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To identify early follicular phase microribonucleic acids (miRNAs) that are altered in serum of women with endometriosis.Case-control study.Large university-affiliated in vitro fertilization center.Women with (n = 21) and without (n = 24) endometriosis.Serum samples were obtained from laparoscopy-confirmed patients with endometriosis.The differential expression of serum miRNAs relative to controls was measured using the NanoString nCounter technology and validated by quantitative real-time polymerase chain reaction in an independent cohort of 27 patients with endometriosis and controls (n = 24). Microribonucleic acid target signaling pathways and genes were analyzed bioinformatically. A chemically modified stable miR-34-3p oligonucleotide was used to examine the effect on proliferation of VK2E6/E7 endometrial cells in vitro.Eighteen miRNAs were significantly up-regulated, and 1 miRNA (hsa-miR-34c-3p) was significantly down-regulated in the follicular phase of patients with endometriosis. The analysis of target signaling pathways using TargetScan predicted regulation of the mitogen-activated protein kinase, phosphoinositide 3-kinase/protein kinase B, Hippo, adenosine monophosphate-activated protein kinase, transforming growth factor beta, and endometrial cancer pathways, which have been implicated in the pathogenesis of endometriosis, by these miRNAs. The analysis of sequence complementarity identified prostaglandin E2 receptor 4, interleukin 6 signal transducer, and polo-like kinase 4 genes as possible direct targets of hsa-miR-34-3p. DSDI-1, a chemically modified stable miR-34-3p oligonucleotide, reduced cell proliferation in VK2E6/E7 endometrial cells in vitro.The follicular phase miRNA levels are altered in serum of women with endometriosis and may be useful as reproducible detection biomarkers for early diagnosis of endometriosis. hsa-miR-34-3p is significantly down-regulated in endometriosis, targets endometriosis genes, and reduces endometrial cell proliferation in vitro. These results support hsa-miR-34-3p as a potential therapeutic target in endometriosis.

The expression of the WWOX tumor suppressor gene is lost or reduced in a large fraction of various cancers, including prostate cancer. We previously reported that Wwox overexpression induced apoptosis and suppressed prostate cancer growth in vitro and in vivo. In this study, pathways through which Wwox contributes to control of prostate cancer cell growth have been investigated. We found that Wwox interacts with Ap2γ and prevents it from entering the nucleus to bind the ERBB2 promoter region to activate transcription of ERBB2, a mediator of androgen receptor activity and prostate cancer cell growth at limiting androgen concentration. Ectopic expression of Wwox reduced ErbB2 protein expression in vitro and expression of Wwox protein inversely correlated with expression of ErbB2 protein in prostate cancer tissues. Furthermore, Wwox suppressed Ap2γ/ErbB2–induced prostate cancer cell growth and suppressed prostate-specific antigen secretion through interaction with Ap2γ and down-modulation of ErbB2, an effect that required functional androgen receptor. (Mol Cancer Res 2007;5(9):957–65)

Supplementary Figure S1. PPP2R2A mRNA levels in the excised tumors from 5637 bearing mouse xenografts. Supplementary Figure S2. PPP2R2A mRNA and protein levels upon transient silencing.Supplementary Figure S3. PPP2R2A protein levels upon transient overexpression. Supplementary Table S1. Bioinformatics prediction of the top networks regulated by PPP2R2A. Supplementary Table S2. Bioinformatics prediction of the top canonical pathways regulated by PPP2R2A. Supplementary Table S3. Xcelligence Cell Index and P values of siPPP2R2RA-treated versus control BC cells. Supplementary Table S4. Xcelligence Cell Index and P values of PPP2R2A-overexpressing versus control BC cells, all treated with miR-21 mimic.

