Gene expression dataset of prostate cells upon MIR205HG/LEADR modulation

Although the role of miR-205 has been widely elucidated, the function of its host gene (MIR205HG) is yet to be clarified. We have recently investigated whether this gene is a simple endorsement for miRNA production or it may act independently, demonstrating its action as nuclear long noncoding RNA able to control basal-luminal differentiation in the human prostate context, thus deserving the reannotation as LEADR, Long Epithelial Alu-interacting Differentiation-related RNA. Here, we describe the loss and gain of function approaches experimentally used to modulate LEADR expression, and the bioinformatic procedures employed to analyze microarray data in our published article \u201cLEADeR role of miR-205 host gene as long noncoding RNA in prostate basal cell differentiation\u201d [1]. The high reproducibility of replicates, the strong concordance with a validation technique, and the coherent behavior observed for differentially expression features, both in terms of single genes and deregulated pathways, not only support the quality of the data, but also endorse their potential reuse. Very relevant are the diverse silencing and overexpression strategies employed (all of which analyzed in multiple biologically independent replicates), which should allow other scientists to analyze our dataset for the specific purpose of their research, may it be the study of MIR205HG function as miRNA host gene, the investigation of its miRNA-independent biological role or again the dissection of Alu sequence involvement in the mechanism of action of long noncoding RNAs, which is a hot topic in the field

Predicting and Understanding Cancer Response to Treatment

Contains fulltext : 196925.pdf (publisher's version ) (Open Access

Clusterin: A potential target for improving response to antiestrogens

Antiestrogens represent the first line of therapy in the treatment of estrogen receptor-positive (ER+) breast cancer patients. Unfortunately, up to 40% of patients develop resistance associated with progression and frequently die for metastatic breast cancer. The molecular events leading to pharmacological resistance are not completely understood. We attempted to verify in an experimental model the role of cytoplasmic clusterin (CLU), a cytoprotective protein found to be up-regulated in antiestrogen-resistant patients, following neoadjuvant treatment with toremifene. The role of cytoplasmic clusterin in modulating response to two antiestrogens (toremifene and tamoxifen) was studied in two ER+ anti-estrogen-sensitive cell lines (MCF-7, 734B) and one ER+ antiestrogen-resistant cell line (T47D) using siRNA strategy. Resistant cells were characterised by higher levels of cytoplasmic clusterin than sensitive cells, and antiestrogen treatments up-regulated clusterin levels in both sensitive and resistant cell lines. Treatment with siRNA completely abolished cytoplasmic clusterin expression in all cell lines, but its down-regulation resulted in a significant decrease of cell growth only In the resistant line. We therefore concluded that: i) basal clusterin levels are higher in antiestrogen resistant cells. ii) clusterin is up-regulated following antiestrogen treatment independently of the sensitivity of the cell line, iii) downregulation of cytoplasmic clusterin restores sensitivity to toremifene in the antiestrogen-resistant cell line. Such results support the concept that targeting CLU could represent a promising therapeutic strategy in association with antiestrogen treatment in breast cancer patients

Reciprocal Metabolic Reprogramming through Lactate Shuttle Coordinately Influences Tumor-Stroma Interplay

Cancer-associated fibroblasts (CAF) engage in tumor progression by promoting the ability of cancer cells to undergo epithelial-mesenchymal transition (EMT), and also by enhancing stem cells traits and metastatic dissemination. Here we show that the reciprocal interplay between CAFs and prostate cancer cells goes beyond the engagement of EMT to include mutual metabolic reprogramming. Gene expression analysis of CAFs cultured ex vivo or human prostate fibroblasts obtained from benign prostate hyperplasia revealed that CAFs undergo Warburg metabolism and mitochondrial oxidative stress. This metabolic reprogramming toward a Warburg phenotype occurred as a result of contact with prostate cancer cells. Intercellular contact activated the stromal fibroblasts, triggering increased expression of glucose transporter GLUT1, lactate production, and extrusion of lactate by de novo expressed monocarboxylate transporter-4 (MCT4). Conversely, prostate cancer cells, upon contact with CAFs, were reprogrammed toward aerobic metabolism, with a decrease in GLUT1 expression and an increase in lactate upload via the lactate transporter MCT1. Metabolic reprogramming of both stromal and cancer cells was under strict control of the hypoxia-inducible factor 1 (HIF1), which drove redox-and SIRT3-dependent stabilization of HIF1 in normoxic conditions. Prostate cancer cells gradually became independent of glucose consumption, while developing a dependence on lactate upload to drive anabolic pathways and thereby cell growth. In agreement, pharmacologic inhibition of MCT1-mediated lactate upload dramatically affected prostate cancer cell survival and tumor outgrowth. Hence, cancer cells allocate Warburg metabolism to their corrupted CAFs, exploiting their byproducts to grow in a low glucose environment, symbiotically adapting with stromal cells to glucose availability. Cancer Res; 72(19); 5130-40. (C)2012 AACR

