Search

Chungyoul Choe,1,* Hyewon Kim,2,* Sol Min,2 Sangkyu Park,2,3 Jeongmin Seo,2,3 Sangho Roh2 1Samsung Biomedical Research Institute, Samsung Medical Center, Sungkyunkwan University, School of Medicine, Seoul, Republic of Korea; 2Laboratory of Cellular Reprogramming and Embryo Biotechnology, School of Dentistry, Seoul National University, Seoul, Republic of Korea; 3Biomedical Research Institute, NeoRegen Biotech Co., Ltd., Gyeonggi-do, Korea *These authors contributed equally to this work Background: Non-small cell lung cancer (NSCLC) is difficult to treat successfully. This intractability is mainly due to the cancer progressing through invasion, metastasis, chemotherapeutic resistance and relapse. Stemness has been linked to the various steps of cancer progression in a variety of tumors, yet little is known regarding its role in NSCLC. Purpose: In this study, we sought to determine the role of SOX2, a master regulator of pluripotency, in the growth of extracellular matrix (ECM)-detached cells during cancer progression. Methods: We established a three-dimensional (3D) Poly-2-hydroxyethyl methacrylate (poly-HEMA) culture of lung adenocarcinoma (LUAD) A549 cells as an ECM-detached cell growth model and examined the role of stemness genes using siRNA and small molecule inhibitor in comparison to standard two dimensional (2D) culture. Results: In poly-HEMA culture, A549 cells formed substratum-detached spheroids with characteristics of intermediate epithelial to mesenchymal transition (EMT) and exhibited greater expression of SOX2 than did control 2D cells. Knockdown of SOX2 markedly suppressed the growth of A549 cell aggregates in poly-HEMA culture conditions and furthermore increased their sensitivity to the anticancer drug vinblastine with concomitant downregulation of the activity of the anti-apoptotic AKT kinase. Interestingly, a small molecule, RepSox, which replaces SOX2, stimulated A549 cell growth in poly-HEMA 3D culture condition. Conclusion: Our findings strongly indicate that SOX2 contributes to anchorage-independent growth and chemoresistance via its downstream signaling mediator AKT kinase during the disease progression of NSCLC. SOX2 may therefore be an invaluable therapeutic target of NSCLC. Keywords: SOX2, lung cancer, NSCLC, EMT, stemness, vinblastine

Chungyoul Choe,1,* Yong-Sung Shin,1,* Changhoon Kim,2 So-Jung Choi,1 Jinseon Lee,1 So Young Kim,1 Yong Beom Cho,3 Jhingook Kim1,41Samsung Biomedical Research Institute, Samsung Medical Center Sungkyunkwan University, School of Medicine, 2Department of Biomedical Science, Graduate School of Biomedical & Engineering, Hanyang University, 3Department of Surgery, 4Department of Thoracic Surgery, Samsung Medical Center, Sungkyunkwan University, School of Medicine, Seoul, Republic of Korea*These authors contributed equally to this workAbstract: Although lung cancers with activating mutations in the epidermal growth factor receptor (EGFR) are highly sensitive to selective EGFR tyrosine kinase inhibitors (TKIs), these tumors invariably develop acquired drug resistance. Host stromal cells have been found to have a considerable effect on the sensitivity of cancer cells to EGFR TKIs. Little is known, however, about the signaling mechanisms through which stromal cells contribute to the response to EGFR TKI in non-small cell lung cancer. This work examined the role of hedgehog signaling in cancer-associated fibroblast (CAF)-mediated resistance of lung cancer cells to the EGFR TKI erlotinib. PC9 cells, non-small cell lung cancer cells with EGFR-activating mutations, became resistant to the EGFR TKI erlotinib when cocultured in vitro with CAFs. Polymerase chain reaction and immunocytochemical assays showed that CAFs induced epithelial to mesenchymal transition phenotype in PC9 cells, with an associated change in the expression of epithelial to mesenchymal transition marker proteins including vimentin. Importantly, CAFs induce upregulation of the 7-transmembrane protein smoothened, the central signal transducer of hedgehog, suggesting that the hedgehog signaling pathway is active in CAF-mediated drug resistance. Indeed, downregulation of smoothened activity with the smoothened antagonist cyclopamine induces remodeling of the actin cytoskeleton independently of Gli-mediated transcriptional activity in PC9 cells. These findings indicate that crosstalk with CAFs plays a critical role in resistance of lung cancer to EGFR TKIs through induction of the epithelial to mesenchymal transition and may be an ideal therapeutic target in lung cancer.Keywords: CAFs, lung cancer, NSCLC, direct coculture, hedgehog signaling, EMT, actin, EGFR TKIs

Creatine buffers cellular ATP via the creatine kinase reaction. Creatine levels are reduced in heart failure, but their contribution to pathophysiology is unclear. Arginine:glycine amidinotransferase (AGAT) in the kidney catalyses both the first step in creatine biosynthesis as well as homoarginine synthesis. AGAT-/- mice fed a creatine-free diet have a whole body creatine-deficiency. We hypothesised that AGAT-/- mice would develop cardiac dysfunction and rescue by dietary creatine would imply causality.Withdrawal of dietary creatine in AGAT-/- mice provided an estimate of myocardial creatine efflux of ∼2.7%/day, however, in vivo cardiac function was maintained despite low levels of myocardial creatine. Using AGAT-/- mice naïve to dietary creatine we confirmed absence of phosphocreatine in the heart, but crucially, ATP levels were unchanged. Potential compensatory adaptations were absent, AMPK was not activated and respiration in isolated mitochondria was normal. AGAT-/- mice had rescuable changes in body water and organ weights suggesting a role for creatine as a compatible osmolyte. Creatine-naïve AGAT-/- mice had haemodynamic impairment with low LV systolic pressure and reduced inotropy, lusitropy and contractile reserve. Creatine supplementation only corrected systolic pressure despite normalisation of myocardial creatine. AGAT-/- mice had low plasma homoarginine and supplementation completely rescued all other haemodynamic parameters. Contractile dysfunction in AGAT-/- was confirmed in Langendorff perfused hearts and in creatine-replete isolated cardiomyocytes, indicating that homoarginine is necessary for normal cardiac function.Our findings argue against low myocardial creatine per se as a major contributor to cardiac dysfunction. Conversely, we show that homoarginine deficiency can impair cardiac function, which may explain why low homoarginine is an independent risk factor for multiple cardiovascular diseases.