Predictive Relevance of Circulating miR-622 in Patients with Newly Diagnosed and Recurrent High-Grade Serous Ovarian Carcinoma.

BACKGROUND: Identifying patients with high-grade serous ovarian cancer (HGSOC) who will respond to treatment remains a clinical challenge. We focused on miR-622, a miRNA involved in the homologous recombination repair (HRR) pathway, and we assessed its predictive value in serum prior to first-line chemotherapy and at relapse. METHODS: Serum miR-622 expression was assessed in serum prior to first-line platinum-based chemotherapy in a prospective multicenter study (miRNA Serum Analysis, miRSA, NCT01391351) and a retrospective cohort (Biological Resource Center, BRC), and was also studied at relapse. Progression-free survival (PFS) and overall survival (OS) were used as primary and secondary endpoints prior to first-line chemotherapy and OS as a primary endpoint at relapse. RESULTS: The group with high serum miR-622 expression was associated with a significantly lower PFS (15.4 versus 24.4 months; adjusted HR 2.11, 95% CI 1.2 3.8, P = 0.015) and OS (29.7 versus 40.6 months; adjusted HR 7.68, 95% CI 2.2-26.2, P = 0.0011) in the miRSA cohort. In the BRC cohort, a high expression of miR-622 was also associated with a significantly lower OS (22.8 versus 35.9 months; adjusted HR 1.98, 95% CI 1.1-3.6, P = 0.026). At relapse, high serum miR-622 was associated with a significantly lower OS (7.9 versus 20.6 months; adjusted HR 3.15, 95% CI 1.4-7.2, P = 0.0062). Serum miR-622 expression is a predictive independent biomarker of response to platinum-based chemotherapy for newly diagnosed and recurrent HGSOC. CONCLUSIONS: These results may open new perspectives for HGSOC patient stratification and monitoring of resistance to platinum-based and poly(ADP-ribose)-polymerase-inhibitor-maintenance therapies, facilitating better and personalized treatment decisions.

Calcium signals inhibition sensitizes ovarian carcinoma cells to anti-Bcl-xL strategies through Mcl-1 down-regulation.

Ovarian carcinoma is the leading cause of death from gynecologic cancer in the developed world and is characterized by acquired chemoresistance leading to an overall 5-year survival rate of about 30 %. We previously showed that Bcl-xL and Mcl-1 cooperatively protect platinum-resistant ovarian cancer cells from apoptosis. Despite BH3-mimetics represent promising drugs to target Bcl-xL, anti-Mcl-1 strategies are still in pre-clinical studies and required new investigations. Calcium is a universal second messenger and dysregulation of calcium signal is often observed during carcinogenesis. As change in cytosolic free calcium concentration [Ca(2+)]i is known to control the fate of the cell by regulating Bcl-2 family members, we wonder if calcium signal could impact on Mcl-1 expression and if its pharmacological inhibition could be useful to sensitize ovarian carcinoma cells to anti-Bcl-xL strategies. We therefore studied the effect of different calcium signals inhibitors in ovarian carcinoma cell lines SKOV3 and IGROV1-R10 and analysed their effects on proliferation and Mcl-1 expression. We also exposed these cells to these inhibitors in combination with anti-Bcl-xL strategies (siRNA or BH3-mimetic: ABT-737). We found that calcium signaling regulates Mcl-1 through translational events and a calmodulin-mediated pathway. BAPTA-AM and calmodulin inhibitor combination with ABT-737 leads to apoptosis, a process that is reversed by Mcl-1 enforced expression. As Mcl-1 represents a crucial hurdle to the success of chemotherapy, these results could open to new area of investigation using calcium modulators to directly or indirectly target Mcl-1 and thus efficiently sensitize ovarian carcinoma cells to anti-Bcl-xL strategies.

Follicular lymphoma-like B cells in healthy individuals: a novel intermediate step in early lymphomagenesis.

