MicroRNA Changes Up to 24 h following Induced Hypoglycemia in Type 2 Diabetes.

Hypoglycemia, as a complication of type 2 diabetes (T2D), causes increased morbidity and mortality but the physiological response underlying hypoglycemia has not been fully elucidated. Small noncoding microRNA (miRNA) have multiple downstream biological effects. This pilot exploratory study was undertaken to determine if induced miRNA changes would persist and contribute to effects seen 24 h post-hypoglycemia. A parallel, prospective study design was employed, involving T2D (n = 23) and control (n = 23) subjects. The subjects underwent insulin-induced hypoglycemia (2 mmol/L; 36 mg/dL); blood samples were drawn at baseline, upon the induction of hypoglycemia, and 4 h and 24 h post-hypoglycemia, with a quantitative polymerase chain reaction analysis of miRNA undertaken. The baseline miRNAs did not differ. In the controls, 15 miRNAs were downregulated and one was upregulated (FDR < 0.05) from the induction of hypoglycemia to 4 h later while, in T2D, only four miRNAs were altered (downregulated), and these were common to both cohorts (miR-191-5p; miR-143-3p; let-7b-5p; let-7g-5p), correlated with elevated glucagon levels, and all were associated with energy balance. From the induction of hypoglycemia to 24 h, 14 miRNAs were downregulated and 5 were upregulated (FDR < 0.05) in the controls; 7 miRNAs were downregulated and 7 upregulated (FDR < 0.05) in T2D; a total of 6 miRNAs were common between cohorts, 5 were downregulated (miR-93-5p, let-7b-5p, miR-191-5p, miR-185-5p, and miR-652-3p), and 1 was upregulated (miR-369-3p). An ingenuity pathway analysis indicated that many of the altered miRNAs were associated with metabolic and coagulation pathways; however, of the inflammatory proteins expressed, only miR-143-3p at 24 h correlated positively with tumor necrosis factor-α (TNFa; p < 0.05 and r = 0.46) and negatively with toll-like receptor-4 (TLR4; p < 0.05 and r = 0.43). The MiRNA levels altered by hypoglycemia reflected changes in counter-regulatory glucagon and differed between cohorts, and their expression at 24 h suggests miRNAs may potentiate and prolong the physiological response. Trial registration: ClinicalTrials.gov NCT03102801.

MiRNA and associated inflammatory changes from baseline to hypoglycemia in type 2 diabetes.

Objective: Hypoglycemia in type 2 diabetes (T2D) increases morbidity and mortality but the underlying physiological response is still not fully understood, though physiological changes are still apparent 24 hours after the event. Small noncoding microRNA (miRNA) have multiple downstream biological effects that may respond rapidly to stress. We hypothesized that hypoglycemia would induce rapid miRNA changes; therefore, this pilot exploratory study was undertaken. Methods: A pilot prospective, parallel study in T2D (n=23) and controls (n=23). Insulin-induced hypoglycemia (2mmol/l: 36mg/dl) was induced and blood sampling performed at baseline and hypoglycemia. Initial profiling of miRNA was undertaken on pooled samples identified 96 miRNA that were differentially regulated, followed by validation on a custom designed 112 miRNA panel. Results: Nine miRNAs differed from baseline to hypoglycemia in control subjects; eight were upregulated: miR-1303, miR-let-7e-5p, miR-1267, miR-30a-5p, miR-571, miR-661, miR-770-5p, miR-892b and one was downregulated: miR-652-3p. None of the miRNAs differed from baseline in T2D subjects. Conclusion: A rapid miRNA response reflecting protective pathways was seen in control subjects that appeared to be lost in T2D, suggesting that mitigating responses to hypoglycemia with blunting of the counter-regulatory response in T2D occurs even in patients with short duration of disease. Clinical trial registration: https://clinicaltrials.gov/ct2/show/NCT03102801?term=NCT03102801&draw=2&rank=1, identifier NCT03102801.

Discovery of new therapeutic targets in ovarian cancer through identifying significantly non-mutated genes.

