Determinación del papel de FGFR1 y FGFR4 en cáncer de pulmón.

El cáncer de pulmón representa uno de los tipos de cáncer con mayor incidencia y mortalidad. Su caracterización molecular ha permitido identificar alteraciones moleculares conductoras de esta patología, algunas de las cuales son abordables terapéuticamente. Éste es el caso de FGFR1 y FGFR4, cuya amplificación o sobreexpresión ha demostrado estar asociada a este tipo de tumores y correlacionada con un peor pronóstico de los pacientes afectos. Se han desarrollado diferentes inhibidores dirigidos frente a los FGFRs, actualmente en ensayos clínicos en diferentes tipos tumorales, incluido el de pulmón. Sin embargo, a pesar de los prometedores resultados obtenidos con estos inhibidores en estudios preclínicos, su eficacia a nivel clínico ha sido más que modesta. La causa de esto se ha atribuido a la ausencia de criterios de selección de pacientes apropiados para recibir este tipo de terapia. Por todo esto, el objetivo de este trabajo de tesis es la elucidación del papel de ambos FGFRs en la tumorogénesis pulmonar y de sus implicaciones clínicas, así como la determinación de factores predictivos de respuesta a sus inhibidores. A lo largo del presente trabajo, mediante la realización de técnicas experimentales in vitro, in vivo y de análisis de muestras tumorales de cohortes de pacientes con cáncer de pulmón, describiremos el papel de FGFR1 y FGFR4 en la biología del cáncer de pulmón dependiente del contexto molecular. La expresión de cualquiera de estos receptores provoca efectos pro-tumorogénicos en dos contextos diferentes. Por un lado, en modelos con sobreactivación de EGFR, ambos FGFRs interaccionan físicamente con EGFR, produciéndose una activación recíproca de ambos tipos de receptores y de las rutas de señalización oncogénicas canónicas de éstos. En línea con estos resultados, los tratamientos combinados con inhibidores de EGFR y FGFR presentan gran eficacia en modelos de xenoinjertos de tumores derivados de pacientes con alta expresión de FGFR1 y/o FGFR4 y activación de EGFR. Además, el análisis de una cohorte de pacientes tratados con inhibidores de EGFR en monoterapia señala que los pacientes con tumores con alta expresión de FGFR1 y/o FGFR4 presentan peor respuesta al tratamiento. Por otro lado, en modelos con sobreexpresión de N-cadherina, esta molécula de adhesión interacciona con ambos receptores provocando su activación y la de su señalización oncogénica. De acuerdo a esto, el tratamiento con inhibidores de FGFR es efectivo solo en los modelos de tumores derivados de pacientes con alta expresión de FGFR1 y/o FGFR4, y de N-cadherina. Además, el análisis de varias cohortes independientes de pacientes indica que la N-cadherina tiene un papel pronóstico en los pacientes afectos de tumores pulmonares con alta expresión de FGFR1 y/o FGFR4, correlacionando con supervivencias más acortadas. Sin embargo, en ausencia de activación de EGFR y de expresión de N-cadherina, ambos FGFRs ejercen un efecto supresor tumoral mediante una disminución de la señalización oncogénica asociada a estos receptores. En vista a los resultados obtenidos en este trabajo de tesis doctoral, se propone que la determinación de la expresión de FGFR1 y FGFR4 no es suficiente para determinar la efectividad de los inhibidores de FGFR. La determinación complementaria de la activación de EGFR y de la expresión de N-cadherina podría ser clave para la selección de la terapia a aplicar a cada paciente.Premio Extraordinario de Doctorado U

MAP17 predicts sensitivity to platinum-based therapy, EGFR inhibitors and the proteasome inhibitor bortezomib in lung adenocarcinoma

