Liat Edry

Hsa-mir-125b-2 is highly expressed in childhood ETV6/RUNX1 (TEL/AML1) leukemias and confers survival advantage to growth inhibitory signals independent of p53

MicroRNAs (miRNAs) regulate the expression of multiple proteins in a dose-dependent manner. We hypothesized that increased expression of miRNAs encoded on chromosome 21 (chr 21) contribute to the leukemogenic function of trisomy 21. The levels of chr 21 miRNAs were quantified by qRT–PCR in four types of childhood acute lymphoblastic leukemia (ALL) characterized by either numerical (trisomy or tetrasomy) or structural abnormalities of chr 21. Suprisingly, high expression of the hsa-mir-125b-2 cluster, consisting of three miRNAs, was identified in leukemias with the structural ETV6/RUNX1 abnormality and not in ALLs with trisomy 21. Manipulation of ETV6/RUNX1 expression and chromatin immunoprecipitation studies showed that the high expression of the miRNA cluster is an event independent of the ETV6/RUNX1 fusion protein. Overexpression of hsa-mir-125b-2 conferred a survival advantage to Ba/F3 cells after IL-3 withdrawal or a broad spectrum of apoptotic stimuli through inhibition of caspase 3 activation. Conversely, knockdown of the endogenous miR-125b in the ETV6/RUNX1 leukemia cell line REH increased apoptosis after Doxorubicin and Staurosporine treatments. P53 protein levels were not altered by miR-125b. Together, these results suggest that the expression of hsa-mir-125b-2 in ETV6/RUNX1 ALL provides survival advantage to growth inhibitory signals in a p53-independent manner.

Automated long-term tracking and social behavioural phenotyping of animal colonies within a semi-natural environment

Social behaviour has a key role in animal survival across species, ranging from insects to primates and humans. However, the biological mechanisms driving natural interactions between multiple animals, over long-term periods, are poorly studied and remain elusive. Rigorous and objective quantification of behavioural parameters within a group poses a major challenge as it requires simultaneous monitoring of the positions of several individuals and comprehensive consideration of many complex factors. Automatic tracking and phenotyping of interacting animals could thus overcome the limitations of manual tracking methods. Here we report a broadly applicable system that automatically tracks the locations of multiple, uniquely identified animals, such as mice, within a semi-natural setting. The system combines video and radio frequency identified tracking data to obtain detailed behavioural profiles of both individuals and groups. We demonstrate the usefulness of these data in characterizing individual phenotypes, interactions between pairs and the collective social organization of groups.

Regulation of cancer aggressive features in melanoma cells by microRNAs

MicroRNAs (miRNAs) are small non-coding RNAs with regulatory roles, which are involved in a broad spectrum of physiological and pathological processes, including cancer. A common strategy for identification of miRNAs involved in cell transformation is to compare malignant cells to normal cells. Here we focus on identification of miRNAs that regulate the aggressive phenotype of melanoma cells. To avoid differences due to genetic background, a comparative high-throughput miRNA profiling was performed on two isogenic human melanoma cell lines that display major differences in their net proliferation, invasion and tube formation activities. This screening revealed two major cohorts of differentially expressed miRNAs. We speculated that miRNAs up-regulated in the more-aggressive cell line contribute oncogenic features, while the down-regulated miRNAs are tumor suppressive. This assumption was further tested experimentally on five candidate tumor suppressive miRNAs (miR-31, -34a, -184, -185 and -204) and on one candidate oncogenic miRNA (miR-17-5p), all of which have never been reported before in cutaneous melanoma. Remarkably, all candidate Suppressive-miRNAs inhibited net proliferation, invasion or tube formation, while miR-17-5p enhanced cell proliferation. miR-34a and miR-185 were further shown to inhibit the growth of melanoma xenografts when implanted in SCID-NOD mice. Finally, all six candidate miRNAs were detected in 15 different metastatic melanoma specimens, attesting for the physiological relevance of our findings. Collectively, these findings may prove instrumental for understanding mechanisms of disease and for development of novel therapeutic and staging technologies for melanoma.