Follicular lymphoma is one of the most common adult lymphoma, and remains virtually incurable despite its relatively indolent nature. t(14;18)(q32;q21) translocation, the genetic hallmark and early initiating event of follicular lymphoma (FL) pathogenesis, is also present at low frequency in the peripheral blood of healthy individuals. It has long been assumed that in healthy individuals t(14;18) is carried by circulating quiescent naive B cells, where its oncogenic potential would be restrained. Here, we question this current view and demonstrate that in healthy individuals, t(14;18) is actually carried by an expanding population of atypical B cells issued from germinal centers, displaying genotypic and phenotypic features of FL, and prone to constitute potent premalignant FL niches. These findings strongly impact both on the current understanding of disease progression and on the proper handling of t(14;18) frequency in blood as a potential early biomarker for lymphoma.

The lncRNA 'UCA1' modulates the response to chemotherapy of ovarian cancer through direct binding to miR-27a-5p and control of UBE2N levels.

Ovarian cancer (OC) is the leading cause of death in patients with gynecologic cancers. Due to late diagnosis and resistance to chemotherapy, the 5-year survival rate in patients with OC is below 40%. We observed that UCA1, a lncRNA previously reported to play an oncogenic role in several malignancies, is overexpressed in the chemoresistant OC cell line OAW42-R compared to their chemotherapy-sensitive counterpart OAW42. Additionally, UCA1 overexpression was related to poor prognosis in two independent patient cohorts. Currently, the molecular mechanisms through which UCA1 acts in OC are poorly understood. We demonstrated that downregulation of the short isoform of UCA1 sensitized OC cells to cisplatin and that UCA1 acted as competing endogenous RNA to miR-27a-5p. Upon UCA1 downregulation, miR-27a-5p downregulated its direct target UBE2N leading to the upregulation of BIM, a proapoptotic protein of the Bcl2 family. The upregulation of BIM is the event responsible for the sensitization of OC cells to cisplatin. In order to model response to therapy in patients with OC, we used several patient-derived organoid cultures, a model faithfully mimicking patient's response to therapy. Inhibition of UBE2N sensitized patient-derived organoids to platinum salts. In conclusion, response to treatment in patients with OC is regulated by the UCA1/miR-27a-5p/UBE2N axis, where UBE2N inhibition could potentially represent a novel therapeutic strategy to counter chemoresistance in OC.

Network-Based Integration of Multi-Omics Data Identifies the Determinants of miR-491-5p Effects.

The identification of miRNAs' targets and associated regulatory networks might allow the definition of new strategies using drugs whose association mimics a given miRNA's effects. Based on this assumption we devised a multi-omics approach to precisely characterize miRNAs' effects. We combined miR-491-5p target affinity purification, RNA microarray, and mass spectrometry to perform an integrated analysis in ovarian cancer cell lines. We thus constructed an interaction network that highlighted highly connected hubs being either direct or indirect targets of miR-491-5p effects: the already known EGFR and BCL2L1 but also EP300, CTNNB1 and several small-GTPases. By using different combinations of specific inhibitors of these hubs, we could greatly enhance their respective cytotoxicity and mimic the miR-491-5p-induced phenotype. Our methodology thus constitutes an interesting strategy to comprehensively study the effects of a given miRNA. Moreover, we identified targets for which pharmacological inhibitors are already available for a clinical use or in clinical trials. This study might thus enable innovative therapeutic options for ovarian cancer, which remains the leading cause of death from gynecological malignancies in developed countries.

Downregulation of Bcl-xL and Mcl-1 is sufficient to induce cell death in mesothelioma cells highly refractory to conventional chemotherapy.

Malignant pleural mesothelioma (MPM) is an aggressive tumor with poor prognosis and limited response to platinum-based chemotherapy. Several lines of evidence support a role for the anti-apoptotic protein Bcl-x(L) in MPM chemoresistance. Since it has been recently suggested that Mcl-1 cooperates with Bcl-x(L) for protection against cell death, we investigated the response of mesothelioma cell lines to the downregulation of Bcl-x(L) (alone or in combination with cisplatin) and the potential interest of its concomitant inhibition with that of Mcl-1. Using RNA interference, we showed that Bcl-x(L) depletion sensitized two highly chemoresistant mesothelioma cell lines to cisplatin and that under this treatment, one cell line, MSTO-211H, displayed an apoptotic type of cell death, whereas the other, NCI-H28, evidenced mainly necrotic-type cell death. Otherwise, the inhibition of Mcl-1 by cisplatin may contribute to this induction of cell death observed after Bcl-x(L) downregulation. Strikingly, we observed that the simultaneous inhibition of Bcl-x(L) and Mcl-1 using small interfering RNA (siRNA) induced a massive cell death in the absence of chemotherapy and was sufficient to avoid escape to treatment in MSTO-211H cells. In NCI-H28, the addition of a low cisplatin concentration allowed to impede the long-term recovery observed after treatment by the siRNA combination. Together, these findings provide a strong molecular basis for the clinical evaluation of therapies targeting both Bcl-x(L) and Mcl-1, alone or in combination with conventional chemotherapy, for the treatment of MPM.