Background The high incidence and mortality of lung tumours is a major health problem. Therefore, the identification both of biomarkers predicting efficacy for therapies in use and of novel efficacious therapeutic agents is crucial to increase patient survival. MAP17 (PDZK1IP1) is a small membrane-bound protein whose upregulation is reported as a common feature in tumours from diverse histological origins. Furthermore, MAP17 is correlated with tumour progression. Go to: Methods We assessed the expression of MAP17 in preclinical models, including cell lines and patient-derived xenografts (PDXs), assessing its correlation with sensitivity to different standard-of-care drugs in lung adenocarcinoma, as well as novel drugs. At the clinical level, we subsequently correlated MAP17 expression in human tumours with patient response to these therapies. Go to: Results We show that MAP17 expression is induced during lung tumourigenesis, particularly in lung adenocarcinomas, and provide in vitro and in vivo evidence that MAP17 levels predict sensitivity to therapies currently under clinical use in adenocarcinoma tumours, including cisplatin, carboplatin and EGFR inhibitors. In addition, we show that MAP17 expression predicts proteasome inhibitor efficacy in this context and that bortezomib, an FDA-approved drug, may be a novel therapeutic approach for MAP17-overexpressing lung adenocarcinomas. Go to: Conclusions Our results indicate a potential prognostic role for MAP17 in lung tumours, with particular relevance in lung adenocarcinomas, and highlight the predictive pot0065ntial of this membrane-associated protein for platinum-based therapy and EGFR inhibitor efficacy. Furthermore, we propose bortezomib treatment as a novel and efficacious therapy for lung adenocarcinomas exhibiting high MAP17 expression

Impact of Heat Shock Protein 90 Inhibition on the Proteomic Profile of Lung Adenocarcinoma as Measured by Two-Dimensional Electrophoresis Coupled with Mass Spectrometry

Heat shock protein 90 (HSP90) is an important chaperone in lung adenocarcinoma, with relevant protein drivers such as EGFR (epidermal growth factor receptor) and EML4-ALK (echinoderm microtubule-associated protein-like protein4 fused to anaplastic lymphoma kinase) depending on it for their correct function, therefore HSP90 inhibitors show promise as potential treatments for lung adenocarcinoma. To study responses to its inhibition, HSP90 was pharmacologically interrupted by geldanamycin and resorcinol derivatives or with combined inhibition of HSP90 plus HSP70 in lung adenocarcinoma cell lines. Two-dimensional electrophoresis was performed to identify proteomic profiles associated with inhibition which will help to understand the biological basis for the responses. HSP90 inhibition resulted in altered protein profiles that differed according the treatment condition studied. Results revealed 254 differentially expressed proteins after treatments, among which, eukaryotic translation initiation factor3 subunit I (eIF3i) and citrate synthase demonstrated their potential role as response biomarkers. The differentially expressed proteins also enabled signalling pathways involved in responses to be identified; these included apoptosis, serine-glycine biosynthesis and tricarboxylic acid cycle. The proteomic profiles identified here contribute to an improved understanding of HSP90 inhibition and open possibilities for the detection of potential response biomarkers which will be essential to maximize treatment efficacy in lung adenocarcinoma.L.P.A. was funded by the Comunidad de Madrid, CAM, (B2017/BMD3884), ISCIII (PIE15/00076, PI17/00778 and DTS17/00089) and CIBERONC (CB16/12/00442), and co-funded by FEDER from Regional Development European Funds (European Union). S.M.P. is funded by the Fundación Mutua Madrileña (2014) Ministry of Health and SocialWelfare of Junta de Andalucía (PI-0046-2012, Nicolas Monardes Program C-0040-2016),ISCIII (PI17/00033), and co-funded by FEDER from Regional Development European Funds (European Union). I.F. is funded by the AECC (AIO2015) and ISCIII (PI16/01311), and co-funded by FEDER from Regional Development European Funds (European Union). AC was funded by grants from the Spanish Ministry of Economy and Competitiveness Plan Estatal de I+D+I 2018 co-funded by FEDER: RTI2018-097455-B-I00; CIBER de Cáncer Cells 2019, 8, 806 17 of 22(CB16/12/00275), co-funded by FEDER from Regional Development European Funds. Especial thanks to the Fundación AECC. L.O. is funded by the Ministerio de Educación, Cultura y Deporte (FPU13/02595).S

Coordinated downregulation of Spinophilin and the catalytic subunits of PP1, PPP1CA/B/C, contributes to a worse prognosis in lung cancer

