Evaluation of the fibroblast growth factor receptor axis as potential therapy target in malignant pleural mesothelioma

Das maligne Pleuramesotheliom (MPM) ist eine aggressive, Asbest-assoziierte Krebsart mit einer generell sehr schlechten Prognose. Bis heute gibt es nur wenige therapeutische Optionen, da Chemo- und Radiotherapie häufig nicht anschlagen. Fibroblasten Wachstumsfaktoren (FGF) und deren Rezeptoren (FGFR) regulieren Wachstum, Wanderungsverhalten und Überleben von Zellen und sind an wichtigen physiologischen Prozessen aber auch an der Entstehung und dem Fortschreiten von Krebs beteiligt. Der FGF Signalweg wurde bereits als potentielles Target für neuartige Therapien in diverse Krebsarten in Erwägung gezogen, aber bisher noch nicht für das maligne Pleuramesotheliom. Ziele: Daher war das Ziel dieser Studie die Evaluierung des FGF/FGFR Signalsystems als mögliches „Therapietarget“ auch für MPM durch eine Analyse der FGF und FGFR Expression, sowie des Einflusses einer Blockade des FGFR1 auf Erscheinungsbild, Wachstum, Spheroidbildung, intrazelluläre Signalkaskaden sowie Sensitivität gegenüber Chemo- und Radiotherapie auf MPM Zellen. Methoden: Die Expression aller vier FGFRs samt Isoformen und der 22 FGFs wurde mittels konventioneller PCR, qRT-PCR, Immunfluoreszenz, genetischen Reporterkonstrukten in MPM Zellinien (n=9) und teilweise durch immunhistochemische Färbungen in Tumorgewebeproben (N=60) nachgewiesen. Zur Stimulierung beziehungsweise Blockierung des FGF Signalwegs wurden rekombinantes FGF2, die synthetischen Inhibitoren SU5402 und PD166866 sowie ein dominant-negativer FGFR1-Adenovirus verwendet. Zellwachstum und Wanderung wurde durch MTT, Clonogenic, Platypus und Transwell Assays ermittelt beziehungsweise durch Färbung mit Hoechst/PI und mikroskopische Betrachtung. Änderungen in intrazellulären Signalwegen wurden mittels Western Blot Analyse dargestellt. Resultate: Die Expressionsanalyse ergab eine starke Überexpression des FGFR1 im Bezug auf die anderen Rezeptoren und die Kontrollzelllinie, sowie eine hohe Expression von FGF2 und FGF18, sowohl in den Zelllinien als auch in Tumorgewebeproben. Die Blockierung des FGFR1 durch spezifische Inhibitoren sowie ein Adenoviruskonstrukt führte zu beträchtlich vermindertem Zellwachstum, Stimulation mit FGF2 hingegen zu einer Erhöhung sowie zu drastischen morphologischen Änderungen. Ähnliche Effekte wurden auch im Bezug auf das Wanderungsverhalten von MPM Zellen beobachtet, begleitet von Änderungen in intrazellulären Signaltransduktionskaskaden. Weiters resultierte die Inhibierung des FGFR1 in stark verminderter Spheroidbildung, das Weglassen von FGF2 hingegen zu weniger dramatischen Effekten. Kombination von FGFR Inhibierung mit herkömmlicher Chemo-und Radiotherapie führte in den meisten Fällen zu einer Verstärkung der Wirkung. Schlussfolgerung: Unsere Daten zeigen, dass der FGF Signalweg in Mesotheliomzellen eine wichtige Rolle für Wachstum, Überleben, Wanderungsverhalten sowie Chemo-und Strahlenresistenz spielt und es daher sinnvoll scheint, die Blockierung dieser Signale für potenzielle neue MPM Therapien weiter zu untersuchen.Malignant pleural mesothelioma (MPM) is a highly aggressive asbestos-related malignancy characterized by frequent resistance to chemo- and radiotherapy with poor outcome and limited therapeutic options. Fibroblast growth factors (FGF) and their receptors (FGFR) comprise a signaling system that is involved in essential physiologic processes, but is also critically involved in tumor development and progression by regulating cell growth, migration and survival. The FGFR signaling pathway has been identified as potential therapy target in several tumor types but has not been systematically investigated in MPM so far. Objectives: The purpose of this study was to provide a systematic analysis of the expressed FGF as well as FGFR molecules in MPM and to investigate the impact of blocking FGFR-mediated signals on MPM cell morphology, growth, spheroid formation, migration, invasion, signaling pathways and sensitivity to irradiation and chemotherapeutic agents to evaluate the FGF/FGFR axis as potential therapy target in MPM. Methods: Expression of all four FGFRs including different isoforms and 22 FGFs was analyzed in a panel of MPM cell lines (n=9) via conventional PCR, qRT-PCR, immunoflourescence and genetic reporter constructs as well as by immunohistochemistry in tissue samples (n=60). The FGFR pathway was stimulated/blocked using recombinant FGF2, a dominant-negative FGFR1 adenoviral construct and the specific small-molecule FGFR inhibitors SU5402 and PD166866. Cell growth, spheroid formation, migration and invasion were assessed by MTT, clonogenic, platypus and transwell assay as well as Hoechst/PI staining and microscopic examination. Changes in FGFR-mediated signaling were evaluated by western blot analysis. Results: Expression analysis revealed high overexpression of FGFR1 in all investigated MPM cell lines compared to the other receptors and the control cell line. Concerning the expression of the ligands, high transcript levels of FGF2 and FGF18 were detected, whereas other FGFs were expressed at more moderate levels. Immunohistochemistry of human tissue samples derived from surgery also showed an overexpression of activated FGFR1, FGF2 and FGF18 compared to the surrounding tissue. Inhibition of FGFR1 by the specific Inhibitor PD166866 led to decreased proliferation, migration and invasion in all cell lines tested, which was further confirmed in selected cases by adenoviral expression of dnFGFR1. In contrast, stimulation with FGF2 showed remarkably increased migration and dramatic changes in morphology accompanied by distinct changes in cellular signal transduction pathways. Inhibition of FGFR signals also markedly reduced spheroid formation ability of MPM cell lines whereas FGF-free conditions showed a more moderate reduction. Combination of FGFR inhibition with irradiation or currently used chemotherapeutic agents e.g. cisplatin led to increased efficacy with respect to cell viability. Conclusion: Our data suggest that FGFR-mediated signals are important for proliferation, survival, migration, chemo- and radioresistance of mesothelioma cells and their inhibition should be further evaluated as a potential new treatment strategy in MPM