Bcl-XL and MCL-1 constitute pertinent targets in ovarian carcinoma and their concomitant inhibition is sufficient to induce apoptosis.

In ovarian carcinomas, recurrence and acquired chemoresistance are the first leading causes of therapeutic failure and are responsible for the poor overall survival rate. Cisplatin exposure of sensitive cells has been previously associated with a down-regulation of Bcl-X(L) expression and apoptosis, whereas recurrence was systematically observed when Bcl-X(L) expression was maintained. Bcl-X(L) down-regulation could thus constitute an interesting chemosensitizing strategy. We showed that a Bcl-X(L)targeted RNA interference strategy efficiently sensitized chemoresistant ovarian carcinoma cells to cisplatin, but some of them were still able to re-proliferate. Considering the possible cooperation between Bcl-X(L)and MCL-1, we investigated the possibility to avoid recurrence in vitro using a multi-targeted RNAi strategy directed against these two anti-apoptotic proteins. We showed that their concomitant inhibition lead to massive apoptosis in absence of cisplatin, this multi-targeted RNAi approach being much more efficient than conventional chemotherapy. We thus demonstrated that Bcl-X(L) and MCL-1 cooperate to constitute together a strong molecular "bolt", which elimination could be sufficient to allow chemoresistant ovarian carcinoma cells apoptosis. Moreover, we demonstrated that in presence of a low concentration of cisplatin, the concomitant down-regulation of Bcl-X(L) and MCL-1 allowed a complete annihilation of tumour cells population thus avoiding subsequent recurrence in vitro in cell lines highly refractory to any type of conventional chemotherapy. Therefore, Bcl-X(L) and MCL-1 targeted strategies could constitute an efficient therapeutic tool for the treatment of chemoresistant ovarian carcinoma, in association with conventional chemotherapy.

Stromal compartment as a survival prognostic factor in advanced ovarian carcinoma.

OBJECTIVE: We investigated the prognostic significance of stromal compartment on the overall survival of patients with advanced epithelial ovarian cancer. METHODS: We evaluated retrospectively the stroma proportion of the tumor surgical specimens of 194 patients with stages III and IV disease, using histochemical staining and fully automatic virtual slide processing. The prognostic significance of stroma proportion and clinical parameters were evaluated using log-rank test and Cox regression. RESULTS: Stroma proportion was found to be an independent prognostic factor by both univariate (P = 0.016) and multivariate analyses (hazards ratio = 1.45, P = 0.011). CONCLUSION: The present data indicate that a high stroma proportion is related to a poor prognosis of stage III and IV ovarian carcinomas.

Functional miRNA Screening Identifies Wide-ranging Antitumor Properties of miR-3622b-5p and Reveals a New Therapeutic Combination Strategy in Ovarian Tumor Organoids.

Novel therapeutic strategies are urgently required for the clinical management of chemoresistant ovarian carcinoma, which is the most lethal of the gynecologic malignancies. miRNAs hold promise because they play a critical role in determining the cell phenotype by regulating several hundreds of targets, which could constitute vulnerabilities of cancer cells. A combination of gain-of-function miRNA screening and real-time continuous cell monitoring allows the identification of miRNAs with robust cytotoxic effects in chemoresistant ovarian cancer cells. Focusing on miR-3622b-5p, we show that it induces apoptosis in several ovarian cancer cell lines by both directly targeting Bcl-xL and EGFR-mediating BIM upregulation. miR-3622b-5p also sensitizes cells to cisplatin by inhibiting Bcl-xL in ovarian cancer cell lines escaping BIM induction. miR-3622b-5p also exerts antimigratory capacities by targeting both LIMK1 and NOTCH1. These wide-ranging antitumor properties of miR-3622b-5p in ovarian cancer cells are mimicked by the associations of pharmacologic inhibitors targeting these proteins. The combination of an EGFR inhibitor together with a BH3-mimetic molecule induced a large decrease in cell viability in a panel of ovarian cancer cell lines and several ovarian patient-derived tumor organoids, suggesting the value of pursuing such a combination therapy in ovarian carcinoma. Altogether, our work highlights the potential of phenotype-based miRNA screening approaches to identify lethal interactions which might lead to new drug combinations and clinically applicable strategies.

