Elisa Latorre

Hippocampal development and neural stem cell maintenance require Sox2 -dependent regulation of Shh

Neural stem cells (NSCs) are controlled by diffusible factors. The transcription factor Sox2 is expressed by NSCs and Sox2 mutations in humans cause defects in the brain and, in particular, in the hippocampus. We deleted Sox2 in the mouse embryonic brain. At birth, the mice showed minor brain defects; shortly afterwards, however, NSCs and neurogenesis were completely lost in the hippocampus, leading to dentate gyrus hypoplasia. Deletion of Sox2 in adult mice also caused hippocampal neurogenesis loss. The hippocampal developmental defect resembles that caused by late sonic hedgehog (Shh) loss. In mutant mice, Shh and Wnt3a were absent from the hippocampal primordium. A SHH pharmacological agonist partially rescued the hippocampal defect. Chromatin immunoprecipitation identified Shh as a Sox2 target. Sox2-deleted NSCs did not express Shh in vitro and were rapidly lost. Their replication was partially rescued by the addition of SHH and was almost fully rescued by conditioned medium from normal cells. Thus, NSCs control their status, at least partly, through Sox2-dependent autocrine mechanisms.

Downregulation of HuR as a new mechanism of doxorubicin resistance in breast cancer cells

BackgroundHuR, an RNA binding protein involved in the post-transcriptional regulation of a wide spectrum of mRNAs, has been demonstrated to be a determinant of carcinogenesis and tumor aggressiveness in several cancer types. In this study, we investigated the role of HuR in the apoptosis and in the chemoresistance induced by the widely used anticancer drug doxorubicin in human breast cancer cells (MCF-7).ResultsWe showed that HuR acts in the early phase of cell response to doxorubicin, being induced to translocate into the cytoplasm upon phosphorylation. Reducing HuR levels diminished the apoptotic response to doxorubicin. Doxorubicin-induced apoptosis was also correlated with the presence of HuR in the cytoplasm. Rottlerin, which was able to block HuR nuclear export, had correspondingly antagonistic effects with doxorubicin on cell toxicity. The proapoptotic activity of HuR was not due to cleavage to an active form, as was previously reported. In in vitro selected doxorubicin resistant MCF-7 cells (MCF-7/doxoR) overexpressing the multidrug resistance (MDR) related ABCG2 transporter, we observed a significant HuR downregulation that was paralleled by a corresponding downregulation of HuR targets and by loss of rottlerin toxicity. Restoration of HuR expression in these cells resensitized MCF-7/doxoR cells to doxorubicin, reactivating the apoptotic response.ConclusionsThe present study shows that HuR is necessary to elicit the apoptotic cell response to doxorubicin and that restoration of HuR expression in resistant cells resensitizes them to the action of this drug, thereby identifying HuR as a key protein in doxorubicin pharmacology.

The Ribonucleic Complex HuR-MALAT1 Represses CD133 Expression and Suppresses Epithelial–Mesenchymal Transition in Breast Cancer

Epithelial-to-mesenchymal transition (EMT) is a core process underlying cell movement during embryonic development and morphogenesis. Cancer cells hijack this developmental program to execute a multi-step cascade, leading to tumorigenesis and metastasis. CD133 (PROM1), a marker of cancer stem cells, has been shown to facilitate EMT in various cancers, but the regulatory networks controlling CD133 gene expression and function in cancer remain incompletely delineated. In this study, we show that a ribonucleoprotein complex including the long noncoding RNA MALAT1 and the RNA-binding protein HuR (ELAVL1) binds the CD133 promoter region to regulate its expression. In luminal nonmetastatic MCF-7 breast cancer cells, HuR silencing was sufficient to upregulate N-cadherin (CDH2) and CD133 along with a migratory and mesenchymal-like phenotype. Furthermore, we found that in the basal-like metastatic cell line MDA-MB-231 and primary triple-negative breast cancer tumor cells, the repressor complex was absent from the CD133-regulatory region, but was present in the MCF-7 and primary ER+ tumor cells. The absence of the complex from basal-like cells was attributed to diminished expression of MALAT1, which, when overexpressed, dampened CD133 levels. In conclusion, our findings suggest that the failure of a repressive complex to form or stabilize in breast cancer promotes CD133 upregulation and an EMT-like program, providing new mechanistic insights underlying the control of prometastatic processes. Cancer Res; 76(9); 2626-36. ©2016 AACR.

Characteristics and Properties of Mesenchymal Stem Cells Derived From Microfragmented Adipose Tissue.

The subcutaneous adipose tissue provides a clear advantage over other mesenchymal stem cell sources due to the ease with which it can be accessed, as well as the ease of isolating the residing stem cells. Human adipose-derived stem cells (hADSCs), localized in the stromal–vascular portion, can be isolated ex vivo using a combination of washing steps and enzymatic digestion. In this study, we report that microfragmented human lipoaspirated adipose tissue is a better stem cell source compared to normal lipoaspirated tissue. The structural composition of microfragments is comparable to the original tissue. Differently, however, this procedure activates the expression of antigens, such as β-tubulin III. The hADSCs derived from microfragmented lipoaspirate tissue were systematically characterized for growth features, phenotype, and multipotent differentiation potential. They fulfill the definition of mesenchymal stem cells, although with a higher neural phenotype profile. These cells also express genes that constitute the core circuitry of self-renewal such as OCT4, SOX2, and NANOG, and neurogenic lineage genes such as NEUROD1, PAX6, and SOX3. Such findings suggest further studies by evaluating Microfrag-AT hADSC action in animal models of neurodegenerative conditions.