BACKGROUND: Mutated and non-mutated genes interact to drive cancer growth and metastasis. While research has focused on understanding the impact of mutated genes on cancer biology, understanding non-mutated genes that are essential to tumor development could lead to new therapeutic strategies. The recent advent of high-throughput whole genome sequencing being applied to many different samples has made it possible to calculate if genes are significantly non-mutated in a specific cancer patient cohort. METHODS: We carried out random mutagenesis simulations of the human genome approximating the regions sequenced in the publicly available Cancer Growth Atlas Project for ovarian cancer (TCGA-OV). Simulated mutations were compared to the observed mutations in the TCGA-OV cohort and genes with the largest deviations from simulation were identified. Pathway analysis was performed on the non-mutated genes to better understand their biological function. We then compared gene expression, methylation and copy number distributions of non-mutated and mutated genes in cell lines and patient data from the TCGA-OV project. To directly test if non-mutated genes can affect cell proliferation, we carried out proof-of-concept RNAi silencing experiments of a panel of nine selected non-mutated genes in three ovarian cancer cell lines and one primary ovarian epithelial cell line. RESULTS: We identified a set of genes that were mutated less than expected (non-mutated genes) and mutated more than expected (mutated genes). Pathway analysis revealed that non-mutated genes interact in cancer associated pathways. We found that non-mutated genes are expressed significantly more than mutated genes while also having lower methylation and higher copy number states indicating that they could be functionally important. RNAi silencing of the panel of non-mutated genes resulted in a greater significant reduction of cell viability in the cancer cell lines than in the non-cancer cell line. Finally, as a test case, silencing ANKLE2, a significantly non-mutated gene, affected the morphology, reduced migration, and increased the chemotherapeutic response of SKOV3 cells. CONCLUSION: We show that we can identify significantly non-mutated genes in a large ovarian cancer cohort that are well-expressed in patient and cell line data and whose RNAi-induced silencing reduces viability in three ovarian cancer cell lines. Targeting non-mutated genes that are important for tumor growth and metastasis is a promising approach to expand cancer therapeutic options.

Signal Transducer and Activator of Transcription 3 (STAT3) Suppresses STAT1/Interferon Signaling Pathway and Inflammation in Senescent Preadipocytes.

Obesity promotes premature aging and dysfunction of white adipose tissue (WAT) through the accumulation of cellular senescence. The senescent cells burden in WAT has been linked to inflammation, insulin-resistance (IR), and type 2 diabetes (T2D). There is limited knowledge about molecular mechanisms that sustain inflammation in obese states. Here, we describe a robust and physiologically relevant in vitro system to trigger senescence in mouse 3T3-L1 preadipocytes. By employing transcriptomics analyses, we discovered up-regulation of key pro-inflammatory molecules and activation of interferon/signal transducer and activator of transcription (STAT)1/3 signaling in senescent preadipocytes, and expression of downstream targets was induced in epididymal WAT of obese mice, and obese human adipose tissue. To test the relevance of STAT1/3 signaling to preadipocyte senescence, we used Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9 (CRISPR/Cas9) technology to delete STAT1/3 and discovered that STAT1 promoted growth arrest and cooperated with cyclic Guanosine Monophosphate-Adenosine Monophosphate (GMP-AMP) synthase-stimulator of interferon genes (cGAS-STING) to drive the expression of interferon ß (IFNß), C-X-C motif chemokine ligand 10 (CXCL10), and interferon signaling-related genes. In contrast, we discovered that STAT3 was a negative regulator of STAT1/cGAS-STING signaling-it suppressed senescence and inflammation. These data provide insights into how STAT1/STAT3 signaling coordinates senescence and inflammation through functional interactions with the cGAS/STING pathway.

Long-term Results After Oncoplastic Surgery for Breast Cancer: A 10-year Follow-up.

OBJECTIVE: The aim of this study was to evaluate the long-term oncologic outcome after oncoplastic surgery (OPS). BACKGROUND: OPS combines wide tumor excision with reduction mammoplasty techniques thus extending breast conserving surgery to large tumors that might else be proposed a mastectomy. Little data are available about the oncologic results for breast conserving surgery of these larger tumors. METHODS: From January 2004 until March 2016, a total of 350 oncoplastic breast reductions were prospectively entered into a database. Patients were included if their breast reshaping included a reduction mammoplasty with skin excision (Level 2 oncoplastic techniques). RESULTS: Histologic subtypes were: invasive ductal carcinoma in 219 cases (62.6%), ductal carcinoma in situ (DCIS) in 88 cases (25.1%), and invasive lobular carcinoma in 43 (12.3%) cases. Seventy-three of the invasive cancers (27.9%) received neoadjuvant chemotherapy. The mean resection weight was 177 grams. The mean pathological tumor size was 26 mm (range 0-180 mm) and varied from 23 mm (4-180 mm) for invasive cancers to 32 mm (0-100 mm) for DCIS. Specimen margins were involved in 12.6% of the cases; 10.5% of invasive ductal, 14.7% of DCIS, and 20.9% of invasive lobular. The overall breast conservation rate was 92% and varied from 87.4% for DCIS to 93.5% for the invasive cancers. Thirty-one patients (8.9%) developed one or more postoperative complications, inducing a delay in postoperative treatments in 4.6% of patients. The median follow up was 55 months. The cumulative 5-year incidences for local, regional, and distant recurrences were 2.2%, 1.1%, and 12.4%, respectively. CONCLUSIONS: Oncoplastic breast reductions allow wide resections with free margins and can be used for large cancers as an alternative to mastectomy.

