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

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

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

Caracterización proteómica de la inhibición de HSP90 en el adenocarcinoma de pulmón

Las proteínas de choque térmico (HSPs) son inducidas, en respuesta a una amplia variedad de factores fisiológicos y ambientales estresantes, permitiendo a las células sobrevivir a condiciones que de otro modo hubieran sido letales. En concordancia con ello, las HSPs se hacen esenciales en el mantenimiento de la célula tumoral, describiéndose una expresión alterada de estas proteínas en casi todos los tipos tumorales. En concreto, la sobreexpresión de HSP90 ha sido relacionada con una pobre prognosis en cáncer, debido a su capacidad para regular y estabilizar un gran número de proteínas oncogénicas, denominadas clientes de HSP90. Particularmente, en el cáncer de pulmón no microcítico (CPNM), el subtipo histológico de adenocarcinoma de pulmón presenta diversas alteraciones moleculares conductoras que son clientes de HSP90 y algunas como EGFR o EML4-ALK exhiben una fuerte dependencia de la chaperona que las hace más sensibles a los inhibidores de HSP90. Sin embargo, tal y como se ha visto en los diferentes ensayos clínicos realizados hasta la fecha en esta patología, no todos los pacientes susceptibles de ser sensibles a la inhibición de HSP90 responden positivamente al tratamiento. Por todo esto, el objetivo de esta tesis doctoral es la identificación de la firma proteica subyacente a la inhibición de HSP90, así como la búsqueda de potenciales biomarcadores de sensibilidad y respuesta, esenciales para maximizar la eficacia del tratamiento en el adenocarcinoma de pulmón..

Inhibition of HSP90 in Driver Oncogene-Defined Lung Adenocarcinoma Cell Lines: Key Proteins Underpinning Therapeutic Efficacy

The use of 90 kDa heat shock protein (HSP90) inhibition as a therapy in lung adenocarcinoma remains limited due to moderate drug efficacy, the emergence of drug resistance, and early tumor recurrence. The main objective of this research is to maximize treatment efficacy in lung adenocarcinoma by identifying key proteins underlying HSP90 inhibition according to molecular background, and to search for potential biomarkers of response to this therapeutic strategy. Inhibition of the HSP90 chaperone was evaluated in different lung adenocarcinoma cell lines representing the most relevant molecular alterations (EGFR mutations, KRAS mutations, or EML4-ALK translocation) and wild-type genes found in each tumor subtype. The proteomic technique iTRAQ was used to identify proteomic profiles and determine which biological pathways are involved in the response to HSP90 inhibition in lung adenocarcinoma. We corroborated the greater efficacy of HSP90 inhibition in EGFR mutated or EML4-ALK translocated cell lines. We identified proteins specifically and significantly deregulated after HSP90 inhibition for each molecular alteration. Two proteins, ADI1 and RRP1, showed independently deregulated molecular patterns. Functional annotation of the altered proteins suggested that apoptosis was the only pathway affected by HSP90 inhibition across all molecular subgroups. The expression of ADI1 and RRP1 could be used to monitor the correct inhibition of HSP90 in lung adenocarcinoma. In addition, proteins such as ASS1, ITCH, or UBE2L3 involved in pathways related to the inhibition of a particular molecular background could be used as potential response biomarkers, thereby improving the efficacy of this therapeutic approach to combat lung adenocarcinoma

Inhibition of HSP90 in Driver Oncogene-Defined Lung Adenocarcinoma Cell Lines: Key Proteins Underpinning Therapeutic Efficacy