Dihydrotanshinone-I interferes with the RNA-binding activity of HuR affecting its post-transcriptional function

Post-transcriptional regulation is an essential determinant of gene expression programs in physiological and pathological conditions. HuR is a RNA-binding protein that orchestrates the stabilization and translation of mRNAs, critical in inflammation and tumor progression, including tumor necrosis factor-alpha (TNF). We identified the low molecular weight compound 15,16-dihydrotanshinone-I (DHTS), well known in traditional Chinese medicine practice, through a validated high throughput screening on a set of anti-inflammatory agents for its ability to prevent HuR:RNA complex formation. We found that DHTS interferes with the association step between HuR and the RNA with an equilibrium dissociation constant in the nanomolar range in vitro (Ki = 3.74 ± 1.63 nM). In breast cancer cell lines, short term exposure to DHTS influences mRNA stability and translational efficiency of TNF in a HuR-dependent manner and also other functional readouts of its post-transcriptional control, such as the stability of selected pre-mRNAs. Importantly, we show that migration and sensitivity of breast cancer cells to DHTS are modulated by HuR expression, indicating that HuR is among the preferential intracellular targets of DHTS. Here, we disclose a previously unrecognized molecular mechanism exerted by DHTS, opening new perspectives to therapeutically target the HuR mediated, post-transcriptional control in inflammation and cancer cells.

Loss of Protein Kinase Cδ/HuR Interaction Is Necessary to Doxorubicin Resistance in Breast Cancer Cell Lines

The protein kinase Cδ (PKCδ) interacts with and phosphorylates HuR, dictating its functionality. We show here that the genotoxic stimulus induced by doxorubicin triggers PKCδ interaction with HuR and leads to HuR phosphorylation on serines 221 and 318 and cytoplasmic translocation. This series of events is crucial to elicit the death pathway triggered by doxorubicin and is necessary to promote HuR function in post-transcriptional regulation of gene expression, because genetic ablation of PKCδ caused the inability of HuR to bind its target mRNAs, topoisomerase IIα (TOP2A) included. In in vitro select doxorubicin-resistant human breast cancer cell lines upregulating the multidrug resistance marker ABCG2, PKCδ, and HuR proteins were coordinately downregulated together with the doxorubicin target TOP2A protein whose mRNA was HuR-regulated. Therefore, we show here that PKCδ, HuR, and TOP2A constitute a network mediating doxorubicin efficacy in breast cancer cells. The importance of these molecular events in cancer therapy is suggested by their being profoundly suppressed in cells selected for doxorubicin resistance.

Recovery from experimental parkinsonism by intrastriatal application of erythropoietin or EPO-releasing neural precursors

ABSTRACT An extensive literature has shown a powerful neuroprotective action of Erythropoietin (EPO) both in vivo and in vitro. This study shows that EPO, whether ectopically administered or released by neural precursors, does reverse MPTP‐induced parkinsonism in mice. Unilateral stereotaxic injection of 2.5 × 105 erythropoietin‐releasing neural precursor cells (Er‐NPCs) rescued degenerating striatal dopaminergic neurons and promoted behavioral recovery as shown by three independent behavioral tests. These effects were replicated through direct intrastriatal administration of recombinant human EPO. At the end of the observational period, most of the transplanted Er‐NPCs were vital and migrated via the striatum to reach Substantia Nigra. The restorative effects appear to be mediated by EPO since co‐injection of anti‐EPO or anti‐EPOR antibodies antagonized the positive outcomes. Furthermore, this report supports the neuroprotective action of EPO, which may also be achieved via administration of EPO‐releasing cells such as Er‐NPCs. HIGHLIGHTSErythropoietin released by Er‐NPCs is responsible for their beneficial effects.Er‐NPCs transplantation improves behavioral performances in parkinsonian mice.Er‐NPCs transplantation promotes the recovery of TH in nigro‐striatal projections.

