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Supplementary Methods - PDF file 95K, Additional information regarding all reagents, antibodies, vectors and primers employed in this study, including experimental procedures

Analysis of miR-214 and miR-148b sponge efficacy

miR-214 and miR-148b have been proposed to antagonize the effects of each other in enabling or blocking metastasis, respectively. In this study, we provide evidence deepening their role and interrelationship in the process of metastatic dissemination. Depleting miR-214 or elevating miR-148b blocked the dissemination of melanoma or breast cancer cells, an effect that could be accentuated by dual alteration. Mechanistic investigations indicated that dual alteration suppressed passage of malignant cells through the blood vessel endothelium by reducing expression of the cell adhesion molecules ITGA5 and ALCAM. Notably, transendothelial migration in vitro and extravasation in vivo impaired by singly alternating miR-214 or miR-148b could be overridden by overexpression of ITGA5 or ALCAM in the same tumor cells. In clinical specimens of primary breast cancer or metastatic melanoma, we found a positive correlation between miR-214 and ITGA5 or ALCAM along with an inverse correlation of miR-214 and miR-148b in the same specimens. Our findings define an antagonistic relationship of miR-214 and miR-148b in determining the dissemination of cancer cells via tumor–endothelial cell interactions, with possible implications for microRNA-mediated therapeutic interventions aimed at blocking cancer extravasation. Cancer Res; 76(17); 5151–62. ©2016 AACR.

miR-214 and miR-148b sponge sequences

Malignant melanoma is one of the most aggressive human cancers, but the mechanisms governing its metastatic dissemination are not fully understood. Upregulation of miR-214 and ALCAM and the loss of TFAP2 expression have been implicated in this process, with TFAP2 a direct target of miR-214. Here, we link miR-214 and ALCAM as well as identify a core role for miR-214 in organizing melanoma metastasis. miR-214 upregulated ALCAM, acting transcriptionally through TFAP2 and also posttranscriptionally through miR-148b (itself controlled by TFAP2), both negative regulators of ALCAM. We also identified several miR-214–mediated prometastatic functions directly promoted by ALCAM. Silencing ALCAM in miR-214–overexpressing melanoma cells reduced cell migration and invasion without affecting growth or anoikis in vitro, and it also impaired extravasation and metastasis formation in vivo. Conversely, cell migration and extravasation was reduced in miR-214–overexpressing cells by upregulation of either miR-148b or TFAP2. These findings were consistent with patterns of expression of miR-214, ALCAM, and miR-148b in human melanoma specimens. Overall, our results define a pathway involving miR-214, miR-148b, TFAP2, and ALCAM that is critical for establishing distant metastases in melanoma. Cancer Res; 73(13); 4098–111. ©2013 AACR.

Supplementary Figure and Table Legends - PDF file 74K, Legend for Supplemental Table S1 and Supplemental Figures S1-S4

Supplementary tables and figure legends

Supplementary Table S1 - PDF file 98K, miR-214 and ALCAM promote melanoma metastasis independently of BRAFV600E mutation status

Supplementary Figures S1-S4 - PDF file 482K, miR-214, miR-148b and ALCAM expression modulations (S1); ALCAM is upregulated by miR-214 and controls cell movement but not proliferation (S2); ALCAM and miR-148b regulations (S3); ALCAM, miR-214, miR-148b and TFAP2 expression in human melanoma samples (S4)

An interactive crosstalk between tumor and stroma cells is essential for metastatic melanoma progression. We evidenced that ESDN/DCBLD2/CLCP1 plays a crucial role in endothelial cells during the spread of melanoma. Precisely, increased extravasation and metastasis formation were revealed in ESDN-null mice injected with melanoma cells, even if the primary tumor growth, vessel permeability, and angiogenesis were not enhanced. Interestingly, improved adhesion of melanoma cells to ESDN-depleted endothelial cells was observed, due to the presence of higher levels of E-selectin transcripts/proteins in ESDN-defective cells. In accordance with these results, anticorrelation was observed between ESDN and E-selectin in human endothelial cells. Most importantly, our data revealed that cimetidine, an E-selectin inhibitor, was able to block cell adhesion, extravasation, and metastasis formation in ESDN-null mice, underlying a major role of ESDN in E-selectin transcription upregulation, which according to our data, may presumably be linked to STAT3. Based on our results, we propose a protective role for ESDN during the spread of melanoma and reveal its therapeutic potential. ispartof: CANCER LETTERS vol:510 pages:13-23 ispartof: location:Ireland status: published

MicroRNAs (miRNAs) are small, non-coding RNAs about 22 nucleotides in length that regulate the expression of target genes post-transcriptionally, and are highly involved in cancer progression. They are able to impact a variety of cell processes such as proliferation, apoptosis and differentiation and can consequently control tumor initiation, tumor progression and metastasis formation. miRNAs can regulate, at the same time, metabolic gene expression which, in turn, influences relevant traits of malignancy such as cell adhesion, migration and invasion. Since the interaction between metabolism and adhesion or cell movement has not, to date, been well understood, in this review, we will specifically focus on miRNA alterations that can interfere with some metabolic processes leading to the modulation of cancer cell movement. In addition, we will analyze the signaling pathways connecting metabolism and adhesion/migration, alterations that often affect cancer cell dissemination and metastasis formation.

