Ras Promotes Growth by Alternative Splicing-Mediated Inactivation of the KLF6 Tumor Suppressor in Hepatocellular Carcinoma

Scott L. Friedman,Josep M. Llovet,Mirko Tarocchi,Xiao Zhao,Goutham Narla,Takehiko Sasazuki,Sigal Tal–Kremer,Johnny Loke,Kunihara Akita,Steven Yea,Augusto Villanueva,Rakhi Garg,John A. Martignetti,Eldad Hod,Senji Shirasawa

doi:10.1053/j.gastro.2008.02.015

Scott L. Friedman, Josep M. Llovet

Open Access

https://doi.org/10.1053/j.gastro.2008.02.015

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Abstract

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. 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. 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. 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.

Highlights

Alternative splicing is a nuclear process that contributes to expanded protein diversity from a limited number of genes
When the level of H-Ras was compared against Krüppel-like Factor 6 (KLF6) splicing by non-parametric chi-square analysis, there was a significant correlation between elevated H-Ras expression and increased KLF6 alternative splicing, supporting an association between Ras activation and KLF6 splicing (Table 1)
We propose a novel mechanism of tumor suppressor inactivation in liver cancer via Rasmediated enhanced alternative splicing of KLF6 into growth-promoting isoforms

Summary

Introduction

Alternative splicing is a nuclear process that contributes to expanded protein diversity from a limited number of genes. Dysregulation of alternative splicing may contribute to degenerative, developmental, and malignant diseases[1,2,3]. Human cancers display alternatively spliced gene products in the absence of genomic mutations, pinpointing the splicing machinery as a cause of disease[1,2,3]. Other examples where alternatively spliced transcripts are increased in human cancers include LKB1 (premature termination codon), KIT (aberrant splicing leading to constitutively activated receptor tyrosine kinase), CDH17 (unknown mechanism), and BRCA1 (exon skipping)[4]. Dysregulation of alternative splicing underlies a number of human diseases, yet its contribution to liver cancer has not been fully explored. The molecular basis for stimulation of KLF6 splicing is unknown

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