The scaffold protein Spinophilin (Spinophilin, PPP1R9B) is one of the regulatory subunits of phosphatase-1 (PP1), directing it to distinct subcellular locations and targets. The loss of Spinophilin reduces PP1 targeting to pRb, thereby maintaining higher levels of phosphorylated pRb. Spinophilin is absent or reduced in approximately 40% of human lung tumors, correlating with the malignant grade. However, little is known about the relevance of the coordinated activity or presence of Spinophilin and its reported catalytic partners in the prognosis of lung cancer. In the present work, we show that the downregulation of Spinophilin, either by protein or mRNA, is related to a worse prognosis in lung tumors. This effect is more relevant in squamous cell carcinoma, SCC, than in adenocarcinoma. Downregulation of Spinophilin is related to a decrease in the levels of its partners PPP1CA/B/C, the catalytic subunits of PP1. A decrease in these subunits is also related to prognosis in SCC and, in combination with a decrease in Spinophilin, are markers of a poor prognosis in these tumors. The analysis of the genes that correlate to Spinophilin in lung tumors showed clear enrichment in ATP biosynthesis and protein degradation GO pathways. The analysis of the response to several common and pathway-related drugs indicates a direct correlation between the Spinophilin/PPP1Cs ratio and the response to oxaliplatin and bortezomib. This finding indicates that this ratio may be a good predictive biomarker for the activity of the drugs in these tumors with a poor prognosis.España, Mineco Plan Estatal de I+D+I 2013-2016España, ISCIII Fis: PI15/00045CIBER de Cáncer CB16/12/00275, CB16/12/00443, CB16/12/00442España, Junta de Andalucía, Consejeria de Ciencia e Innovacion CTS-1848España, Junta de Andalucía, Consejeria de Salud PI-0096-201

Histology-dependent prognostic role of pERK and p53 protein levels in early-stage non-small cell lung cancer

Lung tumors represent a major health problem. In early stage NSCLC tumors, surgical resection is the preferred treatment, but 30-55% of patients will relapse within 5 years after surgery. Thus, the identification of prognostic biomarkers in early stage NSCLC patients, especially those which are therapeutically addressable, is crucial to enhance survival of these patients. We determined the immunohistochemistry expression of key proteins involved in tumorigenesis and oncogenic signaling, p53, EGFR, pAKT and pERK, and correlated their expression level to clinicopathological characteristics and patient outcome. We found EGFR expression is higher in the squamous cell carcinomas than in adenocarcinomas (p=0.043), and that nuclear p53 staining correlated with lower differentiated squamous tumors (p=0.034). Regarding the prognostic potential of the expression of these proteins, high pERK levels proved to be an independent prognostic factor for overall (p<0.001) and progression-free survival (p<0.001) in adenocarcinoma patients, but not in those from the squamous histology, and high p53 nuclear levels were identified as independent prognostic factor for progression-free survival (p=0.031) only in squamous cell carcinoma patients. We propose a role as early prognostic biomarkers for pERK protein levels in adenocarcinoma, and for nuclear p53 levels in squamous cell lung carcinoma. The determination of these potential biomarkers in the adequate histologic context may predict the outcome of early stage NSCLC patients, and may offer a therapeutic opportunity to enhance survival of these patients.L.P.A. was funded by Instituto de Salud Carlos III (PI14/01964, PIE15/00076, CB16/12/00442, and R12/0036/0028) and co-funded by the European Union (ERDF/ESF, “Investing in your future”). The laboratory of A.C. was supported by grants from the Spanish Ministry of Economy and Competitiveness (PN I+D+I 2008-2011 and PE I+D+I 2013-2016), Instituto de Salud Carlos III (PI15/00045 and CB16/12/00275) and co-funded by the European Union (ERDF/ESF, “Investing in your future”), Consejeria de Ciencia e Innovacion (CTS-1848) and Consejeria de Salud of the Junta de Andalucia (PI-0096-2014). S.M.P. is funded by Fundación Mutua Madrileña (2014) and Instituto de Salud Carlos III (PI17/00033) and co-funded by the European Union (ERDF/ESF, “Investing in your future”). I.F. is funded by AECC (AIO2015) and Consejería de Igualdad, Salud y Políticas Sociales de la Junta de Andalucía (PI-0029-2013) and Instituto de Salud Carlos III (PI16/01311) and co-funded by the European Union (ERDF/ESF, “Investing in your future”). A.Q. is funded by Instituto de Salud Carlos III (FI12/00429) and co-funded by the European Union (ERDF/ ESF, “Investing in your future”).S

FGFR1 Cooperates with EGFR in Lung Cancer Oncogenesis, and Their Combined Inhibition Shows Improved Efficacy