microRNAs (miRNAs) are small non-coding RNAs acting as negative regulators of gene expression and involved in tumor progression. We recently showed that inhibition of the pro-metastatic miR-214 and simultaneous overexpression of its downstream player, the anti-metastatic miR-148b, strongly reduced metastasis formation. To explore the therapeutic potential of miR-148b, we generated a conjugated molecule aimed to target miR-148b expression selectively to tumor cells. Precisely, we linked miR-148b to GL21.T, an aptamer able to specifically bind to AXL, an oncogenic tyrosine kinase receptor highly expressed on cancer cells. Axl-148b conjugate was able to inhibit migration and invasion of AXL-positive, but not AXL-negative, cancer cells, demonstrating high efficacy and selectivity in vitro. In parallel, expression of ALCAM and ITGA5, two miR-148b direct targets, was reduced. More importantly, axl-148b chimeric aptamers were able to inhibit formation and growth of 3D-mammospheres, to induce necrosis and apoptosis of treated xenotransplants, as well as to block breast cancer and melanoma dissemination and metastatization in mice. Relevantly, axl aptamer acted as specific delivery tool for miR-148b, but it also actively contributed to inhibit metastasis formation, together with miR-148b. In conclusion, our data show that axl-148b conjugate is able to inhibit tumor progression in an axl- and miR-148b-dependent manner, suggesting its potential development as therapeutic molecule.

Attenuated Listeria monocytogenes (Lmat-LLO) represents a valuable anticancer vaccine and drug delivery platform. Here we show that in vitro Lmat-LLO causes ROS production and, in turn, apoptotic killing of a wide variety of melanoma cells, irrespectively of their stage, mutational status, sensitivity to BRAF inhibitors or degree of stemness. We also show that, when administered in the therapeutic setting to Braf/Pten genetically engineered mice, Lmat-LLO causes a strong decrease in the size and volume of primary melanoma tumors, as well as a reduction of the metastatic burden. At the molecular level, we confirm that the anti-melanoma activity exerted in vivo by Lmat-LLO depends also on its ability to potentiate the immune response of the organism against the infected tumor. Our data pave the way to the preclinical testing of listeria-based immunotherapeutic strategies against metastatic melanoma, using a genetically engineered mouse rather than xenograft models.

Unbalanced immune responses to pathogens can be life-threatening although the underlying regulatory mechanisms remain unknown. Here, we show a hypoxia-inducible factor 1α-dependent microRNA (miR)-210 up-regulation in monocytes and macrophages upon pathogen interaction. MiR-210 knockout in the hematopoietic lineage or in monocytes/macrophages mitigated the symptoms of endotoxemia, bacteremia, sepsis, and parasitosis, limiting the cytokine storm, organ damage/dysfunction, pathogen spreading, and lethality. Similarly, pharmacologic miR-210 inhibition improved the survival of septic mice. Mechanistically, miR-210 induction in activated macrophages supported a switch toward a proinflammatory state by lessening mitochondria respiration in favor of glycolysis, partly achieved by downmodulating the iron-sulfur cluster assembly enzyme ISCU. In humans, augmented miR-210 levels in circulating monocytes correlated with the incidence of sepsis, while serum levels of monocyte/macrophage-derived miR-210 were associated with sepsis mortality. Together, our data identify miR-210 as a fine-tuning regulator of macrophage metabolism and inflammatory responses, suggesting miR-210-based therapeutic and diagnostic strategies. ispartof: SCIENCE ADVANCES vol:7 issue:19 ispartof: location:United States status: published

Simple Summary Ongoing studies demonstrate the importance of metabolism in cancer development and progression. Metabolic alterations occur, not only in cancer cells, but also in the tumor microenvironment (TME). The continuous crosstalk between tumor cells and stromal and immune cells deeply affects metabolic rewiring. miRs, small noncoding RNAs involved in post-transcriptional regulation, are important mediators in these processes. In-depth knowledge of these interactions is crucial in understanding cancer progression and, consequently, for the development of new therapies. Abstract The metabolism of cancer cells is generally very different from what is found in normal counterparts. However, in a tumor mass, the continuous crosstalk and competition for nutrients and oxygen among different cells lead to metabolic alterations, not only in cancer cells, but also in the different stromal and immune cells of the tumor microenvironment (TME), which are highly relevant for tumor progression. MicroRNAs (miRs) are small non-coding RNAs that silence their mRNA targets post-transcriptionally and are involved in numerous physiological cell functions as well as in the adaptation to stress situations. Importantly, miRs can also be released via extracellular vesicles (EVs) and, consequently, take part in the bidirectional communication between tumor and surrounding cells under stress conditions. Certain miRs are abundantly expressed in stromal and immune cells where they can regulate various metabolic pathways by directly suppressing enzymes or transporters as well as by controlling important regulators (such as transcription factors) of metabolic processes. In this review, we discuss how miRs can induce metabolic reprogramming in stromal (fibroblasts and adipocytes) and immune (macrophages and T cells) cells and, in turn, how the biology of the different cells present in the TME is able to change. Finally, we debate the rebound of miR-dependent metabolic alterations on tumor progression and their implications for cancer management.