Preferential Allele Expression Analysis Identifies Shared Germline and Somatic Driver Genes in Advanced Ovarian Cancer.

Identifying genes where a variant allele is preferentially expressed in tumors could lead to a better understanding of cancer biology and optimization of targeted therapy. However, tumor sample heterogeneity complicates standard approaches for detecting preferential allele expression. We therefore developed a novel approach combining genome and transcriptome sequencing data from the same sample that corrects for sample heterogeneity and identifies significant preferentially expressed alleles. We applied this analysis to epithelial ovarian cancer samples consisting of matched primary ovary and peritoneum and lymph node metastasis. We find that preferentially expressed variant alleles include germline and somatic variants, are shared at a relatively high frequency between patients, and are in gene networks known to be involved in cancer processes. Analysis at a patient level identifies patient-specific preferentially expressed alleles in genes that are targets for known drugs. Analysis at a site level identifies patterns of site specific preferential allele expression with similar pathways being impacted in the primary and metastasis sites. We conclude that genes with preferentially expressed variant alleles can act as cancer drivers and that targeting those genes could lead to new therapeutic strategies.

Mesenchymal cell interaction with ovarian cancer cells induces a background dependent pro-metastatic transcriptomic profile.

BACKGROUND: The cross talk between the stroma and cancer cells plays a major role in phenotypic modulation. During peritoneal carcinomatosis ovarian cancer cells interact with mesenchymal stem cells (MSC) resulting in increased metastatic ability. Understanding the transcriptomic changes underlying the phenotypic modulation will allow identification of key genes to target. However in the context of personalized medicine we must consider inter and intra tumoral heterogeneity. In this study we used a pathway-based approach to illustrate the role of cell line background in transcriptomic modification during a cross talk with MSC. METHODS: We used two ovarian cancer cell lines as a surrogate for different ovarian cancer subtypes: OVCAR3 for an epithelial and SKOV3 for a mesenchymal subtype. We co-cultured them with MSCs. Genome wide gene expression was determined after cell sorting. Ingenuity pathway analysis was used to decipher the cell specific transcriptomic changes related to different pro-metastatic traits (Adherence, migration, invasion, proliferation and chemoresistance). RESULTS: We demonstrate that co-culture of ovarian cancer cells in direct cellular contact with MSCs induces broad transcriptomic changes related to enhance metastatic ability. Genes related to cellular adhesion, invasion, migration, proliferation and chemoresistance were enriched under these experimental conditions. Network analysis of differentially expressed genes clearly shows a cell type specific pattern. CONCLUSION: The contact with the mesenchymal niche increase metastatic initiation and expansion through cancer cells' transcriptome modification dependent of the cellular subtype. Personalized medicine strategy might benefit from network analysis revealing the subtype specific nodes to target to disrupt acquired pro-metastatic profile.

Endothelial cells provide a niche for placental hematopoietic stem/progenitor cell expansion through broad transcriptomic modification.