The optogenetic promise for oncology: Episode I

As light-based control of fundamental signaling pathways is becoming a reality, the field of optogenetics is rapidly moving beyond neuroscience. We have recently developed receptor tyrosine kinases that are activated by light and control cell proliferation, epithelial–mesenchymal transition, and angiogenic sprouting—cell behaviors central to cancer progression

Targeting receptor tyrosine kinases in malignant pleural mesothelioma: Focus on FGF-receptors

Fibroblast growth factor receptors (FGFRs) constitute a subfamily of receptor tyrosine kinases. Four different receptors, FGFR1-4, bind 18 different fibroblast growth factors (FGFs) and signal mainly along the mitogen-activated protein kinase (MAPK), the phosphatidylinositol 3 kinase (PI3K) and the phospholipase c gamma (PLC?) pathway. Physiologically, they are major regulators of embryonic development and metabolism. Deregulation of FGFR signals is increasingly recognized to play important roles in malignant diseases and may constitute a feasible therapeutic target. We recently investigated their role in malignant pleural mesothelioma (MPM), an aggressive malignancy mainly caused by asbestos exposure and with currently limited therapeutic options. We demonstrated high expression of several FGFs/FGFRs, especially FGFR1, FGF2 and FGF18 in cultured tumor cells and tissue specimens and identified FGFR-mediated signals as major driver of MPM cell growth, survival and migration. FGFR blockade by a tyrosine kinase inhibitor or by a dominant-negative receptor construct resulted in reduced MPM growth in vitro and in vivo and, furthermore, enhanced the efficacy of chemo- or radiotherapy. Several other receptor tyrosine kinases, including EGFR, MET and AXL were found to be overexpressed in MPM but translation into clinically successful therapeutic approaches has not yet been achieved. Inhibition of FGF-receptors may have the advantage of targeting both the tumor cells as well as the tumor vasculature and should be further evaluated