Made available in DSpace on 2020-12-10T19:59:34Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-02-03 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Melatonin, a hormone secreted by pineal gland, exerts antimetastatic effects by reducing tumor cell proliferation, migration and invasion. MicroRNAs (miRNAs) are small, non-coding RNAs that play a crucial role in regulation of gene expression and biological processes of the cells. Herein, we search for a link between the tumor/metastatic-suppressive actions of melatonin and miRNA expression in triple-negative breast cancer cells. We demonstrated that melatonin exerts its anti-tumor actions by reducing proliferation, migration and c-Myc expression of triple negative breast cancer cells. By using Taqman-based assays, we analyzed the expression levels of a set of miRNAs following melatonin treatment of triple negative breast cancer cells and we identified 17 differentially expressed miRNAs, 6 down-regulated and 11 up-regulated. We focused on the anti-metastatic miR-148b and the oncogenic miR-210 both up-regulated by melatonin treatment and studied the effect of their modulation on melatonin-mediated impairment of tumor progression. Surprisingly, when miR-148b or miR-210 were depleted in triple-negative breast cancer cells, using a specific miR-148b sponge or anti-miR-210, melatonin effects on migration inhibition and c-myc downregulation were still visible suggesting that the increase of miR-148b and miR-210 expression observed following melatonin treatment was not required for the efficacy of melatonin action. Nevertheless, ours results suggest that melatonin exhibit a compound for metastatic trait inhibition, especially in MDA-MB-231 breast cancer cells even if a direct link between modulation of expression of certain proteins or miRNAs and melatonin effects has still to be established. Univ Estadual Paulista, Dept Biol, Sao Jose Do Rio Preto, SP, Brazil Univ Torino, Mol Biotechnol Ctr MBC, Dept Mol Biotechnol & Hlth Sci, Turin, Italy Augusta Univ, Dept Biochem & Mol Biol, Tumor Angiogenesis Lab, Augusta, GA USA Fac Med Sao Jose do Rio Preto, Dept Mol Biol, Sao Jose Do Rio Preto, SP, Brazil Univ Estadual Paulista, Dept Biol, Sao Jose Do Rio Preto, SP, Brazil FAPESP: 2015/04780-6

In the last decades, progresses in medical oncology have ameliorated the treatment of patients and their outcome. However, further improvements are still necessary, in particular for certain types of tumors such as pancreatic, gastric, and lung cancer as well as acute myeloid leukemia where early detection and monitoring of the disease are crucial for final patient outcome. Liquid biopsy represents a great advance in the field because it is less invasive, less time-consuming, and safer compared to classical biopsies and it can be useful to monitor the evolution of the disease as well as the response of patients to therapy. Liquid biopsy allows the detection of circulating tumor cells, nucleic acids, and exosomes not only in blood but also in different biological fluids: urine, saliva, pleural effusions, cerebrospinal fluid, and stool. Among the potential biomarkers detectable in liquid biopsies, microRNAs (miRNAs) are gaining more and more attention, since they are easily detectable, quite stable in biological fluids, and show high sensitivity. Many data demonstrate that miRNAs alone or in combination with other biomarkers could improve the diagnostic and prognostic power for many different tumors. Despite this, standardization of methods, sample preparation, and analysis remain challenging and a huge effort should be made to address these issues before miRNA biomarkers can enter the clinic. This review summarizes the main findings in the field of circulating miRNAs in both solid and hematological tumors.

We previously demonstrated that miR-214 is upregulated in malignant melanomas and triple-negative breast tumors and promotes metastatic dissemination by affecting a complex pathway including the anti-metastatic miR-148b. Importantly, tumor dissemination could be reduced by blocking miR-214 function or increasing miR-148b expression or by simultaneous interventions. Based on this evidence, with the intent to explore the role of miR-214 as a target for therapy, we evaluated the capability of new chemically modified anti-miR-214, R97/R98, to inhibit miR-214 coordinated metastatic traits. Relevantly, when melanoma or breast cancer cells were transfected with R97/R98, anti-miR-214 reduced miR-214 expression and impaired transendothelial migration were observed. Noteworthy, when the same cells were injected in the tail vein of mice, cell extravasation and metastatic nodule formation in lungs were strongly reduced. Thus, suggesting that R97/R98 anti-miR-214 oligonucleotides were able to inhibit tumor cell escaping through the endothelium. More importantly, when R97/R98 anti-miR-214 compounds were systemically delivered to mice carrying melanomas or breast or neuroendocrine pancreatic cancers, a reduced number of circulating tumor cells and lung or lymph node metastasis formation were detected. Similar results were also obtained when AAV8-miR-214 sponges were used in neuroendocrine pancreatic tumors. Based on this evidence, we propose miR-214 as a promising target for anti-metastatic therapies.

