Novel RNA oligonucleotide improves liver function and inhibits liver carcinogenesis in vivo

Authors


  • Potential conflict of interest: Nagy A. Habib, Pål Sætrom, and Steen Lindkær-Jensen own stock in MiNA Therapeutics Ltd. John J. Rossi consults for Calando.

  • This work was funded by MiNA Therapeutics Ltd. MiNA Therapeutics holds licenses for intellectual property related to saRNA technology.

Abstract

Hepatocellular carcinoma (HCC) occurs predominantly in patients with liver cirrhosis. Here we show an innovative RNA-based targeted approach to enhance endogenous albumin production while reducing liver tumor burden. We designed short-activating RNAs (saRNA) to enhance expression of C/EBPα (CCAAT/enhancer-binding protein-α), a transcriptional regulator and activator of albumin gene expression. Increased levels of both C/EBPα and albumin mRNA in addition to a 3-fold increase in albumin secretion and 50% decrease in cell proliferation was observed in C/EBPα-saRNA transfected HepG2 cells. Intravenous injection of C/EBPα-saRNA in a cirrhotic rat model with multifocal liver tumors increased circulating serum albumin by over 30%, showing evidence of improved liver function. Tumor burden decreased by 80% (P = 0.003) with a 40% reduction in a marker of preneoplastic transformation. Since C/EBPα has known antiproliferative activities by way of retinoblastoma, p21, and cyclins, we used messenger RNA (mRNA) expression liver cancer-specific microarray in C/EBPα-saRNA-transfected HepG2 cells to confirm down-regulation of genes strongly enriched for negative regulation of apoptosis, angiogenesis, and metastasis. Up-regulated genes were enriched for tumor suppressors and positive regulators of cell differentiation. A quantitative polymerase chain reaction (PCR) and western blot analysis of C/EBPα-saRNA-transfected cells suggested that in addition to the known antiproliferative targets of C/EBPα, we also observed suppression of interleukin (IL)6R, c-Myc, and reduced STAT3 phosphorylation. Conclusion: A novel injectable saRNA-oligonucleotide that enhances C/EBPα expression successfully reduces tumor burden and simultaneously improves liver function in a clinically relevant liver cirrhosis/HCC model. (Hepatology 2014;58:216–227)

Abbreviations
AFP

alphafetoprotein

ALT

alanine aminotransferase

AST

aspartate aminotransferase

C/EBPα

CCAAT/enhancer binding protein alpha

HCC

hepatocellular carcinoma

OTC

ornithine transcarbamylase

saRNA

short activating RNA

Human hepatocellular carcinoma (HCC) is currently the third most common cause of cancer-related mortality worldwide.[1] The majority of patients with HCC develop malignant tumors from a background of liver cirrhosis. Currently most patients are diagnosed at an advanced disease stage and therefore the 5-year survival rate for the majority of HCC patients remains dismal.[2] Surgical resection, locoregional ablation, and liver transplantation are currently the only therapeutic options which have the potential to cure HCC. However, based on the evaluation of individual liver function and tumor burden, only about 5%-15% of patients are eligible for surgical intervention.[3]

Most eukaryotic cells use RNA-complementarity as a mechanism for regulating gene expression. One example is the classic RNA interference (RNAi) pathway which uses double-stranded short interfering RNAs to knockdown gene expression by way of the RNA-induced silencing complex (RISC).[4] It is now established that short duplex RNA oligonucleotides also have the ability to target the promoter regions of genes and mediate transcriptional activation of these genes and they have been referred to as RNA activation (RNAa), antigene RNAs (agRNAs), and short-activating RNA (saRNA).[5-8] SaRNA-induced activation of genes appears to be conserved in other mammalian species including mouse, rat, and nonhuman primates and is fast becoming a popular method for studying the effects of endogenous up-regulation of genes.[5] SaRNAs have recently been designed to activate expression of genes such as p21 as potential therapy for the treatment of HCC or prostate cancer, thus demonstrating a promising novel approach for adjuvant therapy.[9, 10]

With the same iterative approach that we previously used to design saRNAs specific for Kruppel-like factor 4 (Klf4), c-Myc, and MafA,[7, 11] we generated saRNA molecules to up-regulate transcript levels of the CCAAT/enhancer-binding protein alpha (C/EBPα) gene.