Early evaluation of the effects of chemotherapy with longitudinal FDG small-animal PET in human testicular cancer xenografts: early flare response does not reflect refractory disease.

AIM: We aimed to evaluate the usefulness of FDG PET in the early prediction of the effects of chemotherapy on human testicular cancer xenografts. MATERIAL AND METHODS: Nude rats bearing subcutaneous human embryonal carcinoma xenografts received either cisplatin (5 mg/kg) or saline serum. Small-animal PET studies were performed on days 0, 2, 4 and 7 and compared to immunochemistry studies, flow cytometry studies and hexokinase assays. RESULTS: Cisplatin treatment resulted in biphasic FDG uptake evolution: a peak was observed on day 2, followed by a marked decrease on day 7 despite an insignificant change in tumour volume. Similarly, a peak in cyclin A immunostaining was observed on days 2 and 4), followed by a significant decrease on day 7. Flow cytometry showed that the cyclin A peak was not related to increased cell proliferation but was due to a transient S and G(2)/M cell cycle arrest. A marked increase in cell apoptosis was observed from day 2 to day 7. GLUT-1 showed a significant decrease on day 7. Macrophagic infiltrate remained stable except for an increase observed on day 7. In control tumours, continuous growth was observed, all immunostaining markers remaining stable over time. Hexokinase activity was significantly lower on day 7 in treated tumours than in controls. CONCLUSION: FDG PET may be useful in the early evaluation of treatment in patients with testicular cancer. In our model, a very early increased [(18)F]-FDG uptake was related to a transient cell cycle arrest and early stage apoptosis but did not reveal refractory disease.

Usefulness of automatic quantification of immunochemical staining on whole tumor sections for correlation with oncological small animal PET studies: an example with cell proliferation, glucose transporter 1 and FDG.

AIM: To highlight the use of automatic quantification of immunochemical staining on digitized images of whole tumor sections in preclinical positron emission tomography (PET) studies. MATERIALS AND METHODS: Xenografted human testicular tumors (36) were imaged with 2-deoxy-2[F-18]fluoro-D: -glucose (FDG) small animal PET (SA-PET). Tumor cell proliferation and glucose transportation were assessed with cyclin A and Glut-1 immunostaining. Tumor slides were digitized and processed with PixCyt software enabling whole slide quantification, then compared with junior and senior pathologist manual scoring. Manual and automatic quantification results were correlated to FDG uptake. RESULTS: For cyclin A, inter- and intra-observer agreement for manual scoring was 0.52 and 0.72 and concordance between senior pathologist and automatic quantification was 0.84. Correlations between Tumor/Background ratio and tumor cell proliferation assessed by automatic quantification, junior and senior pathologists were 0.75, 0.55, and 0.61, respectively. Correlation between Tumor/Background ratio and Glut-1 assessed by automatic quantification was 0.74. CONCLUSION: Automatic quantification of immunostaining is a valuable tool to overcome inter- and intra-observer variability for correlation of cell proliferation or other markers with tumor tracer uptake.

Inhibition of store-operated channels by carboxyamidotriazole sensitizes ovarian carcinoma cells to anti-BclxL strategies through Mcl-1 down-regulation.