ErbB2 overexpression is detected in approximately 20% of breast cancers and is correlated with poor survival. It was previously shown that the adaptor protein p130Cas/BCAR1 is a crucial mediator of ErbB2 transformation and that its overexpression confers invasive properties to ErbB2-positive human mammary epithelial cells. We herein prove, for the first time, that the transcriptional repressor Blimp1 is a novel mediator of p130Cas/ErbB2-mediated invasiveness. Indeed, high Blimp1 expression levels are detected in invasive p130Cas/ErbB2 cells and correlate with metastatic status in human breast cancer patients. The present study, by using 2D and 3D breast cancer models, shows that the increased Blimp1 expression depends on both MAPK activation and miR-23b downmodulation. Moreover, we demonstrate that Blimp1 triggers cell invasion and metastasis formation via its effects on focal adhesion and survival signaling. These findings unravel the previously unidentified role that transcriptional repressor Blimp1 plays in the control of breast cancer invasiveness.

The development of resistance remains a major obstacle to long‐term disease control in cancer patients treated with targeted therapies. In BRAF‐mutant mouse models, we demonstrate that although targeted inhibition of either BRAF or VEGF initially suppresses the growth of BRAF‐mutant tumors, combined inhibition of both pathways results in apoptosis, long‐lasting tumor responses, reduction in lung colonization, and delayed onset of acquired resistance to the BRAF inhibitor PLX4720. As well as inducing tumor vascular normalization and ameliorating hypoxia, this approach induces remodeling of the extracellular matrix, infiltration of macrophages with an M1‐like phenotype, and reduction in cancer‐associated fibroblasts. At the molecular level, this therapeutic regimen results in a de novo transcriptional signature, which sustains and explains the observed efficacy with regard to cancer progression. Collectively, our findings offer new biological rationales for the management of clinical resistance to BRAF inhibitors based on the combination between BRAFV 600E inhibitors with anti‐angiogenic regimens.

Aim: Growing evidence shows a strong interplay between post-transcriptional regulation, mediated by miRNAs (miRs) and epigenetic regulation. Nevertheless, the number of experimentally validated miRs (called epi-miRs) involved in these regulatory circuitries is still very small. Material & methods: We propose a pipeline to prioritize candidate epi-miRs and to identify potential epigenetic interactors of any given miR starting from miR transfection experiment datasets. Results & conclusion: We identified 34 candidate epi-miRs: 19 of them are known epi-miRs, while 15 are new. Moreover, using an in-house generated gene expression dataset, we experimentally proved that a component of the polycomb-repressive complex 2, the histone methyltransferase enhancer of zeste homolog 2 (EZH2), interacts with miR-214, a well-known prometastatic miR in melanoma and breast cancer, highlighting a miR-214-EZH2 regulatory axis potentially relevant in tumor progression.

Cancer is a multistep disease based on crucial interactions between tumor cells and the microenvironment (extracellular matrix and stroma/immune cells). In fact, during dissemination, tumor cells have to escape from the primary tumor mass, cross the basal membrane, interact with endothelial cells to enter blood vessels (intravasation), survive in the bloodstream, get in contact with endothelial cells again to exit the bloodstream (extravasation) and seed in distant organs. Interactions between tumor and stroma cells are strongly coordinated by microRNAs (miRNAs), small non-coding RNAs able to silence protein coding genes by binding to specific recognition sites, mostly located at the 3' UTR of mature mRNAs. Relevantly, miRNA expression is often altered (overexpression or downregulation) in tumor cells and influenced by stroma cells. At the same time, miRNAs are abundant and essential in stroma cells during tumor cell dissemination and their expression is influenced by tumor cells. In fact, for instance, conditional ablation of Dicer in the endothelium of tumor bearing-mice leads to reduced tumor growth and microvessel density. In this review, we specifically focus on the role of miRNAs in endothelial cells regarding their positive or negative intervention on tumor angiogenesis or lymphoangiogenesis or when tumor cells detach from the tumor mass and intravasate or extravasate in/out of the blood vessels. Examples of pro-angiogenic miRNAs are miR-9 or miR-494, often overexpressed in tumors, which accumulate in tumor cell microvescicles and, therefore, get transferred to endothelial cells where they induce migration and angiogenesis. Differently, miR-200 and miR-128 are often downregulated in tumors and inhibit angiogenesis and lymphoangiogenesis. Instead, miR-126 controls intravasation while miR-146a, miR-214, miR-148b govern extravasation, in a positive or negative manner. Finally, at the end, we summarize opportunities for therapeutic interventions based on miRNAs acting on endothelial cells.