Umbilical cord blood (UCB) is an attractive source of hematopoietic stem cells (HSCs). However, the number of HSCs in UCB is limited, and attempts to amplify them in vitro remain inefficient. Several publications have documented amplification of hematopoietic stem/progenitor cells (HSPCs) on endothelial or mesenchymal cells, but the lack of homogeneity in culture conditions and HSC definition impairs direct comparison of these results. We investigated the ability of different feeder layers, mesenchymal progenitors (MPs) and endothelial cells (ECs), to amplify hematopoietic stem/progenitor cells. Placental derived HSPCs (defined as Lin(-)CD45(-/dim)CD34(+)CD38(-)CD90(+)) were maintained on confluent feeder layers and the number of cells and their marker expression were monitored over 21 days. Although both types of feeder layers supported hematopoietic expansion, only endothelial cells triggered amplification of Lin(-)CD45(-/dim)CD34(+)CD38(-)CD90(+) cells, which peaked at 14 days. The amplified cells differentiated into all cell lineages, as attested by in vitro colony-forming assays, and were capable of engraftment and multi-lineage differentiation in sub-lethally irradiated mice. Mesenchymal progenitors promoted amplification of CD38(+) cells, previously defined as precursors with more limited differentiation potential. A competitive assay demonstrated that hematopoietic stem/progenitor cells had a preference for interacting with endothelial cells in vitro. Cytokine and transcriptomic analysis of both feeder cell types identified differences in gene expression that correlated with propensity of ECs and MPs to support hematopoietic cell amplification and differentiation respectively. Finally, we used RNA sequencing of endothelial cells and HSPCs to uncover relevant networks illustrating the complex interaction between endothelial cells and HSPCs leading to stem/progenitor cell expansion.

Adaptation of a commonly used, chemically defined medium for human embryonic stem cells to stable isotope labeling with amino acids in cell culture.

Metabolic labeling with stable isotopes is a prominent technique for comparative quantitative proteomics, and stable isotope labeling with amino acids in cell culture (SILAC) is the most commonly used approach. SILAC is, however, traditionally limited to simple tissue culture regimens and only rarely employed in the context of complex culturing conditions as those required for human embryonic stem cells (hESCs). Classic hESC culture is based on the use of mouse embryonic fibroblasts (MEFs) as a feeder layer, and as a result, possible xenogeneic contamination, contribution of unlabeled amino acids by the feeders, interlaboratory variability of MEF preparation, and the overall complexity of the culture system are all of concern in conjunction with SILAC. We demonstrate a feeder-free SILAC culture system based on a customized version of a commonly used, chemically defined hESC medium developed by Ludwig et al. and commercially available as mTeSR1 [mTeSR1 is a trade mark of WiCell (Madison, WI) licensed to STEMCELL Technologies (Vancouver, Canada)]. This medium, together with adjustments to the culturing protocol, facilitates reproducible labeling that is easily scalable to the protein amounts required by proteomic work flows. It greatly enhances the usability of quantitative proteomics as a tool for the study of mechanisms underlying hESCs differentiation and self-renewal. Associated data have been deposited to the ProteomeXchange with the identifier PXD000151.

High-prevalence and broad spectrum of Cell Adhesion and Extracellular Matrix gene pathway mutations in epithelial ovarian cancer.

BACKGROUND: Ovarian cancer is the most deadly gynecological cancer because of late diagnosis, frequently with diffuse peritoneal metastases. Recent findings have shown that serous epithelial ovarian cancer has a narrow mutational spectrum with TP53 being the most frequently targeted when single genes are considered. It is, however, important to understand which pathways as a whole may be targeted for mutation. FINDINGS: Previously published mutational data provided by the cancer genome atlas networks findings on ovarian cancer was searched for statistically significant enrichment of genes in pathways. These pathways were then searched in all patients to identify the spectrum of mutations. Statistical significance was further shown through in-silico permutations of exome sequences using empirically observed mutation rates. We detected mutations in the cell adhesion pathway genes in more than 89% of serous epithelial ovarian cancer patients. This level of near universal mutational targeting of the cell adhesion pathway, including the extracellular matrix pathway, is previously unreported in epithelial ovarian cancer. CONCLUSIONS: Taken together with previous studies on the role of cell adhesion and extracellular matrix gene expression in ovarian cancer and metastasis, our results identify pathways for which the mutational prevalence has previously been overlooked using single gene approaches. Analysis of mutations at the pathway level will be critical in studying heterogeneous diseases such as ovarian cancer.

Signal transduction pathway of TonB-dependent transporters.

Transcription of the ferric citrate import system is regulated by ferric citrate binding to the outer membrane transporter FecA. A signal indicating transporter occupancy is relayed across the outer membrane to energy-transducing and regulatory proteins embedded in the cytoplasmic membrane. Because transcriptional activation is not coupled to ferric citrate import, an allosteric mechanism underlies this complex signaling mechanism. Using evolution-based statistical analysis we have identified a sparse but structurally connected network of residues that links distant functional sites in FecA. Functional analyses of these positions confirm their involvement in the mechanism that regulates transcriptional activation in response to ferric citrate binding at the cell surface. This mechanism appears to be conserved and provides the structural basis for the allosteric signaling of TonB-dependent transporters.