The anti-apoptotic proteins Bcl-xL and Mcl-1 have been identified to play a pivotal role in apoptosis resistance in ovarian cancer and constitute key targets for innovative therapeutic strategies. Although BH3-mimetics (i.e. ABT-737) potently inhibit Bcl-xL activity, targeting Mcl-1 remains a hurdle to the success of these strategies. Calcium signaling is profoundly remodeled during carcinogenesis and was reported to activate the signaling pathway controlling Mcl-1 expression. In this context, we investigated the effect of carboxyamidotriazole (CAI), a calcium channel inhibitor used in clinical trials, on Mcl-1 expression. CAI had an anti-proliferative effect on ovarian carcinoma cell lines and strongly down-regulated Mcl-1 expression. It inhibited store-operated calcium entry (SOCE) and Mcl-1 translation through mTORC1 deactivation. Moreover, it sensitized ovarian carcinoma cells to anti-Bcl-xL strategies as their combination elicited massive apoptosis. Its effect on mTORC1 and Mcl-1 was mimicked by the potent SOCE inhibitor, YM58483, which also triggered apoptosis when combined with ABT-737. As a whole, this study suggests that CAI sensitizes to anti-Bcl-xL strategies via its action on Mcl-1 translation and that modulation of SOCE could extend the therapeutic arsenal for treatment of ovarian carcinoma.

Antiangiogenic and anti-invasive effects of sunitinib on experimental human glioblastoma.

Angiogenesis inhibitors appear to be promising therapies for highly vascularized tumors such as glioblastoma multiforme (GBM). Sunitinib is an oral multitargeted tyrosine kinase inhibitor with both antiangiogenic and antitumor activities due to selective inhibition of various receptor tyrosine kinases, including those important for angiogenesis (vascular endothelial growth factor receptors and platelet-derived growth factor receptors). Here we evaluated the antitumor activities of sunitinib on orthotopic models of GBM in vitro and in vivo. Sunitinib potently inhibited angiogenesis that was stimulated by implantation of U87MG and GL15 cells into organotypic brain slices at concentrations as low as 10 nM. At high dose (10 microM), sunitinib induced direct antiproliferative and proapoptotic effects on GL15 cells and decreased invasion of these cells implanted into brain slices by 49% (p < 0.001). Treatment was associated with decreases in Src (35%) and focal adhesion kinase (44%) phosphorylation. However, anti-invasive activity was not observed in vivo at the highest dose level utilized (80 mg/kg per day). Survival experiments involving athymic mice bearing intracerebral U87MG GBM demonstrated that oral administration of 80 mg/kg sunitinib (five days on, two days off) improved median survival by 36% (p < 0.0001). Sunitinib treatment caused a 74% reduction in microvessel density (p < 0.05), an increase in tumor necrosis, and a decrease in number of GBM cells positive for MIB antibody. Sunitinib exhibited potent antiangiogenic activity that was associated with a meaningful prolongation of survival of mice bearing intracerebral GBM. These data support the potential utility of sunitinib in the treatment of GBM.

Improvement of semi-quantitative small-animal PET data with recovery coefficients: a phantom and rat study.

AIM: To evaluate the accuracy of semi-quantitative small-animal PET data, uncorrected for attenuation, and then of the same semi-quantitative data corrected by means of recovery coefficients (RCs) based on phantom studies. MATERIALS AND METHODS: A phantom containing six fillable spheres (diameter range: 4.4-14 mm) was filled with an 18F-FDG solution (spheres/background activity=10.1, 5.1 and 2.5). RCs, defined as measured activity/expected activity, were calculated. Nude rats harbouring tumours (n=50) were imaged after injection of 18F-FDG and sacrificed. The standardized uptake value (SUV) in tumours was determined with small-animal PET and compared to ex-vivo counting (ex-vivo SUV). Small-animal PET SUVs were corrected with RCs based on the greatest tumour diameter. Tumour proliferation was assessed with cyclin A immunostaining and correlated to the SUV. RESULTS: RCs ranged from 0.33 for the smallest sphere to 0.72 for the largest. A sigmoidal correlation was found between RCs and sphere diameters (r(2)=0.99). Small-animal PET SUVs were well correlated with ex-vivo SUVs (y=0.48x-0.2; r(2)=0.71) and the use of RCs based on the greatest tumour diameter significantly improved regression (y=0.84x-0.81; r(2)=0.77), except for tumours with important necrosis. Similar results were obtained without sacrificing animals, by using PET images to estimate tumour dimensions. RC-based corrections improved correlation between small-animal PET SUVs and tumour proliferation (uncorrected data: Rho=0.79; corrected data: Rho=0.83). CONCLUSION: Recovery correction significantly improves both accuracy of small-animal PET semi-quantitative data in rat studies and their correlation with tumour proliferation, except for largely necrotic tumours.

