Steven Yea

Pivotal Role of mTOR Signaling in Hepatocellular Carcinoma

BACKGROUND & AIMS The advent of targeted therapies in hepatocellular carcinoma (HCC) has underscored the importance of pathway characterization to identify novel molecular targets for treatment. We evaluated mTOR signaling in human HCC, as well as the antitumoral effect of a dual-level blockade of the mTOR pathway. METHODS The mTOR pathway was assessed using integrated data from mutation analysis (direct sequencing), DNA copy number changes (SNP-array), messenger RNA levels (quantitative reverse-transcription polymerase chain reaction and gene expression microarray), and protein activation (immunostaining) in 351 human samples [HCC (n = 314) and nontumoral tissue (n = 37)]. Effects of dual blockade of mTOR signaling using a rapamycin analogue (everolimus) and an epidermal/vascular endothelial growth factor receptor inhibitor (AEE788) were evaluated in liver cancer cell lines and in a xenograft model. RESULTS Aberrant mTOR signaling (p-RPS6) was present in half of the cases, associated with insulin-like growth factor pathway activation, epidermal growth factor up-regulation, and PTEN dysregulation. PTEN and PI3KCA-B mutations were rare events. Chromosomal gains in RICTOR (25% of patients) and positive p-RPS6 staining correlated with recurrence. RICTOR-specific siRNA down-regulation reduced tumor cell viability in vitro. Blockage of mTOR signaling with everolimus in vitro and in a xenograft model decelerated tumor growth and increased survival. This effect was enhanced in vivo after epidermal growth factor blockade. CONCLUSIONS MTOR signaling has a critical role in the pathogenesis of HCC, with evidence for the role of RICTOR in hepato-oncogenesis. MTOR blockade with everolimus is effective in vivo. These findings establish a rationale for targeting the mTOR pathway in clinical trials in HCC.

Ras pathway activation in hepatocellular carcinoma and anti-tumoral effect of combined sorafenib and rapamycin in vivo ☆

BACKGROUND/AIMS The success of sorafenib in the treatment of advanced hepatocellular carcinoma (HCC) has focused interest on the role of Ras signaling in this malignancy. We investigated the molecular alterations of the Ras pathway in HCC and the antineoplastic effects of sorafenib in combination with rapamycin, an inhibitor of mTOR pathway, in experimental models. METHODS Gene expression (qRT-PCR, oligonucleotide microarray), DNA copy number changes (SNP-array), methylation of tumor suppressor genes (methylation-specific PCR) and protein activation (immunohistochemistry) were analysed in 351 samples. Anti-tumoral effects of combined therapy targeting the Ras and mTOR pathways were evaluated in cell lines and HCC xenografts. RESULTS Different mechanisms accounted for Ras pathway activation in HCC. H-ras was up-regulated during different steps of hepatocarcinogenesis. B-raf was overexpressed in advanced tumors and its expression was associated with genomic amplification. Partial methylation of RASSF1A and NORE1A was detected in 89% and 44% of tumors respectively, and complete methylation was found in 11 and 4% of HCCs. Activation of the pathway (pERK immunostaining) was identified in 10.3% of HCC. Blockade of Ras and mTOR pathways with sorafenib and rapamycin reduced cell proliferation and induced apoptosis in cell lines. In vivo, the combination of both compounds enhanced tumor necrosis and ulceration when compared with sorafenib alone. CONCLUSIONS Ras activation results from several molecular alterations, such as methylation of tumor suppressors and amplification of oncogenes (B-raf). Sorafenib blocks signaling and synergizes with rapamycin in vivo, preventing tumor progression. These data provide the rationale for testing this combination in clinical studies.

Functional Linkage of Cirrhosis-Predictive Single Nucleotide Polymorphisms of Toll-like Receptor 4 to Hepatic Stellate Cell Responses

In a recent study, a single nucleotide polymorphism (SNP) of the Toll‐like receptor 4 (TLR4) gene (c.1196C>T [rs4986791, p.T399I]) emerged as conferring protection from fibrosis progression compared to a major, wild‐type (WT) CC allele (p.T399). The present study examined the functional linkage of this SNP, along with another common, highly cosegregated TLR4 SNP (c.896A>G [rs4986790, p.D299G]), to hepatic stellate cell (HSC) responses. Both HSCs from TLR4−/− mice and a human HSC line (LX‐2) reconstituted with either TLR4 D299G and/or T399I complementary DNAs were hyporesponsive to lipopolysaccharide (LPS) stimulation compared to those expressing WT TLR4, as assessed by the expression and secretion of LPS‐induced inflammatory and chemotactic cytokines (i.e., monocyte chemoattractant protein‐1, interleukin‐6), down‐regulation of bone morphogenic protein and the activin membrane‐bound inhibitor expression (an inhibitory transforming growth factor β pseudoreceptor), and activation of a nuclear factor κB (NF‐κB)–responsive luciferase reporter. In addition, spontaneous apoptosis, as well as apoptosis induced by pathway inhibitors of NF‐κB, extracellular signal‐regulated kinase (ERK), and phosphatidylinositol 3‐kinase were greatly increased in HSCs from either TLR4−/− or myeloid differentiation factor 88−/− (a TLR adaptor protein) mice, as well as in murine HSCs expressing D299G and/or T399I SNPs; increased apoptosis in these lines was accompanied by decreased phospho‐ERK and Bcl‐2. Conclusion: TLR4 D299G and T399I SNPs that are associated with protection from hepatic fibrosis reduce TLR4‐mediated inflammatory and fibrogenic signaling and lower the apoptotic threshold of activated HSCs. These findings provide a mechanistic link that explains how specific TLR4 SNPs may regulate the risk of fibrosis progression. (HEPATOLOGY 2009.)