Bladder cancer represents a disease associated with significant morbidity and mortality. MiR-21 has been found to have oncogenic activity in multiple cancers, including bladder cancer, whereas inhibition of its expression suppresses tumor growth. Here, we examine the molecular network regulated by miR-21 in bladder cancer and evaluate the effects of i.v. and i.p. administration of a novel miR-21 chemical inhibitor in vivo. LNA miR-21 reduced the oncogenic potential of bladder cancer cells, whereas the MKAD-21 chemically modified antisense oligo against miR-21 dose-dependently blocked xenograft growth. I.v. administration of LNA miR-21 was more effective in suppressing tumor growth than was i.p. administration. Integration of computational and transcriptomic analyses in a panel of 28 bladder cancer lines revealed a 15-gene signature that correlates with miR-21 levels. Protein Phosphatase 2 Regulatory Subunit Balpha (PPP2R2A) was one of these 15 genes and was experimentally validated as a novel miR-21 direct target gene. Gene network and molecular analyses showed that PPP2R2A is a potent negative regulator of the ERK pathway activation and bladder cancer cell proliferation. Importantly, we show that PPP2R2A acts as a mediator of miR-21–induced oncogenic effects in bladder cancer. Integrative analysis of human bladder cancer tumors and a large panel of human bladder cancer cell lines revealed a novel 15-gene signature that correlates with miR-21 levels. Importantly, we provide evidence that PPP2R2A represents a new miR-21 direct target and regulator of the ERK pathway and bladder cancer cell growth. Furthermore, i.v. administration of the MKAD-21 inhibitor effectively suppressed tumor growth through regulation of the PPP2R2A–ERK network in mice. Mol Cancer Ther; 17(7); 1430–40. ©2018 AACR.

Supplementary Figures S1-S2 from Wwox Suppresses Prostate Cancer Cell Growth through Modulation of ErbB2-Mediated Androgen Receptor Signaling

Purpose: The WWOX gene is down-regulated in breast cancer and loss of Wwox expression correlates with important clinical features of breast cancer. Thus, we have examined the effect of restoration of Wwox expression in breast cancer-derived cells.Experimental Design: Wwox protein expression was restored by the following: (a) infection with a recombinant adenovirus carrying WWOX cDNA (Ad-WWOX) or (b) treatment with the DNA methyltransferase inhibitor, 5-aza-2′-deoxycytidine, to activate the endogenous WWOX gene, in breast cancer-derived cells in vitro and in vivo.Results: Restoration of Wwox expression led to suppression of growth of Wwox-deficient breast cancer-derived cells, through activation of the intrinsic caspase pathway, but did not affect growth of Wwox-sufficient MCF7 cells. Intratumoral Wwox restoration, through Ad-WWOX infection or endogenous Wwox reactivation by 5-aza-2′-deoxycytidine injection, suppressed tumor growth in nude mice by inducing apoptosis. Alteration of global methylation levels was not observed.Conclusions: The results confirm that overexpression of exogenous Wwox inhibits breast cancer cell growth in vitro and in vivo and, perhaps more importantly, shows that restoration of endogenous Wwox expression, and likely other proteins, by treatment with a de novo methyltransferase inhibitor, also inhibits breast cancer cell growth and reverses breast cancer xenograft growth.

Supplementary Data Figure 1 from Inhibition of Breast Cancer Cell Growth In vitro and In vivo: Effect of Restoration of Wwox Expression

ARLTS1 is a tumor suppressor gene initially described as a low-penetrance cancer gene: a truncated Trp149Stop (MUT) polymorphism is associated with general familial cancer aggregation and, particularly, high-risk familial breast cancer. DNA hypermethylation has been identified as a mechanism of ARLTS1 expression down-regulation in lung carcinomas and B-cell chronic lymphocytic leukemia. We found that, in the majority of ovarian carcinomas (61.5%) and in a significant proportion of ovarian and breast cancer cell lines (45%), ARLTS1 is strongly down-regulated due to DNA methylation in its promoter region. After ARLTS1 restoration by adenoviral transduction, only the negative TOV-112 and the homozygously mutated (MUT) MCF7 cells, but not the OV-90 cells expressing a normal ARLTS1 product, underwent apoptosis and inhibition of cell growth. Furthermore, ARLTS1 reexpression significantly reduced the tumorigenic potential of TOV-112 in nude mice. On the contrary, the ARLTS1-MUT induced significantly lower levels of apoptosis in infected cells and reduced in vivo tumorigenesis only partially, supporting the hypothesis that Trp149Stop polymorphism is retained in the general population and predisposes to cancer because of a reduction, but not full loss, of normal ARLTS1 function. (Cancer Res 2006; 66(21): 10287-91)