About 90% of breast cancer mortalities are due to the spread of breast cancer cells (BCCs) from a primary tumor to distant organs, a process known as metastasis. However, the molecular mechanisms underlying metastasis remain poorly understood. Substantial evidence now supports a major role for the tumor microenvironment (TME) in catalyzing breast cancer metastasis. Indeed, observations indicate that proximal interactions between BCCs and cells of the TME induce altered gene expression programs in BCCs, allowing for the navigation of the various steps of the metastatic cascade. Our group and others observed that breast tumors recruit mesenchymal stem cells (MSCs): multipotent fibroblasts that normally exert tissue maintenance functions. We and others have observed that physical interactions of MSCs with BCCs are sufficient to drive their metastatic dissemination in murine xenograft models, via the induction of epithelial-mesenchymal transition (EMT) and dedifferentiation into stem cell-like states (cancer stem cells, or CSCs), states tightly associated with the capacity to seed new tumors (for example in foreign tissues) and with chemotherapeutic resistance. However, the TME-induced molecular pathways regulating such mechanisms remain poorly understood. MicroRNAs (miRNAs, miRs) are small noncoding RNAs that regulate gene expression via base-pair interactions with messenger RNAs (mRNAs), resulting in mRNA degradation or translational inhibition. Due to their ability to interact with large numbers of target mRNAs simultaneously, miRNAs are major regulators of cell identity, and thereby serve critical roles in metastasis. We performed miRnome-wide screening of MSC-stimulated BCCs to determine if TME interactions might contribute to BCC metastasis via the deregulation of miRNAs. We observed that proximal MSCs induce aberrant expression of a specific set of miRNAs in BCCs, which had not been previously implicated in breast cancer pathogenesis. These miRNAs, led by the transcriptionally co-regulated miR-199a-3p and miR-214, were sufficient to actuate the metastasis of weakly metastatic human BCCs in xenograft models. We observed that exogenous expression of the miRNAs provided BCCs with phenotypes and gene markers characteristic of CSCs, including enhanced tumor initiation capacities. Interestingly, we found that the MSC-induced miRNAs function as an interrelated network, and converge upon a common novel target: the speech associated gene FOXP2. Knockdown of FOXP2 phenocopied the metastatic phenotypes observed in MSC-induced miRNA expressing BCCs. Importantly, elevated levels of the MSC-induced miRNAs or depressed levels of FOXP2 could predict patient prognosis in the clinic. Altogether, our results incriminate FOXP2 and it’s MSC-induced miRNA regulatory network as novel determinants of breast cancer metastasis. Citation Format: Benjamin G Cuiffo, Antoine Campagne, George W Bell, Antonio Lembo, Francesca Orso, Evan Lien, Manoj K Bhasin, Monica Raimo, Summer E Hanson, Andriy Marusyk, Peiman Hematti, Kornelia Polyak, Odette Mariani, Stefano Volinia, Anne Vincent-Salomon, Daniela Taverna, Antoine E Karnoub. Mesenchymal stem cell regulated microRNAs converge on the speech gene FOXP2 and regulate breast cancer metastasis [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P1-07-02.

// Sharmila Fagoonee 1, 2 , Gabriele Picco 3, 9 , Francesca Orso 2 , Arrigo Arrigoni 4 , Dario L. Longo 1, 2 , Marco Forni 5 , Irene Scarfo 6 , Adele Cassenti 7 , Roberto Piva 6 , Paola Cassoni 7 , Lorenzo Silengo 1, 2 , Emanuela Tolosano 2 , Silvio Aime 2 , Daniela Taverna 2 , Pier Paolo Pandolfi 2, 8 , Mara Brancaccio 2 , Enzo Medico 3 , Fiorella Altruda 1, 2 1 Institute of Biostructure and Bioimaging, CNR, Department of Molecular Biotechnology and Health Sciences, University of Turin, Italy 2 Molecular Biotechnology Center, Department of Molecular Biotechnology and Health Sciences, University of Turin, Italy 3 Candiolo Cancer Institute-IRCCS, University of Turin, Italy 4 S.C. Gastroenterologia U, Endoscopia San Giovanni A.S., Azienda Citta' della Salute e della Scienza di Torino, Turin, Italy 5 EuroClone S.p.A Research Laboratory, Molecular Biotechnology Centre, University of Turin, Italy 6 Center for Experimental Research and Medical Studies, University of Turin, Italy 7 Department of Medical Sciences, University of Turin, Italy 8 Cancer Research Institute, BIDMC, Harvard Medical School, Boston, USA 9 present address: Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridgeshire CB10 1SA, UK Correspondence to: Sharmila Fagoonee, email: sharmila.fagoonee@unito.it Fiorella Altruda, email: fiorella.altruda@unito.it Keywords: ESRP1, RNA binding protein, proto-oncogene, human colorectal cancer Received: June 08, 2016 Accepted: December 01, 2016 Published: December 28, 2016 ABSTRACT Epithelial splicing regulatory protein 1 (ESRP1) is an epithelial cell-specific RNA binding protein that controls several key cellular processes, like alternative splicing and translation. Previous studies have demonstrated a tumor suppressor role for this protein. Recently, however, a pro-metastatic function of ESRP1 has been reported. We thus aimed at clarifying the role of ESRP1 in Colorectal Cancer (CRC) by performing loss- and gain-of-function studies, and evaluating tumorigenesis and malignancy with in vitro and in vivo approaches. We found that ESRP1 plays a role in anchorage-independent growth of CRC cells. ESRP1-overexpressing cells grown in suspension showed enhanced fibroblast growth factor receptor (FGFR1/2) signalling, Akt activation, and Snail upregulation. Moreover, ESRP1 promoted the ability of CRC cells to generate macrometastases in mice livers. High ESRP1 expression may thus stimulate growth of cancer epithelial cells and promote colorectal cancer progression. Our findings provide mechanistic insights into a previously unreported, pro-oncogenic role for ESRP1 in CRC, and suggest that fine-tuning the level of this RNA-binding protein could be relevant in modulating tumor growth in a subset of CRC patients.

