New oncogenic networks regulated by the RNA binding factor CUGBP1 in melanoma

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 13-05-2016Esta tesis tiene embargado el acceso al texto completo hasta el 13-11-2017Melanoma is a prime example of an aggressive tumor that accumulates a plethora of changes in the transcriptome and the proteome. Consequently, distinguishing drivers from inconsequential passenger events has been a main challenge in this disease. Consequently, and despite great progress in identifying (epi)genetic defects accumulated during melanoma progression, the molecular bases underlying the aggressive behavior of this tumor type are not completely understood. RNA binding proteins remain largely unexplored in melanoma. We considered this lack of information of prime relevance as transcripts of nearly all known oncogenes and tumor suppressors may be regulated by alternative splicing and/or controlled by various mechanisms that define mRNA stability and ultimately, competency for translation. Here we show that a screen for all reported mRNA binding proteins (mRBPs) suggested that these genes are not prime targets of mutation or copy number variation. Instead, a customized microarray revealed a series of RBPs overexpressed in melanoma cells when compared to normal melanocytes. Of those factors, we found particularly attractive the CUGBP1/CELF1 protein. First, CUGBP1 is a multifunctional mRBP with a broad spectrum of functions, ranging from the modulation of alternative splicing to modulation of mRNA decay. Secondly, there is little information on comprehensive genome-wide analyses for CUGBP1 in cancer cells, with no previous report in skin cancer. Therefore this PhD thesis was set to address the following unknown aspects of CUGBP1 in melanoma: (i) expression, (ii) functional requirement, and (iii) mechanism of action. In brief, CUGBP1 was found overexpressed in human melanoma cells and tissue specimens. Targeted gene depletion demonstrated that CUGBP1 is required to sustain melanoma cell proliferation. Mechanistically, genome wide human junction arrays, RNA immunoprecipitation followed by sequencing and iTRAQ-MS/MS proteomics assays were utilized and identified novel and direct targets of CUGBP1. Together, these high throughput approaches (to our knowledge the first in class for this gene) uncovered a coordinated network of tumor-associated cell cycle regulators and chromatin remodelers as CUGBP1 targets. Central in this CUGBP1 regulated networks was the oncogene DEK, a feature we validated by targeted gene depletion, cDNA arrays and histological validation in clinical datasets. Mechanistically, CUGBP1 was found to bind to the 3’UTR of DEK stabilizing its mRNA levels and ultimately allowing for an efficient cell proliferation. These results illustrate the power of comprehensive analyses of RNA regulators in the identification of novel malignant features of cancer cells

Metastatic risk and resistance to BRAF inhibitors in melanoma defined by selective allelic loss of ATG5

Melanoma is a paradigm of aggressive tumors with a complex and heterogeneous genetic background. Still, melanoma cells frequently retain developmental traits that trace back to lineage specification programs. In particular, lysosome-associated vesicular trafficking is emerging as a melanoma-enriched lineage dependency. However, the contribution of other lysosomal functions such as autophagy to melanoma progression is unclear, particularly in the context of metastasis and resistance to targeted therapy. Here we mined a broad spectrum of cancers for a meta-analysis of mRNA expression, copy number variation and prognostic value of 13 core autophagy genes. This strategy identified heterozygous loss of ATG5 at chromosome band 6q21 as a distinctive feature of advanced melanomas. Importantly, partial ATG5 loss predicted poor overall patient survival in a manner not shared by other autophagy factors and not recapitulated in other tumor types. This prognostic relevance of ATG5 copy number was not evident for other 6q21 neighboring genes. Melanocyte-specific mouse models confirmed that heterozygous (but not homozygous) deletion of Atg5 enhanced melanoma metastasis and compromised the response to targeted therapy (exemplified by dabrafenib, a BRAF inhibitor in clinical use). Collectively, our results support ATG5 as a therapeutically relevant dose-dependent rheostat of melanoma progression. Moreover, these data have important translational implications in drug design, as partial blockade of autophagy genes may worsen (instead of counteracting) the malignant behavior of metastatic melanomas.M.S.S. is funded by grants from the Spanish Ministry of Economy and Innovation (projects SAF2011-28317, SAF2014-56868-R and RTC-2014-2442-1), as well as a Team Science Award by the Melanoma Research Alliance, and grants from the Worldwide Cancer Research and the Asociacion Espanola Contra el Cancer (AECC). M.G-F was funded by a Juan de la ~ Cierva postdoctoral fellowship from the Spanish Ministry of Education and P.K and M.C. by predoctoral fellowships from Fundación La Caixa

p62/SQSTM1 Fuels Melanoma Progression by Opposing mRNA Decay of a Selective Set of Pro-metastatic Factors

