classical inducer of EMT. Since epithelial ovarian cancer (EOC) cells have the potential to switch between epithelial and mesenchymal states during metastasis, we predicted that modulation of TGFβ signaling would significantly impact EMT and the malignant potential of EOC spheroid cells. Ovarian cancer patient ascites-derived cells naturally underwent an EMT response when aggregating into spheroids, and this was reversed upon spheroid re-attachment to a substratum. CDH1/E-cadherin expression was markedly reduced in spheroids compared with adherent cells, in concert with an up-regulation of several transcriptional repressors, i.e., SNAI1/Snail, TWIST1/2, and ZEB2. Treatment of EOC spheroids with the TGFβ type I receptor inhibitor, SB-431542, potently blocked the endogenous activation of EMT in spheroids. Furthermore, treatment of spheroids with SB-431542 upon re-attachment enhanced the epithelial phenotype of dispersing cells and significantly decreased cell motility and Transwell migration. Spheroid formation was significantly compromised by exposure to SB-431542 that correlated with a reduction in cell viability particularly in combination with carboplatin treatment. Thus, our findings are the first to demonstrate that intact TGFb signaling is required to control EMT in EOC ascites-derived cell spheroids, and it promotes the malignant characteristics of these structures. As such, we show the therapeutic potential for targeted inhibition of this pathway in ovarian cancer patients with late-stage disease

Spatial and temporal epithelial ovarian cancer cell heterogeneity impacts Maraba virus oncolytic potential

Background: Epithelial ovarian cancer exhibits extensive interpatient and intratumoral heterogeneity, which can hinder successful treatment strategies. Herein, we investigated the efficacy of an emerging oncolytic, Maraba virus (MRBV), in an in vitro model of ovarian tumour heterogeneity. Methods: Four ovarian high-grade serous cancer (HGSC) cell lines were isolated and established from a single patient at four points during disease progression. Limiting-dilution subcloning generated seven additional subclone lines to assess intratumoral heterogeneity. MRBV entry and oncolytic efficacy were assessed among all 11 cell lines. Low-density receptor (LDLR) expression, conditioned media treatments and co-cultures were performed to determine factors impacting MRBV oncolysis. Results: Temporal and intratumoral heterogeneity identified two subpopulations of cells: one that was highly sensitive to MRBV, and another set which exhibited 1000-fold reduced susceptibility to MRBV-mediated oncolysis. We explored both intracellular and extracellular mechanisms influencing sensitivity to MRBV and identified that LDLR can partially mediate MRBV infection. LDLR expression, however, was not the singular determinant of sensitivity to MRBV among the HGSC cell lines and subclones. We verified that there were no apparent extracellular factors, such as type I interferon responses, contributing to MRBV resistance. However, direct cell-cell contact by co-culture of MRBV-resistant subclones with sensitive cells restored virus infection and oncolytic killing of mixed population. Conclusions: Our data is the first to demonstrate differential efficacy of an oncolytic virus in the context of both spatial and temporal heterogeneity of HGSC cells and to evaluate whether it will constitute a barrier to effective viral oncolytic therapy

Evidence for Differential Viral Oncolytic Efficacy in an In Vitro Model of Epithelial Ovarian Cancer Metastasis

Epithelial ovarian cancer is unique among most carcinomas in that metastasis occurs by direct dissemination of malignant cells traversing throughout the intraperitoneal fluid. Accordingly, we test new therapeutic strategies using an in vitro three-dimensional spheroid suspension culture model that mimics key steps of this metastatic process. In the present study, we sought to uncover the differential oncolytic efficacy among three different viruses—Myxoma virus, double-deleted vaccinia virus, and Maraba virus—using three ovarian cancer cell lines in our metastasis model system. Herein, we demonstrate that Maraba virus effectively infects, replicates, and kills epithelial ovarian cancer (EOC) cells in proliferating adherent cells and with slightly slower kinetics in tumor spheroids. Myxoma virus and vaccinia viruses infect and kill adherent cells to a much lesser extent than Maraba virus, and their oncolytic potential is almost completely attenuated in spheroids. Myxoma virus and vaccinia are able to infect and spread throughout spheroids, but are blocked in the final stages of the lytic cycle, and oncolytic-mediated cell killing is reactivated upon spheroid reattachment. Alternatively, Maraba virus has a remarkably reduced ability to initially enter spheroid cells, yet rapidly infects and spreads throughout spheroids generating significant cell killing effects. We show that low-density lipoprotein receptor expression in ovarian cancer spheroids is reduced and this controls efficient Maraba virus binding and entry into infected cells. Taken together, these results are the first to implicate the potential impact of differential viral oncolytic properties at key steps of ovarian cancer metastasis