Supplementary Figure 3 from Alterations of the Tumor Suppressor Gene ARLTS1 in Ovarian Cancer

Expression of the WWOX gene, encompassing the common chromosome fragile site FRA16D, is altered in a large fraction of cancers of various types, including prostate cancer. We have examined expression and biological functions of WWOX in prostate cancer. WWOX mRNA and protein expression were significantly reduced in prostate cancer-derived cells (LNCaP, DU145, and PC-3) compared with noncancer prostate cells (PWR-1E), and WWOX expression was reduced in 84% of prostate cancers, as assessed by immunohistochemical staining. Down-modulation of WWOX expression in the prostate cancer-derived cells is due to DNA hypermethylation in the WWOX regulatory region. Treatment with 5-aza-2′-deoxycytidine (AZA), a DNA methyltransferase inhibitor, and trichostatin A, a histone deacetylase inhibitor, led to increased WWOX mRNA and protein expression in prostate cancer-derived cells, most strikingly in DU145 cells. Transfection-mediated WWOX overexpression in DU145 cells suppressed colony growth (P = 0.0012), and WWOX overexpression by infection with Ad-WWOX virus induced apoptosis through a caspase-dependent mechanism and suppressed cell growth. Lastly, ectopic expression of WWOX by Ad-WWOX infection suppressed tumorigenicity of xenografts in nude mice, and intratumoral AZA treatment halted tumor growth. The data are consistent with a role for WWOX as a prostate cancer tumor suppressor and suggest that WWOX signal pathways should be further investigated in normal and cancerous prostate cells and tissues. (Cancer Res 2006; 66(13): 6477-81)

Supplementary Figure 1 from A Role for the WWOX Gene in Prostate Cancer

Supplementary Figures 1-2 from Inhibition of miR-193a Expression by Max and RXRα Activates K-Ras and PLAU to Mediate Distinct Aspects of Cellular Transformation

Supplementary Table 1 from MicroRNA Signature of Primary Pigmented Nodular Adrenocortical Disease: Clinical Correlations and Regulation of Wnt Signaling

Metformin, the first-line drug for treating diabetes, selectively kills the chemotherapy resistant subpopulation of cancer stem cells (CSC) in genetically distinct types of breast cancer cell lines. In mouse xenografts, injection of metformin and the chemotherapeutic drug doxorubicin near the tumor is more effective than either drug alone in blocking tumor growth and preventing relapse. Here, we show that metformin is equally effective when given orally together with paclitaxel, carboplatin, and doxorubicin, indicating that metformin works together with a variety of standard chemotherapeutic agents. In addition, metformin has comparable effects on tumor regression and preventing relapse when combined with a four-fold reduced dose of doxorubicin that is not effective as a monotherapy. Finally, the combination of metformin and doxorubicin prevents relapse in xenografts generated with prostate and lung cancer cell lines. These observations provide further evidence for the CSC hypothesis for cancer relapse, an experimental rationale for using metformin as part of combinatorial therapy in a variety of clinical settings, and for reducing the chemotherapy dose in cancer patients. Cancer Res; 71(9); 3196–201. ©2011 AACR.

Supplementary Figure 1 from Alterations of the Tumor Suppressor Gene ARLTS1 in Ovarian Cancer

Supplementary Figure 2 from Alterations of the Tumor Suppressor Gene ARLTS1 in Ovarian Cancer