Modulators of mRNA stability are not well understood in melanoma, an aggressive tumor with complex changes in the transcriptome. Here we report the ability of p62/SQSTM1 to extend mRNA half-life of a spectrum of pro-metastatic factors. These include FERMT2 and other transcripts with no previous links to melanoma. Transcriptomic, proteomic, and interactomic analyses, combined with validation in clinical biopsies and mouse models, identified a selected set of RNA-binding proteins (RBPs) recruited by p62, with IGF2BP1 as a key partner. This p62-RBP interaction distinguishes melanoma from other tumors where p62 controls autophagy or oxidative stress. The relevance of these data is emphasized by follow-up analyses of patient prognosis revealing p62 and FERMT2 as adverse determinants of disease-free survival.M.S.S. is funded by grants from the Spanish Ministry of Economy and Innovation (SAF2014-56868-R; SAF2017-89533-R), the Asociación Española Contra el Cáncer (AECC), TV’13-20131430 (Marato de TV3), the Worldwide Cancer Research, an Established Investigator Award by the Melanoma Research Alliance (MRA), and a L'Oreal-Paris USA-MRA Team Science Award for Women in Scientific Research. M.S.S. also acknowledges a donation from “Fundación Causa Alexandra”, Spain. P.K. was a recipient of a predoctoral fellowship from Fundación La Caixa. E.R.-F. was funded by Fundación Mutua Madrileña (FMM-2013) and was a recipient of a fellowship from ‘‘Fundación Científica de la Asociación Española Contra el Cáncer”. The CNIO Proteomics Unit belongs to ProteoRed, PRB2-ISCIII, supported by grant PT13/0001. J.M. is also supported by Ramon y Cajal Programme (MINECO) RYC-2012-10651. J.L.R.-P. is funded by FIS 2014/173711/02568 and CIBERONC, and P.L.O.-R. by FIS 11/17592014/01784, from the Spanish Ministry of Health

RAB7 Controls Melanoma Progression by Exploiting a Lineage-Specific Wiring of the Endolysosomal Pathway

Although common cancer hallmarks are well established, lineage-restricted oncogenes remain less understood. Here, we report an inherent dependency of melanoma cells on the small GTPase RAB7, identified within a lysosomal gene cluster that distinguishes this malignancy from over 35 tumor types. Analyses in human cells, clinical specimens, and mouse models demonstrated that RAB7 is an early-induced melanoma driver whose levels can be tuned to favor tumor invasion, ultimately defining metastatic risk. Importantly, RAB7 levels and function were independent of MITF, the best-characterized melanocyte lineage-specific transcription factor. Instead, we describe the neuroectodermal master modulator SOX10 and the oncogene MYC as RAB7 regulators. These results reveal a unique wiring of the lysosomal pathway that melanomas exploit to foster tumor progression.M.S.S. is funded by Projects SAF2011-28317 and Consolider RNAREG from the Spanish Ministry of Economy and Innovation, R01CA125017 from the NIH, and a Team Science Award by the Melanoma Research Foundation. J.L.R.-P. and P.O.-R. are funded by grants FIS 11/025685 and FIS 11/1759, respectively, from the Spanish Ministry of Health. J.L.R.-P. was also supported by grant FMM-2008-106 of Fundación Mutua Madrileña, and P.O.-R. by Red Tematica de Investigacion Cooperativa en Cancer. D.A.-C. and E.P.-G. are recipients of Scientists in Training predoctoral fellowships from the Spanish Ministry of Science and Innovation. M.C. and P.K. are funded by predoctoral fellowships of Fundación La Caixa. E.R.-F. is the recipient of a postdoctoral fellowship from Fundación Científica de la Asociación Española Contra el Cáncer, and J.A.J. and H.-W.W. are funded by the American Cancer Society (RSG-12-076-01-LIB)

Metastatic risk and resistance to BRAF inhibitors in melanoma defined by selective allelic loss of <i>ATG5</i>

<p>Melanoma is a paradigm of aggressive tumors with a complex and heterogeneous genetic background. Still, melanoma cells frequently retain developmental traits that trace back to lineage specification programs. In particular, lysosome-associated vesicular trafficking is emerging as a melanoma-enriched lineage dependency. However, the contribution of other lysosomal functions such as autophagy to melanoma progression is unclear, particularly in the context of metastasis and resistance to targeted therapy. Here we mined a broad spectrum of cancers for a meta-analysis of mRNA expression, copy number variation and prognostic value of 13 core autophagy genes. This strategy identified heterozygous loss of <i>ATG5</i> at chromosome band 6q21 as a distinctive feature of advanced melanomas. Importantly, partial <i>ATG5</i> loss predicted poor overall patient survival in a manner not shared by other autophagy factors and not recapitulated in other tumor types. This prognostic relevance of <i>ATG5</i> copy number was not evident for other 6q21 neighboring genes. Melanocyte-specific mouse models confirmed that heterozygous (but not homozygous) deletion of <i>Atg5</i> enhanced melanoma metastasis and compromised the response to targeted therapy (exemplified by dabrafenib, a BRAF inhibitor in clinical use). Collectively, our results support ATG5 as a therapeutically relevant dose-dependent rheostat of melanoma progression. Moreover, these data have important translational implications in drug design, as partial blockade of autophagy genes may worsen (instead of counteracting) the malignant behavior of metastatic melanomas.</p