MiR-1227 targets SEC23A to regulate the shedding of large extracellular vesicles

Cancer cells shed a heterogenous mixture of extracellular vesicles (EVs), differing in both size and composition, which likely influence physiological processes in different manners. However, how cells differentially control the shedding of these EV populations is poorly understood. Here, we show that miR-1227, which is enriched in prostate cancer EVs, compared to the cell of origin, but not in EVs derived from prostate benign epithelial cells, induces the shedding of large EVs (such as large oncosomes), while inhibiting the shedding of small EVs (such as exosomes). RNA sequencing from cells stably expressing miR-1227, a modified RISCTRAP assay that stabilizes and purifies mRNA-miR-1227 complexes for RNA sequencing, and in silico target prediction tools were used to identify miR-1227 targets that may mediate this alteration in EV shedding. The COPII vesicle protein SEC23A emerged and was validated by qPCR, WBlot, and luciferase assays as a direct target of miR-1227. The inhibition of SEC23A was sufficient to induce the shedding of large EVs. These results identify a novel mechanism of EV shedding, by which the inhibition of SEC23A by miR-1227 induces a shift in EV shedding, favoring the shedding of large EV over small EV

Core Biopsies from Prostate Cancer Patients in Active Surveillance Protocols Harbor PTEN and MYC Alterations

Background: Genomic characterization of prostate cancer (PCa) biopsies may improve criteria for the selection of patients suitable for active surveillance (AS). Objective: To identify somatic genomic aberrations associated with adverse outcome as AS protocol exclusion indicators. Design, setting and participants: Whole-exome sequencing profiles were generated for Gleason score (GS) = 3 + 3 biopsies obtained from 54 PCa patients enrolled in two AS protocols. Patients were selected as representative of a nonindolent population, consisting of 27 patients who dropped out from AS due to upgrading (ie, finding of GS > 3 + 3 at a follow-up biopsy) within 2 yr, and a potentially indolent population, consisting of 27 patients in AS for 654 yr without any evidence of reclassification. Outcome measurements and statistical analysis: The genomic alteration landscape of core biopsies was analyzed using an integrated computational pipeline and correlated with patient reclassification due to upgrading. Results and limitations: Of all the GS = 3 + 3 biopsies of the study cohort, 34% showed clear evidence of somatic copy number aberrations along the genome. Of these, 39% came from the potentially indolent and 61% from the nonindolent population. Single-nucleotide variants demonstrated low allelic fractions and included a common F133C mutation in the SPOP gene. The minimally altered genomic landscape of the study cohort presented a distinct set of monoallelic deletions, including on 8p, 13q, 16q, and 21q, and rare amplifications of 8q, which were observed in both AS patient populations. Concerning lesions typically associated with adverse outcome, PTEN deletions and MYC amplification, though observed in a small number of cases, were detected exclusively or preferentially, respectively, in nonindolent patients. Such molecular findings were confirmed by immunohistochemistry on the same tissue blocks. The small sample size and the retrospective nature of the analysis represent the main study limitations. Conclusions: Genomic features enriched in aggressive tumors can be detected in GS = 3 + 3 core biopsies of AS patients. Patient summary: PTEN and MYC alterations at the time of diagnosis would deserve investigation in larger cohorts of AS patients to assess their potential as biomarkers for a more precise/earlier identification of patients at risk of reclassification. The presence of adverse outcome-related genomic lesions, such as PTEN deletion and MYC amplification, in GPS = 3 + 3 diagnostic core biopsies of prostate cancer patients could be considered for a more precise/earlier selection of patients not suitable for active surveillance

Estrogen-dependent dynamic profile of eNOS-DNA associations in prostate cancer

In previous work we have documented the nuclear translocation of endothelial NOS (eNOS) and its participation in combinatorial complexes with Estrogen Receptor Beta (ERβ) and Hypoxia Inducible Factors (HIFs) that determine localized chromatin remodeling in response to estrogen (E2) and hypoxia stimuli, resulting in transcriptional regulation of genes associated with adverse prognosis in prostate cancer (PCa). To explore the role of nuclear eNOS in the acquisition of aggressive phenotype in PCa, we performed ChIP-Sequencing on chromatin-associated eNOS from cells from a primary tumor with poor outcome and from metastatic LNCaP cells. We found that: 1. the eNOS-bound regions (peaks) are widely distributed across the genome encompassing multiple transcription factors binding sites, including Estrogen Response Elements. 2. E2 increased the number of peaks, indicating hormone-dependent eNOS re-localization. 3. Peak distribution was similar with/without E2 with ≈ 55% of them in extragenic DNA regions and an intriguing involvement of the 5′ domain of several miRs deregulated in PCa. Numerous potentially novel eNOS-targeted genes have been identified suggesting that eNOS participates in the regulation of large gene sets. The parallel finding of downregulation of a cluster of miRs, including miR-34a, in PCa cells associated with poor outcome led us to unveil a molecular link between eNOS and SIRT1, an epigenetic regulator of aging and tumorigenicity, negatively regulated by miR-34a and in turn activating eNOS. E2 potentiates miR-34a downregulation thus enhancing SIRT1 expression, depicting a novel eNOS/SIRT1 interplay fine-tuned by E2-activated ER signaling, and suggesting that eNOS may play an important role in aggressive PCa