The mTORC1/2 Inhibitor AZD8055 Strengthens the Efficiency of the MEK Inhibitor Trametinib to Reduce the Mcl-1/[Bim and Puma] ratio and to Sensitize Ovarian Carcinoma Cells to ABT-737.

The identification of novel therapeutic strategies is an important urgent requirement for the clinical management of ovarian cancer, which remains the leading cause of death from gynecologic cancer. Several studies have shown that the antiapoptotic proteins Bcl-xL and Mcl-1, as well as the proapoptotic protein Bim, are key elements to be modulated to kill ovarian cancer cells. Pharmacologic inhibition of Bcl-xL is possible by using BH3-mimetic molecules like ABT-737. However, inhibition of Mcl-1 and/or promotion of its BH3-only partners (including Bim, Puma, and Noxa) remains a challenge that may be achieved by modulating the signaling pathways upstream. This study sought whether AZD8055-induced mTOR inhibition and/or trametinib-induced MEK inhibition could modulate Mcl-1 and its partners to decrease the Mcl-1/BH3-only ratio and thus sensitize various ovarian cancer cell lines to ABT-737. AZD8055 treatment inhibited Mcl-1 and increased Puma expression but did not induce massive apoptosis in combination with ABT-737. In contrast, trametinib, which decreased the Mcl-1/BH3-only protein ratio by upregulating Puma and dephosphorylated active Bim, sensitized IGROV1-R10 and OVCAR3 cells to ABT-737. Adding AZD8055 to trametinib further reduced the Mcl-1/BH3-only protein ratio and triggered apoptosis without ABT-737 in IGROV1-R10 cells. Moreover, the AZD8055/trametinib association highly sensitized all cell lines including SKOV3 to ABT-737, the induced dephosphorylated Bim being crucial in this sensitization. Finally, the three-drug combination was also very efficient when replacing AZD8055 by the pan-Akt inhibitor MK-2206. This study thus proposes original multitargeted strategies and may have important implications for the design of novel approaches for ovarian cancer treatment. Mol Cancer Ther; 16(1); 102-15. ©2016 AACR.

Effect of 2-deoxy-D-glucose on various malignant cell lines in vitro.

BACKGROUND: 2-Deoxy-D-glucose (2-DG) is an analog of glucose that is preferentially captured by tumors and is accumulated in transformed cells, because the phosphorylated molecule (2-DG-6P) cannot be metabolized or diffused outside the cells. Targeted with a fluorine atom, 18F-DG is currently used to visualize malignant tumors (PET scan). Although cancer cells have been reported to be strongly dependent on glycolysis (Warburg effect), very few reports have studied the inhibitory effects of 2-DG on cancer. MATERIALS AND METHODS: Our objective was to study, in a large panel of human malignant cells of various origins (ovarian, squamous, cerebral, hepatic, colonic and mesothelial), if the inhibitory activity of 2DG against tumor growth could be considered a general phenomenon and to determine its effect on the cell cycle. RESULTS: Four types of response in the different cell lines were observed when cells were cultured in the presence of 2-DG (5 mM) continuous exposure: proliferation slow down; proliferation arrest without signs of apoptosis; strong cell cycle arrest accompanied by moderate apoptosis induction; massive apoptosis. CONCLUSION: 2-DG appears as an interesting new therapeutic agent against cancer in vitro, and should be tested in in vivo studies.

Characterization of the t(14;18) BCL2-IGH translocation in farmers occupationally exposed to pesticides.

Increasing incidence of non-Hodgkin's lymphoma have been associated repeatedly with farming occupation and particular attention focused on the role of pesticide exposure to potentially explain part of this trend. A genetic hallmark of non-Hodgkin's lymphoma is the presence of recurrent chromosomal translocations involving the immunoglobulin heavy chain gene. Of these, the t(14;18), which deregulates BCL2 expression and inhibits apoptosis, is the most frequent in follicular lymphoma and has been detected consistently in peripheral blood lymphocytes of healthy individuals. As BCL2-IGH translocation represents an early step of the malignant process, we evaluated the occurrence and molecular characteristics of BCL2-IGH translocation in 56 individuals occupationally exposed to pesticides in open field farming They were selected from a representative cohort of farmers with a well-defined assessment of pesticide exposure taking into account potential confounding factors, smoking, sunlight, and age. Our results suggest that occupational exposure to pesticides would increase BCL2-IGH prevalence together with the frequency of BCL2-IGH-bearing cells especially during the high pesticide use period. Distribution of BCL2 or IGH breakpoint positions seemed to be independent of pesticide exposure and was similar to those found in other healthy populations or lymphoma patients. Finally, these results provide additional evidence that BCL2-IGH translocation measurements could be a measure of acquired genetic instability in relation to genotoxic exposure in a gene directly relevant in term of lymphomagenesis.