MicroRNAs comprise a novel group of gene regulators implicated in the development of different types of cancer; however, their role in primary pigmented nodular adrenocortical disease (PPNAD) has not been investigated. PPNAD is a bilateral adrenal hyperplasia often associated with Carney complex, a multiple neoplasia syndrome; both disorders are caused by protein kinase A (PKA) regulatory subunit type 1A (PRKARIA)–inactivating mutations. We identified a 44-microRNA gene signature of PPNAD after comparing PPNAD with normal adrenal samples. Specifically, 33 microRNAs were up-regulated and 11 down-regulated in PPNAD relative to normal tissues. These results were validated by stem loop real-time PCR analysis. Comparison of microRNA microarray data with clinicopathologic variables revealed a negative correlation (r = −0.9499) between let-7b expression and cortisol levels in patients with PPNAD. Integration of microRNA microarray with serial analysis of gene expression data together with bioinformatic algorithm predictions revealed nine microRNA-gene target pairs with a potential role in adrenal pathogenesis. Using a PPNAD cell line, we showed that miR-449 was up-regulated and identified its direct target, WNT1-inducible signaling pathway protein 2 (WISP2); in addition, pharmacologic inhibition of PKA resulted in the up-regulation of miR-449 leading to the suppression of WISP2. Overall, we investigated, for the first time, the microRNA profile and its clinical significance in PPNAD; these data also suggest that PKA, via microRNA regulation, affects the Wnt signaling pathway, which through expression and clinical studies is suspected to be a primary mediator of PRKAR1A-related tumorigenesis. [Cancer Res 2009;69(8):3278–82]

Supplementary Figures 1-18, Methods from Akt2 Regulates All Akt Isoforms and Promotes Resistance to Hypoxia through Induction of miR-21 upon Oxygen Deprivation

Supplementary Table 1 from Alterations of the Tumor Suppressor Gene ARLTS1 in Ovarian Cancer

Supplementary Figure Legend from A Role for the WWOX Gene in Prostate Cancer

Supplementary Figures 1-7 from MicroRNA Signature of Primary Pigmented Nodular Adrenocortical Disease: Clinical Correlations and Regulation of Wnt Signaling

Pancreatic ductal adenocarcinoma (PDAC), the second most prevalent gastrointestinal malignancy and the most common type of pancreatic cancer is linked with poor prognosis and, eventually, with high mortality rates. Early detection is seldom, while tumor heterogeneity and microarchitectural alterations benefit PDAC resistance to conventional therapeutics. Although emerging evidence suggest the core role of cancer stem cells (CSCs) in PDAC aggressiveness, unique stem signatures are poorly available, thus limiting the efforts of anti-CSC-targeted therapy. Herein, we report the findings of the first genome-wide analyses of mRNA/lncRNA transcriptome profiling and co-expression networks in PDAC cell line-derived CD133+/CD44+ cells, which were shown to bear a CSC-like phenotype in vitro and in vivo. Compared to CD133−/CD44− cells, the CD133+/CD44+ population demonstrated significant expression differences in both transcript pools. Using emerging bioinformatic tools, we performed lncRNA target coding gene prediction analysis, which revealed significant Gene Ontology (GO), pathway, and network enrichments in many dyregulated lncRNA nearby (cis or trans) mRNAs, with reported involvement in the regulation of CSC phenotype and functions. In this context, the construction of lncRNA/mRNA networks by ingenuity platforms identified the lncRNAs ATF2, CHEK1, DCAF8, and PAX8 to interact with “hub” SC-associated mRNAs. In addition, the expressions of the above lncRNAs retrieved by TCGA-normalized RNAseq gene expression data of PAAD were significantly correlated with clinicopathological features of PDAC, including tumor grade and stage, nodal metastasis, and overall survival. Overall, our findings shed light on the identification of CSC-specific lncRNA signatures with potential prognostic and therapeutic significance in PDAC.