Introduction: There is substantial evidence for the onco- genic effects of fi broblast growth factor receptor 1 (FGFR1) in many types of cancer, including lung cancer, but the role of this receptor has not been addressed speci fi cally in lung adenocarcinoma. Methods: We performed FGFR1 and EGFR overexpression and co-overexpression assays in adenocarcinoma and in inmortalized lung cell lines, and we also carried out surrogateandinteractionassays.Weperformedmono- therapy and combination EGFR /FGFR inhibitor sensitivity assays in vitro and in vivo in cell line – and patient- derived xenografts. We determined FGFR1 mRNA expression in a cohort of patients with anti – EGFR ther- apy – treated adenocarcinoma. Results: We have reported a cooperative interaction between FGFR1 and EGFR in this context, resulting in increased EGFR activation and oncogenic signaling. We have provided in vitro and in vivo evidence indicating that FGFR1 expression in- creases tumorigenicity in cells with high EGFR activation in EGFR-mutated and EGFR wild-type models. At the clinical level, we have shown that high FGFR1 expression levels pre- dict higher resistance to erlotinib or ge fi tinib in a cohort of patients with tyrosine kinase inhibitor – treated EGFR-mutated and EGFR wild-type lung adenocarcinoma. Dual EGFR and FGFR inhibition in FGFR1-over expressing, EGFR-activated models shows synergistic effects on tumor growth in vitro and in cell line – and patient-derived xenografts, suggesting that patients with tumors bearing these characteristics may bene fi t from combined EGFR/FGFR inhibition. Conclusion: These results support the extended the use of EGFR inhibitors beyond monotherapy in the EGFR-mutated adenocarcinoma setting in combination with FGFR in- hibitors for selected patients with increased FGFR1 over- expression and EGFR activation.ISCIII PI14/01964 PIE15/00076 PI17/00778 DTS17/00089 PI15/00045 PI17/00033 PI16/01311 FI12/00429CIBERONC CD16/12/00442FEDER CD16/12/00442 PI16/01311Spanish Ministry of Economy and Competitiveness PI15/00045Ministry of Health and Social Welfare of Junta de Andalucía PI-0046-2012 C-0040-2016Ministry of Equality, Health and Social Policies of the Junta de Andalucía PI- 0029-2013Comunidad de Madrid B2017/BMD3884Ministry of Education, Culture and Sports FPU13/0259

Identification of Predictive Biomarkers of Response to HSP90 Inhibitors in Lung Adenocarcinoma

Heat shock protein 90 (HSP90) plays an essential role in lung adenocarcinoma, acting as a key chaperone involved in the correct functioning of numerous highly relevant protein drivers of this disease. To this end, HSP90 inhibitors have emerged as promising therapeutic strategies, even though responses to them have been limited to date. Given the need to maximize treatment efficacy, the objective of this study was to use isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomic techniques to identify proteins in human lung adenocarcinoma cell lines whose basal abundances were correlated with response to HSP90 inhibitors (geldanamycin and radicicol derivatives). From the protein profiles identified according to response, the relationship between lactate dehydrogenase B (LDHB) and DNA topoisomerase 1 (TOP1) with respect to sensitivity and resistance, respectively, to geldanamycin derivatives is noteworthy. Likewise, rhotekin (RTKN) and decaprenyl diphosphate synthase subunit 2 (PDSS2) were correlated with sensitivity and resistance to radicicol derivatives. We also identified a relationship between resistance to HSP90 inhibition and the p53 pathway by glucose deprivation. In contrast, arginine biosynthesis was correlated with sensitivity to HSP90 inhibitors. Further study of these outcomes could enable the development of strategies to improve the clinical efficacy of HSP90 inhibition in patients with lung adenocarcinoma

Epigenetics of lung cancer: a translational perspective

[Background] Lung cancer remains the most common cause of cancer-related death, with a 5-year survival rate of only 18%. In recent years, the development of targeted pharmacological agents and immunotherapies has substantially increased the survival of a subset of patients. However, most patients lack such efficacious therapy and are, thus, treated with classical chemotherapy with poor clinical outcomes. Therefore, novel therapeutic strategies are urgently needed. In recent years, the development of epigenetic assays and their application to cancer research have highlighted the relevance of epigenetic regulation in the initiation, development, progression and treatment of lung cancer.[Conclusions] A variety of epigenetic modifications do occur at different steps of lung cancer development, some of which are key to tumor progression. The rise of cutting-edge technologies such as single cell epigenomics is, and will continue to be, crucial for uncovering epigenetic events at a single cell resolution, leading to a better understanding of the biology underlying lung cancer development and to the design of novel therapeutic options. This approach has already led to the development of strategies involving single agents or combined agents targeting epigenetic modifiers, currently in clinical trials. Here, we will discuss the epigenetics of every step of lung cancer development, as well as the translation of these findings into clinical applications.SMP was supported by the Consejería de Salud y Bienestar Social of Junta de Andalucía through the “‘Nicolás Monardes’ program [C-0040-2016] and ISCIII (PI17/00033) and cofounder by FEDER from Regional Development European Funds (European Union).”Peer reviewe

