KLF6 Gene and early melanoma development in a collagen I-rich extracellular environment.
BACKGROUND
A putative tumor suppressor gene at chromosome 10p15, which contains KLF6 and other genes, is predicted to be lost during melanoma development, and its identity is unknown. In this study, we investigated the biological roles and identity of this tumor suppressor gene.


METHODS
The human UACC 903 melanoma cell line containing introduced DNA fragments from the 10p15 region with (10E6/3, 10E6/11, and 10E6/18) and without (10ER4S.2/1) the tumor suppressor gene was used. Xenograft tumors were generated in a total of 40 mice with melanoma cell lines, and tumor size was measured. Cells were cultured on plastic or a gel of type I collagen. Viability, proliferation, and apoptosis were assessed. Expression of KLF6 protein was assessed by immunohistochemistry and immunoblot analysis. Expression of phosphorylated Erk1/2 and cyclin D1 was assessed by immunoblot analysis. Protein expression of KLF6 was inhibited with small interfering RNA (siRNA). KLF6 protein expression was assessed in 17 human nevi and human melanoma specimens from 29 patients. Statistical analyses were adjusted for multiple comparisons by use of Dunnett method. All statistical tests were two-sided.


RESULTS
Melanoma cells containing KLF6 generated smaller subcutaneous xenograft tumors with fewer proliferating cells than control cells. When grown on collagen 1, viability of cells with ectopic KLF6 expression (72%) was lower than that of control cells (100%) (group difference = -28%, 95% confidence interval = -31.3% to -25.2%, P < .001). Viability of melanoma cells with or without the KLF6 tumor suppressor gene on plastic dishes was similar. When KLF6 expression was inhibited with KLF6 siRNA, viability of cells with the tumor suppressor gene on collagen I gel increased compared with that of control cells carrying scrambled siRNA. KLF6 protein was detected in all nevi examined but not in human metastatic melanoma tissue examined. Ectopic expression of KLF6 protein in melanoma cells grown on collagen I decreased levels of phosphorylated Erk1/2 and cyclin D1 in the mitogen-activated protein kinase signaling pathway.


CONCLUSIONS
In melanoma cells, the tumor suppressor gene at 10p15 appears to be KLF6. Signaling from the collagen I-rich extracellular matrix appears to be involved in the tumor suppressive activity of KLF6 protein.
Huh
Sung Jin
SJ
Department of Pharmacology, Pennsylvania State University College of Medicine, Hershey, PA 17033, USA.
Chen
Yen-Liang
YL
Friedman
Scott L
SL
Liao
Jason
J
Huang
H-J Su
HJ
Cavenee
Webster K
WK
Robertson
Gavin P
GP
D000328:Adult; D000368:Aged; D000818:Animals; D017209:Apoptosis / Q000235:genetics; D001483:Base Sequence; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002470:Cell Survival; D002879:Chromosomes, Human, Pair 10; D024042:Collagen Type I / Q000378:metabolism*; D019938:Cyclin D1 / Q000378:metabolism; D053938:DNA Fragmentation; D018076:DNA, Complementary / Q000096:biosynthesis; D005260:Female; D005780:Gelatin; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D000075723:Kruppel-Like Factor 6; D051741:Kruppel-Like Transcription Factors / Q000235:genetics* / Q000378:metabolism; D008297:Male; D020782:Matrix Metalloproteinases; D008545:Melanoma / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D046529:Microscopy, Electron, Transmission; D008875:Middle Aged; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D008969:Molecular Sequence Data; D009506:Nevus / Q000235:genetics; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D011518:Proto-Oncogene Proteins / Q000235:genetics* / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D015398:Signal Transduction; D012878:Skin Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D014183:Transplantation, Heterologous; D025521:Tumor Suppressor Proteins / Q000235:genetics* / Q000378:metabolism
D024042:Collagen Type I; D018076:DNA, Complementary; C105465:KLF6 protein, human; D000075723:Kruppel-Like Factor 6; D051741:Kruppel-Like Transcription Factors; D011518:Proto-Oncogene Proteins; D034741:RNA, Small Interfering; D025521:Tumor Suppressor Proteins; D019938:Cyclin D1; D005780:Gelatin; D048052:Mitogen-Activated Protein Kinase 3; D020782:Matrix Metalloproteinases