Simone Lubrano

Abstract Purpose: Uveal melanoma is the most common eye cancer in adults. Approximately 50% of patients with uveal melanoma develop metastatic uveal melanoma (mUM) in the liver, even after successful treatment of the primary lesions. mUM is refractory to current chemo- and immune-therapies, and most mUM patients die within a year. Uveal melanoma is characterized by gain-of-function mutations in GNAQ/GNA11, encoding Gαq proteins. We have recently shown that the Gαq–oncogenic signaling circuitry involves a noncanonical pathway distinct from the classical activation of PLCβ and MEK–ERK. GNAQ promotes the activation of YAP1, a key oncogenic driver, through focal adhesion kinase (FAK), thereby identifying FAK as a druggable signaling hub downstream from GNAQ. However, targeted therapies often activate compensatory resistance mechanisms leading to cancer relapse and treatment failure. Experimental Design: We performed a kinome-wide CRISPR-Cas9 sgRNA screen to identify synthetic lethal gene interactions that can be exploited therapeutically. Candidate adaptive resistance mechanisms were investigated by cotargeting strategies in uveal melanoma and mUM in vitro and in vivo experimental systems. Results: sgRNAs targeting the PKC and MEK–ERK signaling pathways were significantly depleted after FAK inhibition, with ERK activation representing a predominant resistance mechanism. Pharmacologic inhibition of MEK and FAK showed remarkable synergistic growth-inhibitory effects in uveal melanoma cells and exerted cytotoxic effects, leading to tumor collapse in uveal melanoma xenograft and liver mUM models in vivo. Conclusions: Coupling the unique genetic landscape of uveal melanoma with the power of unbiased genetic screens, our studies reveal that FAK and MEK–ERK cotargeting may provide a new network-based precision therapeutic strategy for mUM treatment. See related commentary by Harbour, p. 2967

// Marianna Vitiello 1, 2 , Andrea Tuccoli 1 , Romina D’Aurizio 3 , Samanta Sarti 1, 4 , Laura Giannecchini 1 , Simone Lubrano 1, 4 , Andrea Marranci 1, 4 , Monica Evangelista 2 , Silvia Peppicelli 5 , Chiara Ippolito 6 , Ivana Barravecchia 7 , Elena Guzzolino 7 , Valentina Montagnani 8 , Michael Gowen 9 , Elisa Mercoledi 1 , Alberto Mercatanti 2 , Laura Comelli 2 , Salvatore Gurrieri 1 , Lawrence W. Wu 10 , Omotayo Ope 10 , Keith Flaherty 11 , Genevieve M. Boland 11 , Marc R. Hammond 11 , Lawrence Kwong 12 , Mario Chiariello 2, 13 , Barbara Stecca 8 , Gao Zhang 10 , Alessandra Salvetti 14 , Debora Angeloni 7 , Letizia Pitto 2 , Lido Calorini 5 , Giovanna Chiorino 15 , Marco Pellegrini 3 , Meenhard Herlyn 10 , Iman Osman 9 , Laura Poliseno 1, 2 1 Oncogenomics Unit, Core Research Laboratory, Istituto Toscano Tumori (ITT), AOUP, Pisa, Italy 2 Institute of Clinical Physiology (IFC), CNR, Pisa, Italy 3 Laboratory of Integrative Systems Medicine (LISM), Institute of Informatics and Telematics (IIT), CNR, Pisa, Italy 4 University of Siena, Italy 5 Section of Experimental Pathology and Oncology, Department of Experimental and Clinical Biomedical Sciences, University of Firenze, Italy 6 Unit of Histology, Department of Clinical and Experimental Medicine, University of Pisa, Italy 7 Scuola Superiore Sant’Anna, Pisa, Italy 8 Tumor Cell Biology Unit, Core Research Laboratory, Istituto Toscano Tumori (ITT), AOUC, Firenze, Italy 9 New York University, New York, NY, USA 10 The Wistar Institute, Philadelphia, PA, USA 11 Massachusetts General Hospital, Boston, MA, USA 12 MD Anderson Cancer Center, Houston, TX, USA 13 Signal Transduction Unit, Core Research Laboratory, Istituto Toscano Tumori (ITT), AOUS, Siena, Italy 14 Unit of Experimental Biology and Genetics, Department of Clinical and Experimental Medicine, University of Pisa, Italy 15 Fondazione Edo and Elvo Tempia, Biella, Italy Correspondence to: Laura Poliseno, email: laura.poliseno@gmail.com Keywords: melanoma, BRAFV600E, ERK pathway, miR-204 family, context-dependency Received: March 11, 2016      Accepted: February 06, 2017      Published: March 06, 2017 ABSTRACT Despite increasing amounts of experimental evidence depicting the involvement of non-coding RNAs in cancer, the study of BRAFV600E-regulated genes has thus far focused mainly on protein-coding ones. Here, we identify and study the microRNAs that BRAFV600E regulates through the ERK pathway. By performing small RNA sequencing on A375 melanoma cells and a vemurafenib-resistant clone that was taken as negative control, we discover miR-204 and miR-211 as the miRNAs most induced by vemurafenib. We also demonstrate that, although belonging to the same family, these two miRNAs have distinctive features. miR-204 is under the control of STAT3 and its expression is induced in amelanotic melanoma cells, where it acts as an effector of vemurafenib’s anti-motility activity by targeting AP1S2. Conversely, miR-211, a known transcriptional target of MITF, is induced in melanotic melanoma cells, where it targets EDEM1 and consequently impairs the degradation of TYROSINASE (TYR) through the ER-associated degradation (ERAD) pathway. In doing so, miR-211 serves as an effector of vemurafenib’s pro-pigmentation activity. We also show that such an increase in pigmentation in turn represents an adaptive response that needs to be overcome using appropriate inhibitors in order to increase the efficacy of vemurafenib. In summary, we unveil the distinct and context-dependent activities exerted by miR-204 family members in melanoma cells. Our work challenges the widely accepted “same miRNA family = same function” rule and provides a rationale for a novel treatment strategy for melanotic melanomas that is based on the combination of ERK pathway inhibitors with pigmentation inhibitors.

