Investigation of SMAD2 and phospho-isoforms in non-small cell lung cancer

Eine abnormale TGFβ-Signalübertragung spielt eine entscheidende Rolle bei der Entwicklung und Progression von Lungenkrebs. Innerhalb des TGFβ-Signalwegs sind SMAD2/3-Proteine die Mediatoren der Signalantwort und direkte Substrate für C-terminale Phosphorylierungen durch den TGFβ-Rezeptor. Anschließend translozieren diese in den Zellkern und wirken als Transkriptionsfaktoren. Zusätzlich besitzen SMAD2/3-Proteine weitere Phosphorylierungsstellen innerhalb der Linker-Domäne, die durch intrazelluläre Kinasen (z. B. GSK3β, JNK) phosphoryliert werden. Linker-Domänen repräsentieren damit Schnittstellen, die den TGFβ-Signalweg mit anderen Signalkaskaden (z. B. MAPK) verbinden und zur Überwindung antiproliferativer Effekte beitragen könnten. Ausgehend von dieser Hypothese wurden SMAD2 Linker-Phosphorylierungen (pS2L) in verschiedenen NSCLC-Zelllinien und Lungenkrebsgeweben untersucht. Es zeigte sich, dass eine hohe Dichte pS2L-positiver Zellen mit einer kürzeren Gesamtüberlebenszeit assoziiert war. Darüber hinaus wurde in fünf NSCLC-Zelllinien und T-Zellen gesunder Spender eine stark phosphorylierte Subpopulation während der Mitose nachgewiesen. Eine Inhibition Zellzyklus-assoziierter Kinasen (CDKs) zeigte zudem eine Modulierbarkeit von pS2L. Aufgrund dieser Ergebnisse wurde eine Substitution von drei Serinresten innerhalb der SMAD2 Linker-Domäne in A549-Zellen (A549Lsub) vorgenommen, was eine verlangsamte Proliferation, Migration und erhöhte Abundanz einer alterantiven SMAD2 Spleißvariante zur Folge hatte. Anreicherungsanalysen differenziell exprimierter Gene zwischen A549Lsub und Wildtyp-Zellen ergaben u. a. einen Zusammenhang mit EGFR-TKI-Resistenz und Embryogenese. Die Ergebnisse weisen darauf hin, dass SMAD2 Phospho-Isoformen eine zentrale Rolle bei Lungenkrebs spielen und diverse zelluläre Prozesse beeinflussen

The multi-modal effect of the anti-fibrotic drug pirfenidone on NSCLC

Although immune checkpoint and targeted therapies offer remarkable benefits for lung cancer treatment, some patients do not qualify for these regimens or do not exhibit consistent benefit. Provided that lung cancer appears to be driven by transforming growth factor beta signaling, we investigated the single drug potency of Pirfenidone, an approved drug for the treatment of lung fibrosis. Five human lung cancer cell lines and one murine line were investigated for transforming growth factor beta inhibition via Pirfenidone by using flow cytometry, In-Cell western analysis, proliferation assays as well as comprehensive analyses of the transcriptome with subsequent bioinformatics analysis. Overall, Pirfenidone induced cell cycle arrest, down-regulated SMAD expression and reduced proliferation in lung cancer. Furthermore, cell stress pathways and pro-apoptotic signaling may be mediated by reduced expression of Survivin. A murine subcutaneous model was used to assess the in vivo drug efficacy of Pirfenidone and showed reduced tumor growth and increased infiltration of T cells and NK cells. This data warrant further clinical evaluation of Pirfenidone with advanced non-small cell lung cancer. The observed in vitro and in vivo effects point to a substantial benefit for using Pirfenidone to reactivate the local immune response and possible application in conjunction with current immunotherapies

DNA methylation profiles of bronchoscopic biopsies for the diagnosis of lung cancer

Background!#!Lung cancer is the leading cause of cancer-related death in most western countries in both, males and females, accounting for roughly 20-25% of all cancer deaths. For choosing the most appropriate therapy regimen a definite diagnosis is a prerequisite. However, histological characterization of bronchoscopic biopsies particularly with low tumor cell content is often challenging. Therefore, this study aims at (a) determining the value of DNA methylation analysis applied to specimens obtained by bronchoscopic biopsy for the diagnosis of lung cancer and (b) at comparing aberrantly CpG loci identified in bronchoscopic biopsy with those identified by analyzing surgical specimens.!##!Results!#!We report the HumanMethylation450-based DNA methylation analysis of paired samples of bronchoscopic biopsy specimens either from the tumor side or from the contralateral tumor-free bronchus in 37 patients with definite lung cancer diagnosis and 18 patients with suspicious diagnosis. A differential DNA methylation analysis between both biopsy sites of patients with definite diagnosis identified 1303 loci. Even those samples were separated by the set of 1303 loci in which histopathological analysis could not unambiguously define the dignity. Further differential DNA methylation analyses distinguished between SCLC and NSCLC. We validated our results in an independent cohort of 40 primary lung cancers obtained by open surgical resection and their corresponding controls from the same patient as well as in publically available DNA methylation data from a TCGA cohort which could also be classified with high accuracy.!##!Conclusions!#!Considering that the prognosis correlates with tumor stage at time of diagnosis, early detection of lung cancer is vital and DNA methylation analysis might add valuable information to reliably characterize lung cancer even in histologically ambiguous sample material