// Valentina Audrito 1, 2, * , Sara Serra 1, 2, * , Aureliano Stingi 1, 2 , Francesca Orso 3 , Federica Gaudino 1, 2 , Cinzia Bologna 1, 2 , Francesco Neri 1 , Giulia Garaffo 3 , Romina Nassini 4 , Gianna Baroni 5 , Eliana Rulli 6 , Daniela Massi 5 , Salvatore Oliviero 1, 7 , Roberto Piva 3 , Daniela Taverna 3 , Mario Mandala 8, ** , Silvia Deaglio 1, 2, ** 1 Human Genetics Foundation (HuGeF), Turin, Italy 2 Department of Medical Sciences, University of Turin, Turin, Italy 3 Department of Molecular Biotechnology and Health Sciences, University of Turin, Turin, Italy 4 Department of Health Sciences, University of Florence, Italy 5 Department of Surgery and Translational Medicine, University of Florence, Italy 6 Methodology for Clinical Research Laboratory, IRCCS – Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy 7 Department of Life Sciences and Systems Biology, University of Turin, Turin, Italy 8 Department of Oncology and Hematology, Papa Giovanni XXIII Hospital, Bergamo, Italy * These authors have contributed equally to this work ** These authors have shared last authorship Correspondence to: Silvia Deaglio, email: silvia.deaglio@unito.it , silvia.deaglio@hugef-torino.org Keywords: melanoma, targeted therapy, resistance to therapy, microRNA, regulation of gene expression Received: August 11, 2016 Accepted: January 06, 2017 Published: February 09, 2017 ABSTRACT PD-L1 is expressed by a subset of patients with metastatic melanoma (MM) with an unfavorable outcome. Its expression is increased in cells resistant to BRAF or MEK inhibitors (BRAFi or MEKi). However, the function and regulation of expression of PD-L1 remain incompletely understood. After generating BRAFi- and MEKi-resistant cell lines, we observed marked up-regulation of PD-L1 expression. These cells were characterized by a common gene expression profile with up-regulation of genes involved in cell movement. Consistently, in vitro they showed significantly increased invasive properties. This phenotype was controlled in part by PD-L1, as determined after silencing the molecule. Up-regulation of PD-L1 was due to post-transcriptional events controlled by miR-17-5p, which showed an inverse correlation with PD-L1 mRNA. Direct binding between miR-17-5p and the 3’-UTR of PD-L1 mRNA was demonstrated using luciferase reporter assays. In a cohort of 80 BRAF-mutated MM patients treated with BRAFi or MEKi, constitutive expression of PD-L1 in the absence of immune infiltrate, defined the patient subset with the worst prognosis. Furthermore, PD-L1 expression increased in tissue biopsies after the metastatic lesions became resistant to BRAFi or MEKi. Lastly, plasmatic miR-17-5p levels were higher in patients with PD-L1 + than PD-L1 - lesions. In conclusion, our findings indicate that PD-L1 expression induces a more aggressive behavior in melanoma cells. We also show that PD-L1 up-regulation in BRAFi or MEKi-resistant cells is partly due to post-transcriptional mechanisms that involve miR-17-5p, suggesting that miR-17-5p may be used as a marker of PD-L1 expression by metastatic lesions and ultimately a predictor of responses to BRAFi or MEKi.

Breast cancer is often fatal during its metastatic dissemination. To unravel the role of microRNAs (miRs) during malignancy, we analyzed miR expression in 77 primary breast carcinomas and identified 16 relapse-associated miRs that correlate with survival and/or distinguish tumor subtypes in different datasets. Among them, miR-148b, down-regulated in aggressive breast tumors, was found to be a major coordinator of malignancy. In fact, it is able to oppose various steps of tumor progression when overexpressed in cell lines by influencing invasion, survival to anoikis, extravasation, lung metastasis formation, and chemotherapy response. miR-148b controls malignancy by coordinating a novel pathway involving over 130 genes and, in particular, it directly targets players of the integrin signaling, such as ITGA5, ROCK1, PIK3CA/p110α, and NRAS, as well as CSF1, a growth factor for stroma cells. Our findings reveal the importance of the identified 16 miRs for disease outcome predictions and suggest a critical role for miR-148b in the control of breast cancer progression.