C/EBPα is a leucine zipper protein that is conserved across humans and rats. This transcription factor is enriched in hepatocytes, myelomonocytes, adipocytes, as well as mammary epithelial cells including other cell types.[12] In the adult liver, C/EBPα is defined as functioning in terminally differentiated hepatocytes, while rapidly proliferating hepatoma cells express only a fraction of C/EBPα.[13] C/EBPα is known to up-regulate p21, a strong inhibitor of cell proliferation through the up-regulation of retinoblastoma and inhibition of Cdk2 and Cdk4.[14, 15] Since the binding sites for the family of C/EBP transcription factors are present in the promoter regions of numerous genes that are involved in the maintenance of normal hepatocyte function and response to injury (including albumin, interleukin (IL)6 response, energy homeostasis, ornithine cycle regulation, and serum amyloid A expression)[16-20]; we determined the therapeutic benefit of up-regulating expression of C/EBPα in cirrhotic rats with compromised liver function and HCC by using saRNA as a safe and potentially clinically translatable method of targeted gene up-regulation.

For targeted in vivo delivery, we complexed C/EBPα-saRNA into the structurally flexible triethanolamine (TEA)-core poly (amidoamine) (PAMAM) dendrimer.[21] The in vivo efficacy of these nanoparticles have previously been evaluated where biodistribution studies show that the dendrimers preferentially accumulate in peripheral blood mononuclear cells and liver with no discernible toxicity.[21] Here we demonstrate the therapeutic effect of intravenously injecting C/EBPα-saRNA-dendrimers in a clinically relevant rat liver tumor model.[44]

After three doses through tail vein injection at 48-hour intervals, the treated cirrhotic rats showed significantly increased serum albumin levels within 1 week. More important was the unexpected observation that liver tumor burden significantly decreased in the C/EBPα-saRNA-dendrimer-treated groups. This study demonstrates, for the first time, that gene targeting by small RNA molecules can be used by systemic intravenous administration to simultaneously ameliorate liver function and reduce tumor burden in cirrhotic rats with HCC.

Materials and Methods

The full methods for designing short-activating RNA, animal experiments, nuclease activity, assessment of tumor burden, immunostaining, quantitative reverse-transcription polymerase chain reaction (qRT-PCR), gene microarray profiling, ChIP-seq analysis, gene ontology enrichment analysis, and gene methylation analysis are available in the online Supporting Information.

Design of Short-Activating RNA Oligonucleotides

The gene sequence of albumin and C/EBPα was selected for designing short-activating RNA molecules for its specific activation using the parameters previously described.[7]

Transfection of SaRNA Oligonucleotides Into HepG2 and Rat Liver Epithelial Cell Lines

HepG2 is a liver cell line derived from a human hepatoblastoma that is free of known hepatotropic viral agents and expresses genes involved in a wide variety of liver-specific metabolic functions.[22] HepG2 cells were cultured in Roswell Park Memorial Institute medium (RPMI) supplemented with 100 units/mL penicillin, 0.1 mg/mL streptomycin, 2 mmol/L glutamine (Sigma), and 10% fetal bovine serum (Labtech International). For C/EBPα-saRNA transfection, cells were grown to 60% confluency in 24-well plates prior to transfection of 5, 10, and 20 nmoles of saRNA using Nanofectamine (PAA, UK) following the manufacturer's protocol. This process was repeated three times at 16-hour intervals before cells were harvested for isolation of total RNA for messenger RNA (mRNA) analysis.

Albumin Enzyme-Linked Immunosorbent Assay (ELISA)

Rat liver epithelial cells and HepG2 cells were cultured in phenol-red free RPMI media in the presence of charcoal-stripped fetal calf serum (FCS). Following three sets of saRNA transfections at 8 hours, 16 hours, and 24 hours, the culture media was collected for total murine albumin ELISA (Assay Max, Albumin ELISA, Assay Pro USA) following the manufacturer's instructions.