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

BACKGROUND & AIMS 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.

Regulation of Krüppel-like Factor 6 Tumor Suppressor Activity by Acetylation

Krüppel-like factor 6 (KLF6) is a zinc finger transcription factor and tumor suppressor that is inactivated in a number of human cancers by mutation, allelic loss, and/or promoter methylation. A key mechanism of growth inhibition by wild-type KLF6 is through p53-independent up-regulation of p21(WAF1/cip1) (CDKN1A), which is abrogated in several tumor-derived mutants. Here we show by chromatin immunoprecipitation that transactivation of p21(WAF1/cip1) by KLF6 occurs through its direct recruitment to the p21(WAF1/cip1) promoter and requires acetylation by histone acetyltransferase activity of either cyclic AMP-responsive element binding protein-binding protein or p300/CBP-associated factor. Direct lysine acetylation of KLF6 peptides can be shown by mass spectrometry. A single lysine-to-arginine point mutation (K209R) derived from prostate cancer reduces acetylation of KLF6 and abrogates its capacity to up-regulate endogenous p21(WAF1/cip1) and reduce cell proliferation. These data indicate that acetylation may regulate KLF6 function, and its loss in some tumor-derived mutants could contribute to its failure to suppress growth in prostate cancer.

A DDX5 S480A Polymorphism Is Associated with Increased Transcription of Fibrogenic Genes in Hepatic Stellate Cells

We recently identified a missense single nucleotide polymorphism (SNP) in DDX5 (rs1140409, p.S480A) that enhances the risk of developing cirrhosis. DDX5 is an ATP-dependent RNA helicase and transcriptional modulator. We hypothesized that the activity of DDX5 in regulating fibrogenic gene transcription in hepatic stellate cells (HSCs) is altered by the S480A SNP. To test this, we employed two approaches: 1) transient overexpression of DDX5 cDNA or siRNA knockdown of endogenous DDX5, with replacement by either DDX5 wild type (WT) or SNP cDNA, or 2) stable expression of exogenous DDX5 WT and SNP in HSC lines. WT DDX5 mRNA in HSCs was inversely correlated with gene expression for α2(I) collagen, tissue inhibitor of metalloproteinase-1, and transforming growth factor-β1. Stable DDX5 SNP-expressing cells had higher basal and transforming growth factor-β1-stimulated expression and enhanced promoter activities of fibrogenic genes. DDX5 variant-expressing cells also had higher Smad3 and AP-1-responsive reporter activities. In a one-hybrid GAL4 system, co-expression of the DDX5 SNP variant with chimeras of GAL4 DNA binding domain linked to JunD or Sp1 displayed higher transactivation of a GAL4-responsive reporter than that of DDX5 WT. Increased fibrogenic gene expression in DDX5 SNP-expressing cells was associated with reduced recruitment of DDX5 homodimers to responsive promoters, but there was no difference in the recruitment of the co-repressor HDAC1 (histone deacetylase 1). These data suggest that DDX5 is a repressor of fibrogenic genes in HSCs through interaction with transcriptional complexes. The enhanced fibrogenic activity of the DDX5 risk variant is linked to a reduced repressive function toward these target genes.

Tumor suppressor activity of KLF6 mediated by downregulation of the PTTG1 oncogene

The tumor suppressor Kruppel‐like factor 6 (KLF6) is frequently inactivated in hepatocellular carcinoma (HCC). To unearth downstream transcriptional targets of KLF6, cDNA microarray analysis of whole liver was compared between KLF6+/+ and KLF6+/− mice. Pituitary tumor transforming gene 1 (PTTG1), an oncogene, was the most up‐regulated transcript in KLF6+/− liver. In human HCCs, KLF6 mRNA was significantly decreased, associated with increased PTTG1. In HepG2, KLF6 transcriptionally repressed PTTG1 by direct promoter interaction. Whereas KLF6 downregulation by siRNA increased HepG2 proliferation, siRNA to PTTG1 was anti‐proliferative. PTTG1 downregulation represents a novel tumor suppressor pathway of KLF6.