The use of 90 kDa heat shock protein (HSP90) inhibition as a therapy in lung adenocarcinoma remains limited due to moderate drug efficacy, the emergence of drug resistance, and early tumor recurrence. The main objective of this research is to maximize treatment efficacy in lung adenocarcinoma by identifying key proteins underlying HSP90 inhibition according to molecular background, and to search for potential biomarkers of response to this therapeutic strategy. Inhibition of the HSP90 chaperone was evaluated in different lung adenocarcinoma cell lines representing the most relevant molecular alterations (EGFR mutations, KRAS mutations, or EML4-ALK translocation) and wild-type genes found in each tumor subtype. The proteomic technique iTRAQ was used to identify proteomic profiles and determine which biological pathways are involved in the response to HSP90 inhibition in lung adenocarcinoma. We corroborated the greater efficacy of HSP90 inhibition in EGFR mutated or EML4-ALK translocated cell lines. We identified proteins specifically and significantly deregulated after HSP90 inhibition for each molecular alteration. Two proteins, ADI1 and RRP1, showed independently deregulated molecular patterns. Functional annotation of the altered proteins suggested that apoptosis was the only pathway affected by HSP90 inhibition across all molecular subgroups. The expression of ADI1 and RRP1 could be used to monitor the correct inhibition of HSP90 in lung adenocarcinoma. In addition, proteins such as ASS1, ITCH, or UBE2L3 involved in pathways related to the inhibition of a particular molecular background could be used as potential response biomarkers, thereby improving the efficacy of this therapeutic approach to combat lung adenocarcinoma.Depto. de MedicinaFac. de MedicinaTRUEpubDescuento UC

The FGFR4-388arg Variant Promotes Lung Cancer Progression by N-Cadherin Induction

The FGFR4-388Arg variant has been related to poor prognosis in several types of cancer, including lung cancer. The mechanism underlying this association has not been addressed in detail in patients with this pathology. Here, we report that this FGFR4 variant induces MAPK and STAT3 activation and causes pro-oncogenic effects in NSCLC in vitro and in vivo. This variant induces the expression of EMT-related genes, such as N-cadherin, vimentin, Snail1 and Twist1. Indeed, the induction of N-cadherin protein expression by this variant is essential for its pro-tumorigenic role. The presence of the FGFR4-388Arg variant correlates with higher N-cadherin expression levels in clinical NSCLC samples and with poorer outcome in patients with FGFR expression. These results support the prognostic role of this FGFR variant in lung cancer and show that these effects may be mediated by the induction of N-cadherin expression and an EMT phenotype.L.P.A. was funded by ISCIII (PI14/01964 and PIE15/00076), CIBER (CB16/12/00442) and RTICC (R12/0036/0028) and co-funded by 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, PE I+D+I 2013-2016, ISCIII (PI15/00045 and CB16/12/00275), 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 Consejería de Salud y Bienestar Social (PI-0046-2012), ISCIII (PI17/00033) and co-funded by European Union (ERDF/ESF, “Investing in your future”), and Fundación Mutua Madrileña (2014). I.F. is funded by AECC (AIO2015) and Consejería de Igualdad, Salud y Políticas Sociales de la Junta de Andalucía (PI-0029-2013) and FIS (PI16/01311). A.Q. is funded by ISCIII (FI12/00429). L.O. is funded by Ministerio de Educación, Cultura y Deporte (FPU13/02595).Peer reviewe

FGFR4 increases EGFR oncogenic signaling in lung adenocarcinoma, and their combined inhibition is highly effective