Unfolding the Secrets of Small Cell Lung Cancer Progression: Novel Approaches and Insights Through Rapid Autopsies

The understanding of small cell lung cancer (SCLC) biology has increased dramatically in recent years, but the processes that allow SCLC to progress rapidly remain poorly understood. Here, we advocate the integration of rapid autopsies and preclinical models into SCLC research as a comprehensive strategy with the potential to revolutionize current treatment paradigms

Characterization of the oncogenic potential of the fibroblast growth factor axis in malignant pleural mesothelioma

Myeloproliferative neoplasms (MPN) are a group of hematologic diseases, characterized by clonal hematopoiesis and elevated number of terminally differentiated blood cells. Polycythemia vera (PV), essential thrombocythemia and primary myelofibrosis are referred to as the three classical BCR-ABL1 negative MPN. The majority of patients suffering from these diseases have mutations in either JAK2, MPL or CALR. Therapies for MPN patients include low-dose aspirin, hydroxyurea, alkylating agents (such as pipobroman) and interferon alpha (IFNa). At present little is known about the genetic influence on MPN treatment outcomes. We performed in vitro screens for drugs used in MPN therapy to identify genes that play a role in therapy resistance. Significant hits were detected that are involved in apoptosis, ubiquitin and DNA damage response pathways for screens with alkylating agents. Moreover, our data suggested that similar resistance mechanisms against diverse alkylating agents could exist. We complemented in vitro findings with an in-depth genetic characterization of 47 MPN patients that developed hydroxyurea and pipobroman resistance. We observed chromosomal aberrations that are typically seen in chronic phase MPN. For six pipobroman resistant patients we performed whole exome sequencing and detected 2 to 17 somatic mutations per patient. Each of the identified somatic changes could potentially be involved in resistance mechanisms. As no recurrent mutation was found besides JAK2 mutations and most clones seemed stable over time, it was difficult to pinpoint exact resistance driver mutations. Furthermore, we evaluated the effect of chromosomal aberrations on IFNa treatment outcomes in a cohort of 51 PV patients. We detected a similar profile of cytogenetic lesions for responding and non-responding patients. These results suggested that responses to IFNa were achieved independent of type and number of chromosomal aberrations. Additionally, patients with complex karyotypes may equally profit from IFNa therapy. We next assessed whether germline variants could explain therapeutic outcomes of IFNa treatment. We identified a haplotype on chromosome 3q13.11 that significantly associated with molecular responses to IFNa therapy. If validated in an independent cohort, this haplotype represents the first marker for molecular response in MPN treatment, which will help to identify MPN patients that benefit most from IFNa therapy. We detected several genetic changes that could influence MPN therapy outcomes and a variety of factors seem to be involved in these processes, such as somatic mutations and germline variants. Future research will help to further dissect the genetic basis of treatment responses, which can support personalized, precision medicine in MPN.Myeloproliferative Neoplasien (MPN) sind eine Gruppe hämatologischer Erkrankungen, welche durch eine klonale Hämatopoese und eine erhöhte Anzahl terminal differenzierter Blutzellen charakterisiert ist. Polycythaemia vera (PV), essentielle Thrombozythämie und primäre Myelofibrose werden als die drei klassischen BCR-ABL1 negativen MPN bezeichnet. Die meisten Patienten, die an diesen Erkrankungen leiden, haben Mutationen in entweder JAK2, MPL oder CALR. Therapien für MPN Patienten inkludieren niedrig dosiertes Aspirin, Hydroxyurea, Alkylanzien (zum Beispiel Pipobroman) und Interferon Alpha (IFNa). Zurzeit ist über den genetischen Einfluss auf den Ausgang der verabreichten MPN Therapien wenig bekannt. Wir führten in vitro „Screens” für Medikamente die zur MPN Therapie verwendet werden durch, um Gene zu identifizieren die in der Therapieresistenz eine Rolle spielen. Aus den „Screens“ mit Alkylanzien wurden signifikante Hits detektiert, die im Apoptose-, Ubiquitin- und DNA-Reparatur-Signalweg involviert sind. Zudem deuteten unsere Daten an, dass ähnliche Resistenzmechanismen gegen verschiedene Alkylanzien existieren könnten. Wir ergänzten die in vitro Ergebnisse mit einer detaillierten genetischen Charakterisierung von 47 MPN Patienten, die Hydroxyurea- und Pipobromanresistenzen entwickelten. Wir fanden chromosomale Veränderungen die typisch für die chronische-MPN-Phase sind. Für sechs Pipobroman-resistente Patienten führten wir „whole exome“ Sequenzierungen durch und detektierten 2 bis 17 somatische Mutationen pro Patient. Jede der identifizierten somatischen Veränderungen könnte potentiell in Resistenzmechanismen involviert sein. Da außer JAK2 Mutationen keine wiederkehrende Mutation gefunden wurde und die meisten Klone über die Zeit stabil erschienen, war es schwierig eine exakte resistenzverursachende Mutation festzulegen. Des Weiteren evaluierten wir in einer Kohorte von 51 PV Patienten den Effekt von chromosomalen Veränderungen auf den IFNa-Therapieausgang. Wir detektierten ein ähnliches Profil an zytogenetischen Läsionen für therapieansprechende und nichtansprechende Patienten. Diese Ergebnisse deuteten darauf hin, dass ein Ansprechen auf die IFNa-Therapie unabhängig von der Art und Anzahl chromosomaler Veränderungen erreicht wurde. Zusätzlich könnten Patienten mit einem komplexen Karyotyp ebenso von einer IFNa-Therapie profitieren. Als Nächstes bestimmten wir, ob Keimbahnvarianten den therapeutischen Ausgang von IFNa erklären könnten. Wir identifizierten einen Haplotyp auf Chromosom 3q13.11, der signifikant mit der molekularen Auswirkung der IFNa-Therapie assoziiert war. Falls dieser in einer unabhängigen Kohorte validiert werden kann, repräsentiert dieser Haplotyp den ersten Marker für das molekulare Ansprechen auf eine Therapie in MPN, dies wird dabei helfen Patienten zu identifizieren, die am meisten von einer IFNa-Therapie profitieren. Wir detektierten mehrere genetische Veränderungen, die die MPN Therapieausgänge beeinflussen könnten und eine Vielfalt an Faktoren scheint in diesen Prozessen involviert zu sein, zum Beispiel somatische Mutationen und Keimbahnvarianten. Die zukünftige Forschung wird dabei helfen die genetische Grundlage der Therapieausgänge weiter zu verstehen, was die personalisierte Präzisionsmedizin für MPN Patienten unterstützen kann.submitted by Karin SchelchZsfassung in dt. SpracheWien, Med. Univ., Diss., 2015OeBB(VLID)522273