Many diseases that affect modern humans fall in the category of complex diseases, thus called because they result from a combination of multiple aetiological and pathogenic factors. Regardless of the organ or system affected, complex diseases present major challenges in diagnosis, classification, and management. Current forms of therapy are usually applied in an indiscriminate fashion based on clinical information, but even the most advanced drugs only benefit a limited number of patients and to a variable and unpredictable degree. This ‘one measure does not fit all’ situation has spurred the notion that therapy for complex disease should be tailored to individual patients or groups of patients, giving rise to the notion of ‘precision medicine’ [PM]. Inflammatory bowel disease [IBD] is a prototypical complex disease where the need for PM has become increasingly clear. This prompted the European Crohn’s and Colitis Organisation to focus the Seventh Scientific Workshop on this emerging theme. The articles in this special issue of the Journal address the various complementary aspects of PM in IBD, including what PM is; why it is needed and how it can be used; how PM can contribute to prediction and prevention of IBD; how IBD PM can aid in prognosis and improve response to therapy; and the challenges and future directions of PM in IBD. This first article of this series is structured on three simple concepts [what, why, and how] and addresses the definition of PM, discusses the rationale for the need of PM in IBD, and outlines the methodology required to implement PM in IBD in a correct and clinically meaningful way.

The present experimental model aimed to investigate the possible effect of endometriosis on ovarian function by altering follicular maturation and development. This single-blind, randomized study included twenty-four female Sprague Dawley mice, 2.5 months old, weighing 160-200 grams. The animals were randomly separated into four groups on the day of the surgery. Each group consisted of 6 mice. The first group (A) consisted of healthy female mice (control group). The second group (B) consisted of mice subjected to surgical insertion of ovarian endometrioma. The third group (C) consisted of mice subjected to surgically induced diffuse intraperitoneal endometriosis, and the fourth group (D) consisted of mice subjected to surgically induced extraperitoneal endometriosis. According to our experimental model, endometriosis may affect ovarian function by increasing the number of luteinized unruptured follicles (follicles that have undergone luteinization without prior rupture).

Paragangliomas are rare tumors of chromaffin cells arising from an extra-adrenal location. Unlike pheochromocytomas, they are seldom functional. We present a case of pericardial paraganglioma incidentally encountered on an echocardiographic study, focusing on the characteristic features the tumor demonstrates on different imaging modalities. (Level of Difficulty: Intermediate.)

The myeloid differentiation factor Schlafen4 (Slfn4) marks a subset of myeloid-derived suppressor cells (MDSCs) in the stomach during Helicobacter-induced spasmolytic polypeptide-expressing metaplasia (SPEM).ObjectiveTo identify the gene products expressed by Slfn4+-MDSCs and to determine how they promote SPEM.DesignWe performed transcriptome analyses for both coding genes (mRNA by RNA-Seq) and non-coding genes (microRNAs using NanoString nCounter) using flow-sorted SLFN4+ and SLFN4– cells from Helicobacter-infected mice exhibiting metaplasia at 6 months postinfection. Thioglycollate-elicited myeloid cells from the peritoneum were cultured and treated with IFNα to induce the T cell suppressor phenotype, expression of MIR130b and SLFN4. MIR130b expression in human gastric tissue including gastric cancer and patient sera was determined by qPCR and in situ hybridisation. Knockdown of MiR130b in vivo in Helicobacter-infected mice was performed using Invivofectamine. Organoids from primary gastric cancers were used to generate xenografts. ChIP assay and Western blots were performed to demonstrate NFκb p65 activation by MIR130b.ResultsMicroRNA analysis identified an increase in MiR130b in gastric SLFN4+ cells. Moreover, MIR130b colocalised with SLFN12L, a human homologue of SLFN4, in gastric cancers. MiR130b was required for the T-cell suppressor phenotype exhibited by the SLFN4+ cells and promoted Helicobacter-induced metaplasia. Treating gastric organoids with the MIR130b mimic induced epithelial cell proliferation and promoted xenograft tumour growth.ConclusionTaken together, MiR130b plays an essential role in MDSC function and supports metaplastic transformation.

