Ras promotes growth by alternative splicing-mediated inactivation of the KLF6 tumor suppressor in hepatocellular carcinoma.
OBJECTIVE
Hepatocellular carcinoma (HCC) is the fifth most prevalent cancer worldwide and the third most lethal. Dysregulation of alternative splicing underlies a number of human diseases, yet its contribution to liver cancer has not been explored fully. The Krüppel-like factor 6 (KLF6) gene is a zinc finger transcription factor that inhibits cellular growth in part by transcriptional activation of p21. KLF6 function is abrogated in human cancers owing to increased alternative splicing that yields a dominant-negative isoform, KLF6 splice variant 1 (SV1), which antagonizes full-length KLF6-mediated growth suppression. The molecular basis for stimulation of KLF6 splicing is unknown.


METHODS
In human HCC samples and cell lines, we functionally link oncogenic Ras signaling to increased alternative splicing of KLF6 through signaling by phosphatidylinositol-3 kinase and Akt, mediated by the splice regulatory protein ASF/SF2.


RESULTS
In 67 human HCCs, there is a significant correlation between activated Ras signaling and increased KLF6 alternative splicing. In cultured cells, Ras signaling increases the expression of KLF6 SV1, relative to full-length KLF6, thereby enhancing proliferation. Abrogation of oncogenic Ras signaling by small interfering RNA (siRNA) or a farnesyl-transferase inhibitor decreases KLF6 SV1 and suppresses growth. Growth inhibition by farnesyl-transferase inhibitor in transformed cell lines is overcome by ectopic expression of KLF6 SV1. Down-regulation of the splice factor ASF/SF2 by siRNA increases KLF6 SV1 messenger RNA levels. KLF6 alternative splicing is not coupled to its transcriptional regulation.


CONCLUSIONS
Our findings expand the role of Ras in human HCC by identifying a novel mechanism of tumor-suppressor inactivation through increased alternative splicing mediated by an oncogenic signaling cascade.
Yea
Steven
S
Division of Liver Diseases and Department of Medicine, Mount Sinai School of Medicine, New York, New York, USA.
Narla
Goutham
G
Zhao
Xiao
X
Garg
Rakhi
R
Tal-Kremer
Sigal
S
Hod
Eldad
E
Villanueva
Augusto
A
Loke
Johnny
J
Tarocchi
Mirko
M
Akita
Kunihara
K
Shirasawa
Senji
S
Sasazuki
Takehiko
T
Martignetti
John A
JA
Llovet
Josep M
JM
Friedman
Scott L
SL
D015153:Blotting, Western; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D015972:Gene Expression Regulation, Neoplastic; D011905:Genes, ras / Q000235:genetics*; D006801:Humans; D000075723:Kruppel-Like Factor 6; D051741:Kruppel-Like Transcription Factors / Q000235:genetics* / Q000378:metabolism; D008113:Liver Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D009154:Mutation; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D020033:Protein Isoforms; D011518:Proto-Oncogene Proteins / Q000235:genetics* / Q000378:metabolism; D012334:RNA, Neoplasm / Q000235:genetics*; D015398:Signal Transduction; D025521:Tumor Suppressor Proteins
C105465:KLF6 protein, human; D000075723:Kruppel-Like Factor 6; D051741:Kruppel-Like Transcription Factors; D020033:Protein Isoforms; D011518:Proto-Oncogene Proteins; D012334:RNA, Neoplasm; D025521:Tumor Suppressor Proteins