An overview of long non-coding RNAs in ovarian cancers.

As with miRNAs a decade ago, the scientific community recently understood that lncRNAs represent a new layer of complexity in the regulation of gene expression. Although only a subset of lncRNAs has been functionally characterized, it is clear that they are deeply involved in the most critical physiological and pathological biological processes. This review shows that in ovarian carcinoma, data already available testify to the importance of lncRNAs and that the demonstration of an ever-growing role of lncRNAs in the biology of this malignancy can be expected from future studies. We also underline the importance of their relationship with associated protein partners and miRNAs. Together, the available information suggests that the emerging field of lncRNAs will pave the way for a better understanding of ovarian cancer biology and might lead to the development of innovative therapeutic approaches. Moreover, lncRNAs expression signatures either alone or in combination with other types of markers (miRNAs, mRNAs, proteins) could prove useful to predict outcome or treatment follow-up in order to improve the therapeutic care of ovarian carcinoma patients.

Towards a new standardized method for circulating miRNAs profiling in clinical studies: Interest of the exogenous normalization to improve miRNA signature accuracy.

Circulating miRNAs are promising biomarkers in oncology but have not yet been implemented in the clinic given the lack of concordance across studies. In order to increase the cross-studies reliability, we attempted to reduce and to control the circulating miRNA expression variability between patients. First, to maximize profiling signals and to reduce miRNA expression variability, three isolation kits were compared and the NucleoSpin(®) kit provided higher miRNA concentrations than the other widely used kits. Second, to control inter-sample variability during the profiling step, the exogenous miRNAs normalization method commonly used for RT-qPCR validation step was adapted to microarray experiments. Importantly, exogenous miRNAs presented two-fold lower inter-sample variability than the widely used endogenous miR-16-5p reflecting that the latter is subject to both biological and technical variability. Although Caenorhabditis elegans miRNAs isolation yields were heterogeneous, they correlated to each other and to their geometrical mean across samples. The normalization based on the geometrical mean of three exogenous miRNAs increased the correlation up-to 0.97 between the microarrays and individual RT-qPCR steps of circulating miRNAs expression. Overall, this new strategy open new avenue to identify reliable circulating miRNA signatures for translation into clinical practice.

alpha(v) integrins regulate cell proliferation through integrin-linked kinase (ILK) in ovarian cancer cells.

Integrins regulate both adhesion and signaling processes involved in proliferation and survival. alpha(v)beta(3) and alpha(v)beta(5) integrins have been shown to mediate cell adhesion and migration. Here we used human ovarian cancer cell lines (IGROV1, SKOV-3) that express alpha(v)beta(3) and alpha(v)beta(5) to study their role in cell proliferation and the signaling pathways involved. We found that alpha(v) integrins regulate cell proliferation through activation of integrin-linked kinase (ILK). An anti-alpha(v)-blocking antibody specifically inhibits the growth of IGROV1 and SKOV-3. The inhibition of cell proliferation involves alpha(v)beta(3) in IGROV1 cells, and both alpha(v)beta(3) and alpha(v)beta(5) in SKOV-3 cells. The reduced growth rate induced by alpha(v) integrin blockade is linked in both cell lines to G1/S cell cycle arrest. alpha(v) integrin blockade by neutralizing antibody as well as cyclic-RGD peptide caused an inhibition of ILK activity and phosphorylation of PKB/Akt on serine-473 but not on threonine-308, and was accompanied by an increase in p27(Kip1) expression. Overexpression of wild-type ILK rescued the phosphorylation of PKB/Akt on serine-473 in cells treated with anti-alpha(v) antibody. Inhibition of ILK by a pharmacological inhibitor results in inhibition of cell proliferation, PKB/Akt phosphorylation and increase of p27(Kip1). These results demonstrate that alpha(v) integrins regulate ovarian cancer cell proliferation through ILK.