The EPHB6 receptor tyrosine kinase is a metastasis suppressor that is frequently silenced by promoter DNA hypermethylation in non-small cell lung cancer.
OBJECTIVE
Loss of EPHB6 receptor tyrosine kinase expression in early-stage non-small cell lung carcinoma (NSCLC) is associated with the subsequent development of distant metastasis. Here, we analyzed the regulation and function of EPHB6 in lung cancer metastasis.


METHODS
The expression levels of EPHB6 were compared among normal lung tissue (n = 9), NSCLC without metastasis (n = 39), and NSCLC with metastasis (n = 39) according to the history of the patients. In addition, EPHB6 expression levels of matched tumor-normal pairs from 24 NSCLC patients were analyzed. The promoter DNA methylation status and its association with the expression levels of EPHB6 were determined among 14 pairs of tumor-normal samples. Metastatic potential of EPHB6 was assessed in vitro and in vivo in a metastasis mouse model. Overexpression and RNA interference (RNAi) approaches were used for analysis of the biological functions of EPHB6.


RESULTS
EPHB6 mRNA and protein levels were significantly reduced in NSCLC tumors compared with matched normal lung tissue. Decreased EPHB6 expression levels were associated with an increased risk for metastasis development in NSCLC patients. Loss of expression correlated with EPHB6 hypermethylation. EPHB6 expression was induced by 5-aza-2'-deoxycytidine treatment in an NSCLC cell line. Restoration of EPHB6 expression in lung adenocarcinoma cells increased adhesion and decreased migration. Reexpression of EPHB6 in lung cancer cells almost entirely abolished metastasis formation in non obese diabetic (NOD)/severe combined immunodeficient mice.


CONCLUSIONS
Taken together, these analyses show that EPHB6 is a metastasis inhibitory gene that is frequently silenced by hypermethylation of its promoter in NSCLC.
Yu
Jun
J
Department of Medicine A--Hematology, Oncology and Pneumology, University of Münster, Münster, Germany.
Bulk
Etmar
E
Ji
Ping
P
Hascher
Antje
A
Tang
Moying
M
Metzger
Ralf
R
Marra
Alessandro
A
Serve
Hubert
H
Berdel
Wolfgang E
WE
Wiewroth
Rainer
R
Koschmieder
Steffen
S
Müller-Tidow
Carsten
C
D000818:Animals; D015153:Blotting, Western; D002289:Carcinoma, Non-Small-Cell Lung / Q000235:genetics* / Q000378:metabolism / Q000556:secondary; D045744:Cell Line, Tumor; D018899:CpG Islands; D019175:DNA Methylation; D044127:Epigenesis, Genetic; D005260:Female; D020868:Gene Silencing; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D011379:Prognosis; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics* / Q000378:metabolism; D036081:Receptors, Eph Family; D020133:Reverse Transcriptase Polymerase Chain Reaction
D012333:RNA, Messenger; C106797:EPHB6 protein, human; D020794:Receptor Protein-Tyrosine Kinases; D036081:Receptors, Eph Family