Combination of AKT inhibition with autophagy blockade effectively reduces ascites-derived ovarian cancer cell viability

Recent genomics analysis of the high-grade serous subtype of epithelial ovarian cancer (EOC) show aberrations in the phosphatidylinositol 3-kinase (PI3K)/AKT pathway that result in upregulated signaling activity. Thus, the PI3K/AKT pathway represents a potential therapeutic target for aggressive high-grade EOC. We previously demonstrated that treatment of malignant ascites-derived primary human EOC cells and ovarian cancer cell lines with the allosteric AKT inhibitor Akti-1/2 induces a dormancy-like cytostatic response but does not reduce cell viability. In this report, we show that allosteric AKT inhibition in these cells induces cytoprotective autophagy. Inhibition of autophagy using chloroquine (CQ) alone or in combination with Akti-1/2 leads to a significant decrease in viable cell number. In fact, Akti-1/2 sensitizes EOC cells to CQ-induced cell death by exhibiting markedly reduced EC50 values in combination-treated cells compared with CQ alone. In addition, we evaluated the effects of the novel specific and potent autophagy inhibitor-1 (Spautin-1) and demonstrate that Spautin-1 inhibits autophagy in a Beclin-1-independent manner in primary EOC cells and cell lines. Multicellular EOC spheroids are highly sensitive to Akti-1/2 and CQ/Spautin-1 cotreatments, but resistant to each agent alone. Indeed, combination index analysis revealed strong synergy between Akti-1/2 and Spautin-1 when both agents were used to affect cell viability; Akti-1/2 and CQ cotreatment also displayed synergy in most samples. Taken together, we propose that combination AKT inhibition and autophagy blockade would prove efficacious to reduce residual EOC cells for supplying ovarian cancer recurrence. © The Author 2014. Published by Oxford University Press. All rights reserved

A comprehensive analysis of common genetic variation in prolactin (PRL) and PRL receptor (PRLR) genes in relation to plasma prolactin levels and breast cancer risk: the Multiethnic Cohort