Insulin-like Growth Factor-I Receptor (IGF-IR) Translocates to Nucleus and Autoregulates IGF-IR Gene Expression in Breast Cancer Cells

The insulin-like growth factor (IGF) system plays an important role in mammary gland biology as well as in the etiology of breast cancer. The IGF-I receptor (IGF-IR), which mediates the biological actions of IGF-I and IGF-II, has emerged in recent years as a promising therapeutic target. The IGF and estrogen signaling pathways act in a synergistic manner in breast epithelial cells. The present study was aimed at investigating 1) the putative translocation of IGF-IR and the related insulin receptor (IR) to the nucleus in breast cancer cells, 2) the impact of IGF-IR and IR levels on IGF-IR biosynthesis in estrogen receptor (ER)-positive and ER-depleted breast cancer cells, and 3) the potential transcription factor role of IGF-IR in the specific context of IGF-IR gene regulation. We describe here a novel mechanism of autoregulation of IGF-IR gene expression by cellular IGF-IR, which is seemingly dependent on ER status. Regulation of the IGF-IR gene by IGF-IR protein is mediated at the level of transcription, as demonstrated by 1) binding assays (DNA affinity chromatography and ChIP) showing specific IGF-IR binding to IGF-IR promoter DNA and 2) transient transfection assays showing transactivation of the IGF-IR promoter by exogenous IGF-IR. The IR is also capable of translocating to the nucleus and binding the IGF-IR promoter in ER-depleted, but not in ER-positive, cells. However, transcription factors IGF-IR and IR display diametrically opposite activities in the context of IGF-IR gene regulation. Thus, whereas IGF-IR stimulated IGF-IR gene expression, IR inhibited IGF-IR promoter activity. In summary, we have identified a novel mechanism of IGF-IR gene autoregulation in breast cancer cells. The clinical implications of these findings and, in particular, the impact of IGF-IR/IR nuclear localization on targeted therapy require further investigation.

Mesenchymal Stem Cell Transplantation Promotes Neurogenesis and Ameliorates Autism Related Behaviors in BTBR Mice

Autism spectrum disorders (ASD) are characterized by social communication deficits, cognitive rigidity, and repetitive stereotyped behaviors. Mesenchymal stem cells (MSC) have a paracrine regenerative effect, and were speculated to be a potential therapy for ASD. The BTBR inbred mouse strain is a commonly used model of ASD as it demonstrates robust behavioral deficits consistent with the diagnostic criteria for ASD. BTBR mice also exhibit decreased brain‐derived neurotrophic factor (BDNF) signaling and reduced hippocampal neurogenesis. In the current study, we evaluated the behavioral and molecular effects of intracerebroventricular MSC transplantation in BTBR mice. Transplantation of MSC resulted in a reduction of stereotypical behaviors, a decrease in cognitive rigidity and an improvement in social behavior. Tissue analysis revealed elevated BDNF protein levels in the hippocampus accompanied by increased hippocampal neurogenesis in the MSC‐transplanted mice compared with sham treated mice. This might indicate a possible mechanism underpinning the behavioral improvement. Our study suggests a novel therapeutic approach which may be translatable to ASD patients in the future. Autism Res 2015.

Involvement of IGF-1R regulation by miR-515-5p modifies breast cancer risk among BRCA1 carriers.

Several lines of evidence indicate that sequence alterations within microRNA (miRNA)-binding sites can modify the binding to its target gene resulting in altered expression patterns. We hypothesized that a single nucleotide polymorphism (SNP) located in the miR-515-5p binding site of igf-1r gene may alter IGF-1R regulation, with consequent effects on breast cancer risk in BRCA1 mutation carriers. Computational prediction revealed that the rs28674628 SNP in the igf-1r 3′ UTR is located within a predicted binding site for miR-515-5p. The effect of this SNP on breast cancer risk was evaluated by genotyping 115 Jewish Ashkenazi carriers of the 185delAG mutation in the BRCA1 gene using the Sequenom platform followed by Kaplan–Meier analysis. Additional data set of 378 Jewish BRCA1 carriers was analyzed to validate our results. MiRNA transfection, Western blot analysis, luciferase reporter assay, real time PCR, and immunohistochemistry were performed to assess direct regulation of igf-1r by miR-515-5p. We show direct regulation of IGF-1R by miR-515-5p. We identified that disrupting miR-515-5p and igf-1r 3′ UTR binding by SNP may cause elevated IGF-1R protein levels. Interestingly, miR-515-5p is downregulated in tumor tissue compared to its non-neoplastic surrounding tissue while IGF-1R levels are elevated. This igf-1r SNP was found to be significantly associated with age at diagnosis of breast cancer in Jewish Ashkenazi BRCA1 mutation carriers. These findings support the hypothesis that a SNP located in igf-1r gene may alter miRNA regulation of IGF-1R, with a putative effect on BRCA1 penetrance and breast cancer risk.

