Fibulin 2 Is Hypermethylated and Suppresses Tumor Cell Proliferation through Inhibition of Cell Adhesion and Extracellular Matrix Genes in Non-Small Cell Lung Cancer

Fibulins (FBLNs), interacting with cell adhesion receptors and extracellular matrix (ECM) components, play multiple roles in ECM structures and tissue functions. Abnormal expression of FBLN2, one of the fibulin family members, contributes to tumor initiation and development. However, the function of FBLN2 in human non-small cell lung cancer (NSCLC) has not yet been elucidated. In this study, we found that FBLN2 was downregulated in 9 out of 11 lung cancer cell lines compared to normal bronchial epithelial cells, which was associated with DNA hypermethylation. Primary lung squamous cell carcinoma expressed significantly more FBLN2 protein compared to adenocarcinoma ( p = 0.047). Ectopic expression of FBLN2 led to decreased cell proliferation, migration and invasion, accompanied by inactivated MAPK/ERK and AKT/mTOR pathways, while FBLN2 siRNA knockdown resulted in an opposite biological behaviour in NSCLC cells. Additionally, overexpression of FBLN2 led to dysregulation of cell adhesion molecules, ECM markers and a panel of lysate/exosome-derived-microRNAs, which are involved in cell adhesion and ECM remodelling. Taken together, our data indicate that FBLN2 is methylated and exerts a tumor suppressor function through modulation of MAPK/ERK and AKT pathways and regulation of cell adhesion and ECM genes. Moreover, FBLN2 might be a potential biomarker for the sub-classification of NSCLC

Epithelial Membrane Protein 2 Suppresses Non-Small Cell Lung Cancer Cell Growth by Inhibition of MAPK Pathway

Epithelial membrane proteins (EMP1-3) are involved in epithelial differentiation and carcinogenesis. Dysregulated expression of EMP2 was observed in various cancers, but its role in human lung cancer is not yet clarified. In this study, we analyzed the expression of EMP1-3 and investigated the biological function of EMP2 in non-small cell lung cancer (NSCLC). The results showed that lower expression of EMP1 was significantly correlated with tumor size in primary lung tumors (p = 0.004). Overexpression of EMP2 suppressed tumor cell growth, migration, and invasion, resulting in a G1 cell cycle arrest, with knockdown of EMP2 leading to enhanced cell migration, related to MAPK pathway alterations and disruption of cell cycle regulatory genes. Exosomes isolated from transfected cells were taken up by tumor cells, carrying EMP2-downregulated microRNAs (miRNAs) which participated in regulation of the tumor microenvironment. Our data suggest that decreased EMP1 expression is significantly related to increased tumor size in NSCLC. EMP2 suppresses NSCLC cell growth mainly by inhibiting the MAPK pathway. EMP2 might further affect the tumor microenvironment by regulating tumor microenvironment-associated miRNAs

Funktionsanalyse des epithelialen Membranproteins 2 (EMP2) in menschlichen Lungenkrebszellen

The epithelial membrane proteins (EMP1-3), which belong to the family of peripheral myelin proteins 22-kDa (PMP22), are involved in epithelial differentiation. EMP2 was found to be a downstream target gene of the tumor suppressor gene HOPX, a homeobox-containing gene. Additionally, a dysregulation of EMP2 has been observed in various cancers, but the function of EMP2 in human lung cancer has not yet been clarified. In this study, a real-time RT-PCR, Western blot and cytoblock analysis were performed to analyze the expression of EMP2. Gain-of-function was achieved by stable transfection with an EMP2 expression vector and loss-of-function by siRNA knockdown. Stable transfection led to overexpression of EMP2 at both mRNA and protein levels in the transfected cell lines H1299 and H2170. Functional assays including proliferation, colony formation, migration and invasion assays as well as cell cycle analyzes were performed after stable transfection and it was found that the ectopic EMP2 expression resulted in a reduced cell proliferation, migration and invasion as well as a G1 cell cycle arrest. After the EMP2 gene was silenced by the siRNA knockdown, inhibition of the cell invasive property was observed. These phenomena were accompanied by reduced AKT, mTor and p38 activities. Taken together, the data suggest that the epithelial membrane protein 2 (EMP2) is a tumor suppressor and exerts its tumor suppressive function by inhibiting AKT and MAPK signaling pathways in human lung cancer cells.Die Epithel-Membranproteine (EMP1-3), die zur Genfamilie der peripheren Myelinproteine 22-kDa (PMP22) gehören, sind an der epithelialen Differenzierung beteiligt. Es wurde festgestellt, dass das EMP2 ein Zielgen des Tumorsuppressorgens HOPX ist. Außerdem wurde eine Dysregulation von EMP2 bei verschiedenen Krebsarten beobachtet, jedoch ist die Funktion von EMP2 in menschlichem Lungenkrebs noch nicht geklärt. In dieser Studie wurden neben einer real-time RT-PCR auch ein Western Blot und eine Cytoblock-Analyse durchgeführt, um die Expression von EMP2 zu analysieren. Die Überexpression wurde durch stabile Transfektion mit einem EMP2-Expressionsvektor und der Genverlusst durch siRNA Knockdown erzielt. Die stabile Transfektion führte zu einer Überexpression von EMP2 sowohl auf mRNA- als auch auf Proteinebene in den transfizierten Zelllinien H1299 und H2170. Funktionelle Assays einschließlich Proliferations-, Koloniebildungs-, Migrations- und Invasionsassays sowie Zellzyklusanalysen wurden nach stabiler Transfektion durchgeführt und dabei stellte sich heraus, dass die ektopische EMP2-Expression zu einer reduzierten Zellproliferation, Migration und Invasion sowie zu einem Rückgang des G1-Zellzyklus führte. Nachdem das EMP2-Gen durch den siRNA Knockdown stillgelegt wurde, konnte eine Hemmung der zellinvasiven Eigenschaft beobachtet werden. Diese Phänomene wurden von reduzierten AKT-, mTor- und p38-Aktivitäten begleitet. Zusammengenommen deuten die Daten darauf hin, dass das Epithel-Membranprotein 2 ein Tumorsuppressor ist und seine tumorsuppressive Funktion durch die Hemmung der AKT- und MAPK-Signalwege in menschlichen Lungenkrebszellen ausübt

Epithelial Membrane Protein 2 Suppresses Non-Small Cell Lung Cancer Cell Growth by Inhibition of MAPK Pathway

Epithelial membrane proteins (EMP1-3) are involved in epithelial differentiation and carcinogenesis. Dysregulated expression of EMP2 was observed in various cancers, but its role in human lung cancer is not yet clarified. In this study, we analyzed the expression of EMP1-3 and investigated the biological function of EMP2 in non-small cell lung cancer (NSCLC). The results showed that lower expression of EMP1 was significantly correlated with tumor size in primary lung tumors (p = 0.004). Overexpression of EMP2 suppressed tumor cell growth, migration, and invasion, resulting in a G1 cell cycle arrest, with knockdown of EMP2 leading to enhanced cell migration, related to MAPK pathway alterations and disruption of cell cycle regulatory genes. Exosomes isolated from transfected cells were taken up by tumor cells, carrying EMP2-downregulated microRNAs (miRNAs) which participated in regulation of the tumor microenvironment. Our data suggest that decreased EMP1 expression is significantly related to increased tumor size in NSCLC. EMP2 suppresses NSCLC cell growth mainly by inhibiting the MAPK pathway. EMP2 might further affect the tumor microenvironment by regulating tumor microenvironment-associated miRNAs