<p>Abstract</p> <p>Background</p> <p>Studies in animals and humans clearly indicate a role for prolactin (PRL) in breast epithelial proliferation, differentiation, and tumorigenesis. Prospective epidemiological studies have also shown that women with higher circulating PRL levels have an increase in risk of breast cancer, suggesting that variability in PRL may also be important in determining a woman's risk.</p> <p>Methods</p> <p>We evaluated genetic variation in the PRL and PRL receptor (PRLR) genes as predictors of plasma PRL levels and breast cancer risk among African-American, Native Hawaiian, Japanese-American, Latina, and White women in the Multiethnic Cohort Study (MEC). We selected single nucleotide polymorphisms (SNPs) from both the public (dbSNP) and private (Celera) databases to construct high density SNP maps that included up to 20 kilobases (kb) upstream of the transcription initiation site and 10 kb downstream of the last exon of each gene, for a total coverage of 59 kb in PRL and 210 kb in PRLR. We genotyped 80 SNPs in PRL and 173 SNPs in PRLR in a multiethnic panel of 349 unaffected subjects to characterize linkage disequilibrium (LD) and haplotype patterns. We sequenced the coding regions of PRL and PRLR in 95 advanced breast cancer cases (19 of each racial/ethnic group) to uncover putative functional variation. A total of 33 and 60 haplotype "tag" SNPs (tagSNPs) that allowed for high predictability (R_h²≥ 0.70) of the common haplotypes in PRL and PRLR, respectively, were then genotyped in a multiethnic breast cancer case-control study of 1,615 invasive breast cancer cases and 1,962 controls in the MEC. We also assessed the association of common genetic variation with circulating PRL levels in 362 postmenopausal controls without a history of hormone therapy use at blood draw. Because of the large number of comparisons being performed we used a relatively stringent type I error criteria (p < 0.0005) for evaluating the significance of any single association to correct for performing approximately 100 independent tests, close to the number of tagSNPs genotyped for both genes.</p> <p>Results</p> <p>We observed no significant associations between PRL and PRLR haplotypes or individual SNPs in relation to breast cancer risk. A nominally significant association was noted between prolactin levels and a tagSNP (tagSNP 44, rs2244502) in intron 1 of PRL. This SNP showed approximately a 50% increase in levels between minor allele homozygotes vs. major allele homozygotes. However, this association was not significant (p = 0.002) using our type I error criteria to correct for multiple testing, nor was this SNP associated with breast cancer risk (p = 0.58).</p> <p>Conclusion</p> <p>In this comprehensive analysis covering 59 kb of the PRL locus and 210 kb of the PRLR locus, we found no significant association between common variation in these candidate genes and breast cancer risk or plasma PRL levels. The LD characterization of PRL and PRLR in this multiethnic population provide a framework for studying these genes in relation to other disease outcomes that have been associated with PRL, as well as for larger studies of plasma PRL levels.</p

Antiinflammatory Therapy with Canakinumab for Atherosclerotic Disease

Background: Experimental and clinical data suggest that reducing inflammation without affecting lipid levels may reduce the risk of cardiovascular disease. Yet, the inflammatory hypothesis of atherothrombosis has remained unproved. Methods: We conducted a randomized, double-blind trial of canakinumab, a therapeutic monoclonal antibody targeting interleukin-1β, involving 10,061 patients with previous myocardial infarction and a high-sensitivity C-reactive protein level of 2 mg or more per liter. The trial compared three doses of canakinumab (50 mg, 150 mg, and 300 mg, administered subcutaneously every 3 months) with placebo. The primary efficacy end point was nonfatal myocardial infarction, nonfatal stroke, or cardiovascular death. RESULTS: At 48 months, the median reduction from baseline in the high-sensitivity C-reactive protein level was 26 percentage points greater in the group that received the 50-mg dose of canakinumab, 37 percentage points greater in the 150-mg group, and 41 percentage points greater in the 300-mg group than in the placebo group. Canakinumab did not reduce lipid levels from baseline. At a median follow-up of 3.7 years, the incidence rate for the primary end point was 4.50 events per 100 person-years in the placebo group, 4.11 events per 100 person-years in the 50-mg group, 3.86 events per 100 person-years in the 150-mg group, and 3.90 events per 100 person-years in the 300-mg group. The hazard ratios as compared with placebo were as follows: in the 50-mg group, 0.93 (95% confidence interval [CI], 0.80 to 1.07; P = 0.30); in the 150-mg group, 0.85 (95% CI, 0.74 to 0.98; P = 0.021); and in the 300-mg group, 0.86 (95% CI, 0.75 to 0.99; P = 0.031). The 150-mg dose, but not the other doses, met the prespecified multiplicity-adjusted threshold for statistical significance for the primary end point and the secondary end point that additionally included hospitalization for unstable angina that led to urgent revascularization (hazard ratio vs. placebo, 0.83; 95% CI, 0.73 to 0.95; P = 0.005). Canakinumab was associated with a higher incidence of fatal infection than was placebo. There was no significant difference in all-cause mortality (hazard ratio for all canakinumab doses vs. placebo, 0.94; 95% CI, 0.83 to 1.06; P = 0.31). Conclusions: Antiinflammatory therapy targeting the interleukin-1β innate immunity pathway with canakinumab at a dose of 150 mg every 3 months led to a significantly lower rate of recurrent cardiovascular events than placebo, independent of lipid-level lowering. (Funded by Novartis; CANTOS ClinicalTrials.gov number, NCT01327846.