Targeting receptor tyrosine kinases in malignant pleural mesothelioma: Focus on FGF-receptors

Fibroblast growth factor receptors (FGFRs) constitute a subfamily of receptor tyrosine kinases. Four different receptors, FGFR1-4, bind 18 different fibroblast growth factors (FGFs) and signal mainly along the mitogen-activated protein kinase (MAPK), the phosphatidylinositol 3 kinase (PI3K) and the phospholipase c gamma (PLCγ) pathway. Physiologically, they are major regulators of embryonic development and metabolism. Deregulation of FGFR signals is increasingly recognized to play important roles in malignant diseases and may constitute a feasible therapeutic target. We recently investigated their role in malignant pleural mesothelioma (MPM), an aggressive malignancy mainly caused by asbestos exposure and with currently limited therapeutic options. We demonstrated high expression of several FGFs/FGFRs, especially FGFR1, FGF2 and FGF18 in cultured tumor cells and tissue specimens and identified FGFR-mediated signals as major driver of MPM cell growth, survival and migration. FGFR blockade by a tyrosine kinase inhibitor or by a dominant-negative receptor construct resulted in reduced MPM growth in vitro and in vivo and, furthermore, enhanced the efficacy of chemo- or radiotherapy. Several other receptor tyrosine kinases, including EGFR, MET and AXL were found to be overexpressed in MPM but translation into clinically successful therapeutic approaches has not yet been achieved. Inhibition of FGF-receptors may have the advantage of targeting both the tumor cells as well as the tumor vasculature and should be further evaluated

Transcriptional suppression of the miR-15/16 family by c-Myc in malignant pleural mesothelioma