MiR-205 enhances radiation sensitivity of prostate cancer cells by impairing DNA damage repair through PKCϵ and ZEB1 inhibition

Background: Radiotherapy is one of the main treatment options for non-metastatic prostate cancer (PCa). Although treatment technical optimization has greatly improved local tumor control, a considerable fraction of patients still experience relapse due to the development of resistance. Radioresistance is a complex and still poorly understood phenomenon involving the deregulation of a variety of signaling pathways as a consequence of several genetic and epigenetic abnormalities. In this context, cumulative evidence supports a functional role of microRNAs in affecting radioresistance, suggesting the modulation of their expression as a novel radiosensitizing approach. Here, we investigated for the first time the ability of miR-205 to enhance the radiation response of PCa models. Methods: miR-205 reconstitution by a miRNA mimic in PCa cell lines (DU145 and PC-3) was used to elucidate miR-205 biological role. Radiation response in miRNA-reconstituted and control cells was assessed by clonogenic assay, immunofluorescence-based detection of nuclear \u3b3-H2AX foci and comet assay. RNAi was used to silence the miRNA targets PKC\u3f5 or ZEB1. In addition, target-protection experiments were carried out using a custom oligonucleotide designed to physically disrupt the pairing between the miR-205 and PKC\u3f5. For in vivo experiments, xenografts generated in SCID mice by implanting DU145 cells stably expressing miR-205 were exposed to 5-Gy single dose irradiation using an image-guided animal micro-irradiator. Results: miR-205 reconstitution was able to significantly enhance the radiation response of prostate cancer cell lines and xenografts through the impairment of radiation-induced DNA damage repair, as a consequence of PKC\u3f5 and ZEB1 inhibition. Indeed, phenocopy experiments based on knock-down of either PKC\u3f5 or ZEB1 reproduced miR-205 radiosensitizing effect, hence confirming a functional role of both targets in the process. At the molecular level, miR-205-induced suppression of PKC\u3f5 counteracted radioresistance through the impairment of EGFR nuclear translocation and the consequent DNA-PK activation. Consistently, disruption of miR-205-PKC\u3f5 3'UTR pairing almost completely abrogated the radiosensitizing effect. Conclusions: Our results uncovered the molecular and cellular mechanisms underlying the radiosensitizing effect of miR-205. These findings support the clinical interest in developing a novel therapeutic approach based on miR-205 reconstitution to increase PCa response to radiotherapy

Large oncosomes mediate intercellular transfer of functional microRNA

Prostate cancer cells release atypically large extracellular vesicles (EVs), termed large oncosomes, which may play a role in the tumor microenvironment by transporting bioactive molecules across tissue spaces and through the blood stream. In this study, we applied a novel method for selective isolation of large on cosomes applicable to human plateletpoor plasma, where the presence of caveolin-l-positive large oncosomes identified patients with metastatic disease. This procedure was also used to validate results of a miRNA array performed on heterogeneous populations of EVs isolated from tumorigenic RWPE-2 prostate cells and from isogenic non-tumorigenic RWPE-1 cells. The results showed that distinct classes of miRNAs are expressed at higher levels in EVs derived from the tumorigenic cells in comparison to their non-tumorigenic counterpart. Large oncosomes enhanced migration of cancer-associated fibroblasts (CAFs), an effect that was increased by miR-1227, a miRNA abundant in large oncosomes produced by RWPE-2 cells. Our findings suggest that large oncosomes in the circulation report metastatic disease in patients with prostate cancer, and that this class of EV harbors functional molecules that may play a role in conditioning the tumor microenvironment

Anti-tumor activity of selective inhibitors of XPO1/CRM1-mediated nuclear export in diffuse malignant peritoneal mesothelioma : the role of survivin

Survivin, which is highly expressed and promotes cell survival in diffuse malignant peritoneal mesothelioma (DMPM), exclusively relies on exportin 1 (XPO1/ CRM1) to be shuttled into the cytoplasm and perform its anti-apoptotic function. Here, we explored the efficacy of Selective Inhibitors of Nuclear Export (SINE), KPT-251, KPT-276 and the orally available, clinical stage KPT-330 (selinexor), in DMPM preclinical models. Exposure to SINE induced dose-dependent inhibition of cell growth, cell cycle arrest at G1-phase and caspase-dependent apoptosis, which were consequent to a decrease of XPO1/CRM1 protein levels and the concomitant nuclear accumulation of its cargo proteins p53 and CDKN1a. Cell exposure to SINE led to a time-dependent reduction of cytoplasmic survivin levels. In addition, after an initial accumulation, the nuclear protein abundance progressively decreased, as a consequence of an enhanced ubiquitination and proteasome-dependent degradation. SINE and the survivin inhibitor YM155 synergistically cooperated in reducing DMPM cell proliferation. Most importantly, orally administered SINE caused a significant antitumor effect in subcutaneous and orthotopic DMPM xenografts without appreciable toxicity. Overall, we have demonstrated a marked efficacy of SINE in DMPM preclinical models that may relay on the interference with survivin intracellular distribution and function. Our study suggests SINE-mediated XPO1/ CRM1 inhibition as a novel therapeutic option for DMPM