Degenerative aortic valve stenosis is the most common primary valve disease and a significant cause of cardiovascular morbidity and mortality. In an era when new techniques for the management of aortic stenosis are gaining ground, the understanding of this disease is more important than ever to optimize treatment. So far, the focus has been placed on the assessment of the valve itself. However, the role that the arterial system plays in the pathogenesis and natural history of the disease needs to be further elucidated. Arteriosclerosis, when it coexists with a stenotic valve, augments the load posed on the left ventricle contributing to greater impairment of cardiovascular function. Arterial stiffness, a well-established predictor for cardiovascular disease and all-cause mortality, could play a role in the prognosis and quality of life of this population. Several studies using a variety of indices to assess arterial stiffness have tried to address the potential utility of arterial function assessment in the case of aortic stenosis. Importantly, reliable data identify a prognostic role of arterial biomarkers in aortic stenosis and stress their possible use to optimize timing and method of treatment. This review aims at summarizing the existing knowledge on the interplay between the heart and the vessels in the presence of degenerative aortic stenosis, prior, upon and after interventional management. Further, it discusses the evidence supporting the potential clinical application of arterial biomarkers for the assessment of progression, severity, management and prognosis of aortic stenosis.

Background Systems biology is a rapidly advancing field of science that allows us to look into disease mechanisms, patient diagnosis and stratification, and drug development in a completely new light. It is based on the utilization of unbiased computational systems free of the traditional experimental approaches based on personal choices of what is important and what select experiments should be performed to obtain the expected results. Methods Systems biology can be applied to inflammatory bowel disease (IBD) by learning basic concepts of omes and omics and how omics-derived “big data” can be integrated to discover the biological networks underlying highly complex diseases like IBD. Once these biological networks (interactomes) are identified, then the molecules controlling the disease network can be singled out and specific blockers developed. Results The field of systems biology in IBD is just emerging, and there is still limited information on how to best utilize its power to advance our understanding of Crohn disease and ulcerative colitis to develop novel therapeutic strategies. Few centers have embraced systems biology in IBD, but the creation of international consortia and large biobanks will make biosamples available to basic and clinical IBD investigators for further research studies. Conclusions The implementation of systems biology is indispensable and unavoidable, and the patient and medical communities will both benefit immensely from what it will offer in the near future.

Paragangliomas are rare tumors of chromaffin cells arising from an extra-adrenal location. Unlike pheochromocytomas, they are seldom functional. We present a case of pericardial paraganglioma incidentally encountered on an echocardiographic study, focusing on the characteristic features the tumor demonstrates on different imaging modalities. (Level of Difficulty: Intermediate.), Graphical abstract

Background & aimsAlterations in microRNA (miRNA) and in the intestinal barrier are putative risk factors for irritable bowel syndrome (IBS). We aimed to identify differentially expressed colonic mucosal miRNAs, their targets in IBS compared to healthy controls (HCs), and putative downstream pathways.MethodsTwenty-nine IBS patients (15 IBS with constipation [IBS-C], 14 IBS with diarrhea [IBS-D]), and 15 age-matched HCs underwent sigmoidoscopy with biopsies. A nCounter array was used to assess biopsy specimen-associated miRNA levels. A false discovery rate (FDR) < 10% was considered significant. Real-time polymerase chain reaction (PCR) was used to validate differentially expressed genes. To assess barrier function, trans-epithelial electrical resistance (TEER) and dextran flux assays were performed on Caco-2 intestinal epithelial cells that were transfected with miRNA-inhibitors or control inhibitors. Protein expression of barrier function associated genes was confirmed using western blots.ResultsFour out of 247 miRNAs tested were differentially expressed in IBS compared to HCs (FDR < 10%). Real-time PCR validation suggested decreased levels of miR-219a-5p and miR-338-3p in IBS (P = .026 and P = .004), and IBS-C (P = .02 and P = .06) vs. HCs as the strongest associations. Inhibition of miR-219a-5p resulted in altered expression of proteasome/barrierfunction genes. Functionally, miR-219a-5p inhibition enhanced the permeability of intestinal epithelial cells as TEER was reduced (25-50%, P < .05) and dextran flux wasincreased (P < .01). Additionally, inhibition of miR-338-3p incells caused alterations in the mitogen-activated protein kinase (MAPK) signaling pathway genes.ConclusionTwomicroRNAs that potentially affect permeability and visceral nociception were identified to be altered in IBS patients. MiR-219a-5p and miR-338-3p potentially alter barrier function and visceral hypersensitivity via neuronal and MAPK signaling and could be therapeutic targets in IBS.