3-Phosphoinositide-dependent protein kinase 1 (PDK1) is the pivotal element of the phosphatidylinositol 3 kinase (PI3K) signaling pathway because it phosphorylates Akt/PKB through interactions with phosphatidylinositol 3,4,5 phosphate. Recent data indicate that PDK1 is overexpressed in many breast carcinomas and that alterations of PDK1 are critical in the context of oncogenic PI3K activation. However, the role of PDK1 in tumor progression is still controversial. Here, we show that PDK1 is required for anchorage-independent and xenograft growth of breast cancer cells harboring either PI3KCA or KRAS mutations. In fact, PDK1 silencing leads to increased anoikis, reduced soft agar growth, and pronounced apoptosis inside tumors. Interestingly, these phenotypes are reverted by PDK1 wild-type but not kinase-dead mutant, suggesting a relevant role of PDK1 kinase activity, even if PDK1 is not relevant for Akt activation here. Indeed, the expression of constitutively active forms of Akt in PDK1 knockdown cells is unable to rescue the anchorage-independent growth. In addition, Akt down-regulation and pharmacological inhibition do not inhibit the effects of PDK1 overexpression. In summary, these results suggest that PDK1 may contribute to breast cancer, even in the absence of PI3K oncogenic mutations and through both Akt-dependent and Akt-independent mechanisms.

Malignant melanoma is fatal in its metastatic stage. It is therefore essential to unravel the molecular mechanisms that govern disease progression to metastasis. MicroRNAs (miRs) are endogenous non-coding RNAs involved in tumourigenesis. Using a melanoma progression model, we identified a novel pathway controlled by miR-214 that coordinates metastatic capability. Pathway components include TFAP2C, homologue of a well-established melanoma tumour suppressor, the adhesion receptor ITGA3 and multiple surface molecules. Modulation of miR-214 influences in vitro tumour cell movement and survival to anoikis as well as extravasation from blood vessels and lung metastasis formation in vivo. Considering that miR-214 is known to be highly expressed in human melanomas, our data suggest a critical role for this miRNA in disease progression and the establishment of distant metastases.

Introduction Morgana is a chaperone protein encoded by the CHORDC1 gene. Its deletion is embryonic lethal due to apoptosis of the cells of the inner cell mass. We recently characterised the role of Morgana in myeloid malignancies, as the haploinsufficiency of the protein in mice is able to induce a fatal and transplantable myeloproliferative disease resembling human Atypical Chronic Myeloid Leukaemia (aCML). 5 out of 5 aCML patients and 16% of Philadelphia-positive CML patients express low/undetectable levels of Morgana in their bone marrow. As we never found mutations or deletions of CHORDC1 gene, we decided to investigate if Morgana can be targeted by miRNAs. Five miRNAs are predicted to target Morgana: miR-15a/b and miR-16 sharing the same seed sequence and miR-26a/b. Material and methods HEK-293T cells were used to overexpress miRNAs predicted to target Morgana mRNA. The level of miRNAs overexpression and Morgana mRNA was assessed with qRT-PCR and Morgana protein level with Western Blot at different time points. The seed sequences for the selected miRNAs in the 3’UTR of CHORDC1 gene were than mutagenized to validate the specificity of the binding. Bioinformatic analysis were used to correlate miRNAs and Morgana expression levels in leukaemia and lymphoma. Results and discussions We demonstrated that miRNA-15a/b and miRNA-26a/b are able to bind to Morgana 3’-UTR and, in this way, mediate its mRNA deregulation leading to a reduction of Morgana, both at mRNA and protein level. We were able to highlight an anti-correlation between Morgana and miRNAs expression in haematological tumours: in particular miR-15b in Chronic Lymphocytic Leukaemia and Lymphomas, miR-15a in aCML and CML and miR-26a and miR-16 in Lymphomas. Conclusion Morgana is able to act both as proto-oncogene and as oncosuppressor depending on tissue type and levels of expression as it is frequently found both overexpressed and downregulated. Different approaches to elucidate its mechanisms of regulation failed and we believe that miRNAs are just one of them. Further investigations are needed to clirify how Morgana expression is regulated in different type of tumours.

Objective— Circulating angiogenic cells (CACs) expansion is a multistage process requiring sequential activation of transcriptional factors, including STAT5. STAT5, in concert with peroxisome proliferator-activated receptors (PPARs), seems to induce discrete biological responses in different tissues. In the present study we investigated the role of STAT5 and PPARγ in regulating CAC expansion in normal and diabetic settings. Methods and Results— Normal and diabetic CACs were used. siRNA technology, EMSA, and chromatin immunoprecipitation (ChIP) assay as well as site-directed mutagenesis of the STAT5 response element in the PPARγ promoter enabled us to demonstrate that STAT5 transcriptional activity controls PPARγ expression. Moreover, FACS analysis, coimmunoprecipitation experiments, and ChIP assay revealed that a STAT5/PPARγ transcriptional complex controls cyclin D1 expression and CAC progression into the cell-cycle. Conversely, PPARγ agonists, by preventing the expression of STAT5 and the formation of the STAT5/PPARγ heterodimeric complex failed to promote CAC expansion. Finally, we demonstrated that diabetic CAC functional capability can be recovered by molecules able to activate the STAT5/PPARγ transcriptional complex. Conclusions— Our data identify the STAT5/PPARγ heterodimers as landmark of CAC expansion and provide evidences for a mechanism that partially rescues CAC bioavailability in diabetic setting.