MicroRNA downregulation is frequent in malignant pleural mesothelioma (MPM), but the mechanisms responsible for loss of miR-15/16 and miR-193a are yet to be elucidated and were investigated in this study. Copy Number Variation (CNV) of microRNA-coding genes was analyzed in MPM cells by digital droplet PCR (ddPCR) and revealed heterozygous loss of miR-193a and miR-15a/16-1, but no change in miR-15b/16-2. Epigenetic control of microRNA expression was inferred following decitabine and Trichostatin A (TSA) treatment which did not substantially affect microRNA expression. Knockdown of c-Myc expression led to upregulation of SMC4, miR-15b and 16, and to a lesser extent DLEU2 and miR-15a, whereas c-Myc overexpression repressed microRNA expression. Chromatin immunoprecipitation (ChIP) assays confirmed the interaction of c-Myc with the DLEU2 and SMC4 promoters. Tumor microRNA expression was determined in samples from MPM patients, with samples of pleura from cardiac surgery patients used as controls. In tumor samples, a strong correlation was observed between the expression of miR-15b and 16 (R$^{2}$=0.793), but not miR-15a and 16. Our data suggest that in MPM, the downregulation of miR-15/16 is due to transcriptional repression by c-Myc, primarily via control of the miR-15b/16-2 locus, while miR-193a-3p loss is due to genomic deletion

A link between the fibroblast growth factor axis and the miR‐16 family reveals potential new treatment combinations in mesothelioma

Malignant pleural mesothelioma (MPM) is an aggressive malignancy with very limited therapeutic options. Fibroblast growth factor (FGF) signals play important roles in mesothelioma cell growth. Several FGFs and FGF receptors (FGFRs) are predicted targets of the miR‐15/16 family, which is downregulated in MPM. The aim of this study was to explore the link between the miR‐15/16 family and the FGF axis in MPM. Expression analyses via RT‐qPCR showed downregulation of the FGF axis after transfection with miR‐15/16 mimics. Direct interaction was confirmed by luciferase reporter assays. Restoration of miR‐15/16 led to dose‐dependent growth inhibition in MPM cell lines, which significantly correlated with their sensitivity to FGFR inhibition. Treatment with recombinant FGF2 prevented growth inhibition and further reduced the levels of FGF/R‐targeting microRNAs, indicating a vicious cycle between miR‐15/16 down‐ and FGF/FGFR signaling upregulation. Combined inhibition of two independent miR‐15/16 targets, the FGF axis and Bcl‐2, resulted in additive or synergistic activity. Our data indicate that post‐transcriptional repression of FGF‐mediated signals contributes to the tumor suppressor function of the microRNA‐15/16 family. Inhibiting hyperactivated FGF signals and Bcl‐2 might serve as a novel therapeutic combination strategy in MPM

Expression of FGFR1-4 in malignant pleural mesothelioma tissue and corresponding cell lines and its relationship to patient survival and FGFR inhibitor sensitivity

Malignant pleural mesothelioma (MPM) is a devastating malignancy with limited therapeutic options. Fibroblast growth factor receptors (FGFR) and their ligands were shown to contribute to MPM aggressiveness and it was suggested that subgroups of MPM patients could benefit from FGFR-targeted inhibitors. In the current investigation, we determined the expression of all four FGFRs (FGFR1-FGFR4) by immunohistochemistry in tissue samples from 94 MPM patients. From 13 of these patients, we were able to establish stable cell lines, which were subjected to FGFR1-4 staining, transcript analysis by quantitative RT-PCR, and treatment with the FGFR inhibitor infigratinib. While FGFR1 and FGFR2 were widely expressed in MPM tissue and cell lines, FGFR3 and FGFR4 showed more restricted expression. FGFR1 and FGFR2 showed no correlation with clinicopathologic data or patient survival, but presence of FGFR3 in 42% and of FGFR4 in 7% of patients correlated with shorter overall survival. Immunostaining in cell lines was more homogenous than in the corresponding tissue samples. Neither transcript nor protein expression of FGFR1-4 correlated with response to infigratinib treatment in MPM cell lines. We conclude that FGFR3 and FGFR4, but not FGFR1 or FGFR2, have prognostic significance in MPM and that FGFR expression is not suffcient to predict FGFR inhibitor response in MPM cell lines