The GSK3β inhibitor BIS I reverts YAP-dependent EMT signature in PDAC cell lines by decreasing SMADs expression level

The Yes-associated protein, YAP, is a transcriptional co-activator, mediating the Epithelial to Mesenchymal Transition program in pancreatic ductal adenocarcinoma (PDAC). With the aim to identify compounds that can specifically modulate YAP functionality in PDAC cell lines, we performed a small scale, drug-based screening experiment using YAP cell localization as the read-out. We identified erlotinib as an inducer of YAP cytoplasmic localization, an inhibitor of the TEA luciferase reporter system and the expression of the bona fide YAP target gene, Connective Tissue Growth Factor CTGF. On the other hand, BIS I, an inhibitor of PKCδ and GSK3β, caused YAP accumulation into the nucleus. Activation of β-catenin reporter and interfering experiments show that inhibition of the PKCδ/GSK3β pathway triggers YAP nuclear accumulation inducing YAP/TEAD transcriptional response. Inhibition of GSK3β by BIS I reduced the expression levels of SMADs protein and reduced YAP contribution to EMT. Notably, BIS I reduced proliferation, migration and clonogenicity of PDAC cells in vitro, phenocopying YAP genetic down-regulation. As shown by chromatin immunoprecipitation experiments and YAP over-expressing rescue experiments, BIS I reverted YAP-dependent EMT program by modulating the expression of the YAP target genes E-cadherin, vimentin, CTGF and of the newly identified target, CD133. In conclusion, we identified two different molecules, erlotinib and BIS I, modulating YAP functionality although via different mechanisms of action, with the second one specifically inhibiting the YAP-dependent EMT program in PDAC cell lines.

Human Antigen R Binding and Regulation of SOX2 mRNA in Human Mesenchymal Stem Cells

Since 2005, sex determining region y–box 2 (SOX2) has drawn the attention of the scientific community for being one of the key transcription factors responsible for pluripotency induction in somatic stem cells. Our research investigated the turnover regulation of SOX2 mRNA in human adipose-derived stem cells, considered one of the most valuable sources of somatic stem cells in regenerative medicine. Mitoxantrone is a drug that acts on nucleic acids primarily used to treat certain types of cancer and was recently shown to ameliorate the outcome of autoimmune diseases such as multiple sclerosis. In addition, mitoxantrone has been shown to inhibit the binding of human antigen R (HuR) RNA-binding protein to tumor necrosis factor-α mRNA. Our results show that HuR binds to the 3′-untranslated region of SOX2 mRNA together with the RNA-induced silencing complex miR145. The HuR binding works by stabilizing the interaction between the 3′-untranslated region and the RNA-induced silencing complex. Cell exposure to mitoxantrone leads to HuR detachment and the subsequent prolongation of the SOX2 mRNA half-life. The prolonged SOX2 half-life allows improvement of the spheroid-forming capability of the adipose-derived stem cells. The silencing of HuR confirmed the above observations and illustrates how the RNA-binding protein HuR may be a required molecule for regulation of SOX2 mRNA decay.

Abstract 3384: An internal ribosomal entry site in the 5’-untranslated region of p16INK4a mRNA provides a novel mechanism for the regulation of its translation

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA In mammalian systems, controlled progression through the cell cycle is essential for normal proliferation and its loss is a hallmark of malignancy. p16INK4a inhibits the cyclin-dependent kinases CDK4 and CDK6, thereby keeping the retinoblastoma protein (pRB) in a hypo-phosphorylated state, which can lead to G1/S checkpoint activation. In addition p16INK4a plays a crucial role in the process of replicative senescence. p16INK4a loss or inactivation is associated with predisposition to melanoma, pancreatic cancer and other malignancies, highlighting its recognized function as a tumor suppressor. Given its critical role in cell homeostasis, there is much interest in understanding the molecular regulators of p16INK4a expression. Here we report that p16 belongs to the expanding group of proteins whose translation is influenced by sequence/structural features of the 5’UTR mRNA that are endowed of cellular Internal Ribosome Entry Site (IRES) activity. To study the potential for p16INK4a 5’UTR to drive cap-independent translation we developed a dual-luciferase assay using a bicistronic vector (named pRuF), where wild type or deletion mutants of the p16INK4a 5’UTRs were cloned as intervening sequence between Renilla and Firefly luciferase cDNAs. The p16INK4a 5’UTR sequence in inverted orientation was included as additional control. Results of reporters’ relative activity coupled to control analyses of actual bicistronic mRNA transcription, indicated that the wild type p16INK4a 5’UTR could stimulate Firefly luciferase translation. The IRES-like activity could not be mapped to a specific region of the 5’UTR based on results with deletion constructs, and was two-fold stronger compared to the cMYC 5’UTR, a known cellular IRES used as a control. Notably, hypoxic stress in particular, but also the treatment with mTOR inhibitors, enhanced the translation-stimulating property of the p16INK4a 5’UTR. RNA immuno-precipitation (RIP) assays performed in the p16-positive melanoma-derived cell line SK-Mel-28 suggest that the RNA-binding protein YBX-1, known to act in translation control, can participate in p16 INK4a translation. Experiments were YBX-1 was over-expressed or knocked-down by si-RNA confirmed its involvement in p16INK4a cap-independent translational regulation. Taken collectively, our results suggest that the 5’UTR region can modulate p16INK4a mRNA translation efficiency. Citation Format: Alessandra Bisio, Elisa Latorre, Virginia Andreotti, Alessandro Provenzani, Giovanna Bianchi- Scarra, Paola Ghiorzo, Alberto Inga. An internal ribosomal entry site in the 5’-untranslated region of p16INK4a mRNA provides a novel mechanism for the regulation of its translation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3384. doi:10.1158/1538-7445.AM2014-3384