BACKGROUND: Understanding the intricate signaling network involved in triple-negative breast cancer (TNBC) represents a challenge for developing novel therapeutic approaches. Here, we aim to provide novel mechanistic insights on the function of the S100A8/A9-RAGE system in TNBC. METHODS: TNM plot analyzer, Kaplan-Meier plotter, Meta-analysis, GEPIA2 and GOBO publicly available datasets were used to evaluate the clinical significance of S100A8/A9 and expression levels of S100A8/A9, RAGE and Filamin family members in breast cancer (BC) subtypes. METABRIC database and Cox proportional hazard model defined the clinical impact of high RAGE expression in BC patients. Multiple bioinformatics programs identified the main enriched pathways within high RAGE expression BC cohorts. By lentiviral system, TNBC cells were engineered to overexpress RAGE. Western blotting, immunofluorescence, nucleus/cytoplasm fractionation, qRT-PCR, gene silencing and luciferase experiments were performed to identify signal transduction mediators engaged by RAGE upon stimulation with S100A8/A9 in TNBC cells. Proliferation, colony formation and transwell migration assays were carried out to evaluate the growth and migratory capacity of TNBC cells. Statistical analysis was performed by ANOVA and independent t-tests. RESULTS: We found a remarkable high expression of S100A8 and S100A9 in BC, particularly in HER2-positive and TNBC, with the latter associated to worst clinical outcomes. In addition, high RAGE expression correlated with a poor overall survival in BC. Next, we determined that the S100A8/A9-RAGE system triggers FAK activation by engaging a cytoskeleton mechanosensing complex in TNBC cells. Through bioinformatics analysis, we identified the Hippo pathway as the most enriched in BC patients expressing high RAGE levels. In accordance with these data, we demonstrated the involvement of S100A8/A9-RAGE-FAK signaling in the control of Hippo/YAP activities, and we established the crucial contribution of RAGE-FAK-YAP circuitry in the growth and migratory effects initiated by S100A8/A9 in TNBC cells. CONCLUSIONS: The present study provides novel mechanistic insights on RAGE actions in TNBC. Moreover, our findings suggest that RAGE-FAK-YAP transduction pathway could be exploited as a druggable system halting the aggressive TNBC subtype.

BACKGROUND: The BRAF protein kinase is widely studied as a cancer driver and therapeutic target. However, the regulation of its expression is not completely understood. RESULTS: Taking advantage of the RNA-seq data of more than 4800 patients belonging to 9 different cancer types, we show that BRAF mRNA exists as a pool of 3 isoforms (reference BRAF, BRAF-X1, and BRAF-X2) that differ in the last part of their coding sequences, as well as in the length (BRAF-ref: 76 nt; BRAF-X1 and BRAF-X2: up to 7 kb) and in the sequence of their 3'UTRs. The expression levels of BRAF-ref and BRAF-X1/X2 are inversely correlated, while the most prevalent among the three isoforms varies from cancer type to cancer type. In melanoma cells, the X1 isoform is expressed at the highest level in both therapy-naïve cells and cells with acquired resistance to vemurafenib driven by BRAF gene amplification or expression of the Δ[3-10] splicing variant. In addition to the BRAF-ref protein, the BRAF-X1 protein (the full length as well as the Δ[3-10] variant) is also translated. The expression levels of the BRAF-ref and BRAF-X1 proteins are similar, and together they account for BRAF functional activities. In contrast, the endogenous BRAF-X2 protein is hard to detect because the C-terminal domain is selectively recognized by the ubiquitin-proteasome pathway and targeted for degradation. CONCLUSIONS: By shedding light on the repertoire of BRAF mRNA and protein variants, and on the complex regulation of their expression, our work paves the way to a deeper understanding of a crucially important player in human cancer and to a more informed development of new therapeutic strategies.