[Objectives] Lung adenocarcinoma accounts for approximately half of lung cancer cases. Twenty to 50% of tumors of this type harbor mutations affecting epidermal growth factor receptor (EGFR) expression or activity, which can be therapeutically targeted. EGFR inhibitors in this context exhibit high efficacy and are currently used in the clinical setting. However, not all adenocarcinomas harboring EGFR mutations respond to therapy, so predictive biomarkers of therapeutic outcomes, as well as novel therapies sensitizing these tumors to EGFR inhibition, are needed.[Materials and methods] We performed in vitro gene overexpression/silencing and tumorigenic surrogate assays, as well as in vitro and in vivo combination treatments with Fibroblast Growth Factor Receptor (FGFR)/EGFR inhibitors. At the clinical level, we determined FGFR4 expression levels in tumors from patients treated with EGFR inhibitors and correlated these with treatment response.[Results] We describe a cooperative interaction between EGFR and FGFR4, which results in their reciprocal activation with pro-oncogenic consequences in vitro and in vivo. This cooperation is independent of EGFR activating mutations and increases resistance to different EGFR inhibitors. At the therapeutic level, we provide evidence of the synergistic effects of the combination of EGFR and FGFR inhibitors in high FGFR4-expressing, EGFR-activated tumors in vitro and in vivo. Correlated with these results, we found that patients treated with EGFR inhibitors relapse earlier when their tumors exhibit high FGFR4 expression.[Conclusions] We propose a novel predictive biomarker for EGFR-targeted therapy, and a highly efficacious combinatory therapeutic strategy to treat EGFR-dependent; this may may extend the use of appropriate inhibitors beyond EGFR-mutated adenocarcinoma patients.L.P.A. was funded by the Comunidad de Madrid, CAM, (B2017/BMD3884), 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). I.F. is funded by the AECC (AIO2015) and Consejería de Igualdad, Salud y Políticas Sociales de la Junta de Andalucía (PI-0029-2013) and ISCIII (PI16/01311), and co-funded by FEDER from Regional Development European Funds (European Union). AC was supported by grants from the Spanish Ministry of Economy and Competitiveness Plan Estatal de I + D+I 2013–2016, ISCIII (PI15/00045) and CIBERONC (CD16/12/00275), and co-funded by FEDER from Regional Development European Funds (European Union). S.M.P. is funded by the Consejería de Salud y Bienestar Social (PI-0046-2012), the Fundación Mutua Madrileña (2014) and ISCIII (PI17/00033). A.Q is funded by the ISCIII (FI12/00429). L.O. is funded by the Ministerio de Educación, Cultura y Deporte (FPU13/02595).Peer reviewe

FGFR1 and FGFR4 oncogenicity depends on n-cadherin and their co-expression may predict FGFR-targeted therapy efficacy.

Fibroblast growth factor receptor (FGFR)1 and FGFR4 have been associated with tumorigenesis in a variety of tumour types. As a therapeutic approach, their inhibition has been attempted in different types of malignancies, including lung cancer, and was initially focused on FGFR1-amplified tumours, though with limited success. In vitro and in vivo functional assessments of the oncogenic potential of downregulated/overexpressed genes in isogenic cell lines were performed, as well as inhibitor efficacy tests in vitro and in vivo in patient-derived xenografts (PDXs). mRNA was extracted from FFPE non-small cell lung cancer samples to determine the prognostic potential of the genes under study. We provide in vitro and in vivo evidence showing that expression of the adhesion molecule N-cadherin is key for the oncogenic role of FGFR1/4 in non-small cell lung cancer. According to this, assessment of the expression of genes in different lung cancer patient cohorts showed that FGFR1 or FGFR4 expression alone showed no prognostic potential, and that only co-expression of FGFR1 and/or FGFR4 with N-cadherin inferred a poorer outcome. Treatment of high-FGFR1 and/or FGFR4-expressing lung cancer cell lines and patient-derived xenografts with selective FGFR inhibitors showed high efficacy, but only in models with high FGFR1/4 and N-cadherin expression. Our data show that the determination of the expression of FGFR1 or FGFR4 alone is not sufficient to predict anti-FGFR therapy efficacy; complementary determination of N-cadherin expression may further optimise patient selection for this therapeutic strategy.This work was funded by the Community of Madrid, the ISCIII co-funded by FEDER from Regional Development European Funds (European Union), the Spanish Ministry of Economy and Competitiveness, the Mutua Madrilena Foundation, the Ministry of Health and Social Welfare of Junta de Andalucia, the AECC scientific foundation and the Spanish Ministry of Education, Culture and Sport.S