Neuro-Epigenetic Indications of Acute Stress Response in Humans: The Case of MicroRNA-29c

Stress research has progressively become more integrative in nature, seeking to unfold crucial relations between the different phenotypic levels of stress manifestations. This study sought to unravel stress-induced variations in expression of human microRNAs sampled in peripheral blood mononuclear cells and further assess their relationship with neuronal and psychological indices. We obtained blood samples from 49 healthy male participants before and three hours after performing a social stress task, while undergoing functional magnetic resonance imaging (fMRI). A seed-based functional connectivity (FC) analysis was conducted for the ventro-medial prefrontal cortex (vmPFC), a key area of stress regulation. Out of hundreds of microRNAs, a specific increase was identified in microRNA-29c (miR-29c) expression, corresponding with both the experience of sustained stress via self-reports, and alterations in vmPFC functional connectivity. Explicitly, miR-29c expression levels corresponded with both increased connectivity of the vmPFC with the anterior insula (aIns), and decreased connectivity of the vmPFC with the left dorso-lateral prefrontal cortex (dlPFC). Our findings further revealed that miR-29c mediates an indirect path linking enhanced vmPFC-aIns connectivity during stress with subsequent experiences of sustained stress. The correlative patterns of miR-29c expression and vmPFC FC, along with the mediating effects on subjective stress sustainment and the presumed localization of miR-29c in astrocytes, together point to an intriguing assumption; miR-29c may serve as a biomarker in the blood for stress-induced functional neural alterations reflecting regulatory processes. Such a multi-level model may hold the key for future personalized intervention in stress psychopathology.

Abstract 5235: Uncovering tumor-host molecular and cellular interactions involved in tumor dormancy using microRNAs

There is an increasing interest in elucidating the mechanisms of the earlier stages in tumor progression, the point at which a dormant tumor acquires the ability to grow and metastasize. Although the tumor dormancy phenomenon has important implications for early detection and treatment of cancer, its biology and genetic characteristics are poorly understood. microRNAs (miRs), small non-coding RNA molecules which regulate gene expression at the post transcriptional level, are emerging as key regulators in physiological and pathological processes, including tumorigenesis. We hypothesized that specific miRs may also play a key role in the regulation of tumor dormancy and the angiogenic switch. We therefore established a pair of cell lines that generate dormant avascular and fast-growing angiogenic osteosarcomas in SCID mice (Saos-2-D and Saos-2-E respectively). Although these cells share similar growth rate in vitro, when inoculated into mice, Saos-2-E cells establish vascularized and palpable tumors within one month, while Saos-2-D cells remain avascular and non-palpable for a year. Following our hypothesis, miR array of Saos-2-D and Saos-2-E cells, was performed. Several miRs that were differentially-expressed in the dormant versus fast-growing-tumor-forming cells were selected for further evaluation. Out of those miRs, miR200c and miR93 (representing either a mild or a substantial difference in the expression profile respectively) reverted cells forming fast-growing tumors to a dormant non-angiogenic phenotype, both in vitro and in vivo. Preliminary in vitro analysis of human brain, breast and prostate cancer cell lines revealed a similar phenotypic effect following overexpression of those miRs. We therefore propose that miR200c and miR93 play a role in the switch from dormancy. Further analysis on the target genes of those miRs is required in order to fully elucidate their role in tumor dormancy. Uncovering new molecular targets involved in the tumor dormancy phenomenon could provide important tools for novel dormancy-directed tumor therapy strategies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5235. doi:1538-7445.AM2012-5235