Ripensando il "welfare"

Sommario: 1. "Welfare senza risorse: emergenza o sistema. - 2. Economia reale e finanza speculativa. - 3. Eurozona e crisi globale. - 4. Solidariet\ue0 sociale e solidariet\ue0 familiare. - 5. Persona e salute. - 6. Vecchiaia e pensioni. - 7. Occupazione e ammortizzatori sociali. - 8. Previdenza e assistenza. - 9. "Welfare" ed autosufficienza. - 10. Sicurezza sociale e cittadinanza

RAB7 Controls Melanoma Progression by Exploiting a Lineage-Specific Wiring of the Endolysosomal Pathway

SummaryAlthough common cancer hallmarks are well established, lineage-restricted oncogenes remain less understood. Here, we report an inherent dependency of melanoma cells on the small GTPase RAB7, identified within a lysosomal gene cluster that distinguishes this malignancy from over 35 tumor types. Analyses in human cells, clinical specimens, and mouse models demonstrated that RAB7 is an early-induced melanoma driver whose levels can be tuned to favor tumor invasion, ultimately defining metastatic risk. Importantly, RAB7 levels and function were independent of MITF, the best-characterized melanocyte lineage-specific transcription factor. Instead, we describe the neuroectodermal master modulator SOX10 and the oncogene MYC as RAB7 regulators. These results reveal a unique wiring of the lysosomal pathway that melanomas exploit to foster tumor progression

Whole-body imaging of lymphovascular niches identifies pre-metastatic roles of midkine

Cutaneous melanoma is a type of cancer with an inherent potential for lymph node colonization, which is generally preceded by neolymphangiogenesis1,2,3. However, sentinel lymph node removal does not necessarily extend the overall survival of patients with melanoma4,5. Moreover, lymphatic vessels collapse and become dysfunctional as melanomas progress6,7. Therefore, it is unclear whether (and how) lymphangiogenesis contributes to visceral metastasis. Soluble and vesicle-associated proteins secreted by tumours and/or their stroma have been proposed to condition pre-metastatic sites in patients with melanoma8,9,10,11,12,13,14. Still, the identities and prognostic value of lymphangiogenic mediators remain unclear2,14. Moreover, our understanding of lymphangiogenesis (in melanomas and other tumour types) is limited by the paucity of mouse models for live imaging of distal pre-metastatic niches15. Injectable lymphatic tracers have been developed7, but their limited diffusion precludes whole-body imaging at visceral sites16. Vascular endothelial growth factor receptor 3 (VEGFR3) is an attractive ‘lymphoreporter’17 because its expression is strongly downregulated in normal adult lymphatic endothelial cells, but is activated in pathological situations such as inflammation and cancer17,18. Here, we exploit this inducibility of VEGFR3 to engineer mouse melanoma models for whole-body imaging of metastasis generated by human cells, clinical biopsies or endogenously deregulated oncogenic pathways. This strategy revealed early induction of distal pre-metastatic niches uncoupled from lymphangiogenesis at primary lesions. Analyses of the melanoma secretome and validation in clinical specimens showed that the heparin-binding factor midkine is a systemic inducer of neo-lymphangiogenesis that defines patient prognosis. This role of midkine was linked to a paracrine activation of the mTOR pathway in lymphatic endothelial cells. These data support the use of VEGFR3 reporter mice as a ‘MetAlert’ discovery platform for drivers and inhibitors of metastasis.M.S.S. is funded by grants from the Spanish Ministry of Economy and Innovation (project SAF2014-56868-R), the Asociación Española Contra el Cáncer (AECC), the Worldwide Cancer Research, an Established Investigator Award from the Melanoma Research Alliance (MRA), and a L’Oréal Paris USA-MRA Team Science Award for Woman in Scientific Research. The CNIO Proteomics Unit belongs to ProteoRed, PRB2-ISCIII, supported by grant PT13/0001. N.I. and J.M. are funded by SAF2013-45504-R (MINECO). J.M. is also supported by Ramon y Cajal Programme (MINECO) RYC-2012-10651. J.L.R.-P and P.O.-R are funded by grants FIS 2014/1737, 11/02568 and FIS 2014/01784, 11/1759, respectively, from the Spanish Ministry of Health. F.M. is funded by the AMIT Project/CDTI/CENIT Programme (MICINN), S.O. by SAF2013-44866-R (MINECO), and J.J.B.-C. by an NCI K22CA196750 grant and the TCI Young Scientist Cancer Research Award JJR Fund (P30 CA196521). J.D.M. is the recipient of a postdoctoral fellowship from the ARC Foundation and E.R.-F. from Fundación Científica de la Asociación Española Contra el Cáncer. D.C.-W. is the recipient of a predoctoral fellowship from Fundación La Caixa, and M.C.-A. and X.C. are recipients of the Immutrain Marie Skłodowska-Curie ITN Grant.Peer reviewe