miR-214 and miR-148b have been proposed to antagonize the effects of each other in enabling or blocking metastasis, respectively. In this study, we provide evidence deepening their role and interrelationship in the process of metastatic dissemination. Depleting miR-214 or elevating miR-148b blocked the dissemination of melanoma or breast cancer cells, an effect that could be accentuated by dual alteration. Mechanistic investigations indicated that dual alteration suppressed passage of malignant cells through the blood vessel endothelium by reducing expression of the cell adhesion molecules ITGA5 and ALCAM. Notably, transendothelial migration in vitro and extravasation in vivo impaired by singly alternating miR-214 or miR-148b could be overridden by overexpression of ITGA5 or ALCAM in the same tumor cells. In clinical specimens of primary breast cancer or metastatic melanoma, we found a positive correlation between miR-214 and ITGA5 or ALCAM along with an inverse correlation of miR-214 and miR-148b in the same specimens. Our findings define an antagonistic relationship of miR-214 and miR-148b in determining the dissemination of cancer cells via tumor–endothelial cell interactions, with possible implications for microRNA-mediated therapeutic interventions aimed at blocking cancer extravasation. Cancer Res; 76(17); 5151–62. ©2016 AACR.

Molecular biology experiments allow to obtain reliable data about the expression of different classes of molecules involved in several cellular processes. This information is mostly static and does not give much clue about the causal relationships (i.e., regulation) among the different molecules. A typical scenario is the presence of a set of modulated mRNAs (up or down regulated) along with an over expression of one or more small non-coding RNAs molecules like miRNAs. To computationally identify the presence of transcriptional or post-transcriptional regulatory modules between one or more miRNAs and a set of target modulated genes, we propose a computational pipeline designed to integrate data from multiple online data repositories. The pipeline produces a set of three types of putative regulatory motifs involving coding genes, intronic miRNAs, and transcription factors. We used this pipeline to analyze the results of a set of expression experiments on a melanoma cell line that showed an over expression of miR-214 along with the modulation of a set of 73 other genes. The results suggest the presence of 27 putative regulatory modules involving miR-214, NFKB1, SREBPF2, miR-33a and 9 out of the 73 miR-214 modulated genes (ALCAM, POSTN, TFAP2A, ADAM9, NCAM1, SEMA3A, PVRL2, JAG1, EGFR1). As a preliminary experimental validation we focused on 9 out of the 27 identified regulatory modules that involve miR-33a and SREBF2. The results confirm the importance of the predictions obtained with the presented computational approach.

The activator protein 2 (AP-2) transcription factors are essential proteins for oestrogenic repression of the ERBB2 proto-oncogene in breast cancer cells. In the present study, we have examined the possible oestrogenic regulation of AP-2 genes themselves in breast-tumour-derived lines. As early as 1 h after oestrogen treatment, AP-2gamma mRNA was markedly increased, whereas AP-2alpha was down-regulated, but with slower kinetics, and AP-2beta was not affected at all. Addition of anti-oestrogens ablated these effects. Modulation of the protein levels corresponded to changes in the transcript levels, thus suggesting that in oestrogen-treated cells, an inversion of the balance between AP-2alpha and AP-2gamma isoforms occurs. The 5'-untranslated region (5'-UTR) of the human AP-2gamma gene contains one consensus and one degenerate oestrogen-responsive element (ERE). Reporter constructs carrying the AP-2gamma promoter and the 5'-UTR were up-regulated by oestrogens in transient transfection assays. Deletion of the most conserved (but not of the degenerate) ERE from reporter constructs abrogated the oestrogenic response, although both ERE-containing segments were footprinted in DNaseI protection assays. In vitro binding assays demonstrated the ability of oestrogen receptor alpha (ERalpha) to bind to this site, and chromatin immunoprecipitation analysis of the endogenous gene showed that ERalpha occupies this region in response to oestrogens. We conclude that AP-2gamma is a primary oestrogen-responsive gene and suggest that AP-2 proteins may mediate some oestrogenic responses.

SummaryMesenchymal stem/stromal cells (MSCs) are progenitor cells shown to participate in breast tumor stroma formation and to promote metastasis. Despite expanding knowledge of their contributions to breast malignancy, the underlying molecular responses of breast cancer cells (BCCs) to MSC influences remain incompletely understood. Here, we show that MSCs cause aberrant expression of microRNAs, which, led by microRNA-199a, provide BCCs with enhanced cancer stem cell (CSC) properties. We demonstrate that such MSC-deregulated microRNAs constitute a network that converges on and represses the expression of FOXP2, a forkhead transcription factor tightly associated with speech and language development. FOXP2 knockdown in BCCs was sufficient in promoting CSC propagation, tumor initiation, and metastasis. Importantly, elevated microRNA-199a and depressed FOXP2 expression levels are prominent features of malignant clinical breast cancer and are associated significantly with poor survival. Our results identify molecular determinants of cancer progression of potential utility in the prognosis and therapy of breast cancer.