A patent review of FGFR4 selective inhibition in cancer (2007-2018)

[Introduction] FGFR4 is a tyrosine kinase receptor which, under physiological conditions, is activated upon ligand binding in a highly regulated manner. This triggers downstream signaling related to proliferation and apoptosis resistance as well as other physiological processes. Many molecular alterations of the receptor and its ligands, specially FGF19, have been reported in several types of cancer, with special relevance in hepatocellular carcinoma. In addition, these have also been detected in other solid malignancies, including lung, breast, or colon cancer, among others.[Areas covered] This review covers patent literature on specific FGFR4 inhibitors and their applications, published from 2007 to June 2018.[Expert opinion] FGFR4 inhibition has gained relevance in oncology. A considerable number of patents disclosing different approaches to inhibit this receptor have been reported, displaying promising preclinical results for different cancer models. Currently, the safety and preliminary efficacy of several small molecule inhibitors targeting FGFR4 are under early phase clinical assessment, mainly in hepatocellular carcinoma patients. If positive results are derived from these trials, they will open the door for the application of FGFR4 small molecule inhibitors to a wide population of tumors of different types that harbor FGFR4-FGF19 signaling dysregulation.[Trial registration] ClinicalTrials.gov identifier: NCT02325739.[Trial registration] ClinicalTrials.gov identifier: NCT02834780.[Trial registration] ClinicalTrials.gov identifier: NCT03144661.[Trial registration] ClinicalTrials.gov identifier: NCT02508467.L Paz Ares was supported by ISCIII (PI14/01964, PIE15/00076, PI17/00778 and DTS17/00089) and CIBERONC (CD16/12/00442), and co-funded by FEDER from Regional Development European Funds (European Union). L Paz Ares also reports personal fees from Roche, Lilly, MSD, BMS, Astra Zeneca, Boehringer Ing., Pfizer, Takeda, Novartis, Merck Serono and Amgem, outside the submitted work. In addition, L Paz Ares has patent P201730928 and patent PCT/ES2018/070502 pending. I Ferrer was supported by AECC (AIO2015) and Consejería de Igualdad, Salud y Políticas Sociales de la Junta de Andalucía (PI-0029-2013) and ISCIII (PI16/01311) and cofunded by FEDER from Regional Development European Funds (European Union). Also, I Ferrer has patent P201730928 and patent PCT/ES2018/070502 pending. S Molina-Pinelo was supported by the Consejería de Salud y Bienestar Social of Junta de Andalucía through the ‘“Nicolás Monardes”’ program [C-0040-2016] and ISCIII (PI17/00033) and co-funded by FEDER from Regional Development European Funds (European Union). Also, S Molina-Pinelo has patent P201730928 and patent PCT/ES2018/070502 pending.Peer reviewe

N-Cadherina y FGFR1 y/o FGFR4 para uso en la predicción de la respuesta de los pacientes a un tratamiento del cancer de pulmón y método y kit basado en dicho uso

N-cadherina y FGFR1 y/o FGFR4 para uso en la predicción de la respuesta de los pacientes a un tratamiento del cáncer de pulmón y método y kit basados en dicho uso. La presente invención describe un método para predecir la respuesta de un sujeto que sufre cáncer de pulmón al tratamiento con inhibidores de FGFR. La relación de la expresión de los biomarcadores descritos con la respuesta al tratamiento, permite clasificar a los sujetos como respondedores o norespondedores al tratamiento , lo que facilita la decisión terapéutica al responsable clínico . El presente método describe los biomarcadores Ncadherina, FGFR1 y FGFR4, cómo analizarlos y cómo interpretar los resultados obtenidos , con el fin de administrar inhibidores de FGFR únicamente a los sujetos que van a responder a dicho tratamiento, optimizando el mismo, lo que permite que los sujetos no-respondedores puedan ser tratados con terapias alternativas a la de los inhibidores de FGFR.Peer reviewedFundación de investigación Hospital 12 De Octubre, Servicio Andaluz de Salud, Consejo Superior de Investigaciones Científicas (España)B2 Patente con examen previ