WST-1 Assay

Cell proliferation was quantified at 16, 24, and 96 hours following C/EBPα-saRNA transfection by mitochondrial dehydrogenase expression analysis, using WST-1 reagent following the manufacturer's guideline (Roche, UK). Briefly, the WST-1 reagent was used at 1:100 dilution to plates and incubated for 1 hour. The enzymatic reaction was measured at 450 nm using the Bio-Tek ELISA reader.

Isolation of Total RNA From Cell Lines

Total RNA extraction from cell lines was performed using the RNAqueous-Micro kit (Ambion, UK) following the manufacturer's instructions. Briefly, the cells were gently centrifuged followed by three pulses of sonication at Output 3 in Lysis buffer (Ambion, UK). The cell lysates were then processed through an RNA binding column, followed by multiple washes and elution. The total RNA isolated was quantified by a Nanodrop 2000 spectrophotometer. 500 ng of total extracted RNA was processed for elimination of genomic DNA followed by reverse transcription using the QuantiTect Reverse Transcription kit from Qiagen.

Animal Experiments

We used a clinically relevant rat liver tumor model previously described.[23] For in vivo therapy C/EBPα-saRNA was reconstituted with 100 μL of RNase/Dnase free H2O; 50 μL of 20 nM saRNA oligonucleotide and 50 μL of (TEA) core PAMAM dendrimer, previously described.[24, 25] Ten cirrhotic animals were treated with 3× doses by way of tail vein injections in the first week. Control animals (n = 10) were injected with an equal volume of phosphate-buffered saline (PBS) or scramble-saRNA. All animals received humane care according to the criteria outlined in the “Guide for the Care and Use of Laboratory Animals” prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH publication 86-23 revised 1985).

Results

Expression Level of C/EBPα and Albumin in HepG2 Cells Transfected With C/EBPα-saRNA

We assessed the effect of transfecting C/EBPα-saRNA on C/EBPα and albumin transcript levels. Both C/EBPα (Fig. 1A) and albumin transcripts (Fig. 1B) increased over 2-fold. Increasing the amounts of C/EBPα-saRNA (5, 10, and 20 nM) dose-dependently enhanced C/EBPα transcript levels (Fig. 1C). The maximum expression of albumin was achieved with 5 nM of C/EBPα-saRNA, with no further dose-dependent increase at higher saRNA levels (Fig. 1D). Analysis of the promoter regions of C/EBPα (Fig. 1E), the binding box of albumin (DBP) (Fig. 1F), and albumin (Fig. 1G) showed the presence of the core C/EBPα binding motifs (GCAAT), thus supporting targeting of both transcripts by C/EBPα-saRNA-induced up-regulation of C/EBPα. An EpiTect Methyl PCR assay also demonstrated reduced methylation at the CpG-island of both C/EBPA and DBP promoters following transfection of C/EBPα-saRNA (Fig. 2A,B).

Figure 1.

Transfection of C/EBPα-saRNA in HepG2 cells regulates expression of C/EBPα and albumin. (A) HepG2 cells were transfected with 20 nM C/EBPα-saRNA and harvested for total RNA extraction and reverse transcription for quantitative analysis of C/EBPα and (B) albumin gene expression. (C) A dose escalation of C/EBPα-saRNA demonstrates its effect over 96 hours (hr) on C/EBPα gene expression and (D) albumin gene expression. Data represent mean ± SD. The panels show the genomic region containing (E) C/EBPα, (F) DBP, and (G) albumin (ALB) 2000 nucleotides upstream and downstream of each gene where all have one or more C/EBPα binding sites. The figure panels show the chromosomal coordinates (“Scale” and chromosome identifier), C/EBPα binding sites (“C/EBPA”; black boxes), occurrence of the C/EBPα binding motif (“GCAAT motif”; black vertical lines), and RefSeq genes (blue boxes and lines) within the genomic regions.

Figure 2.

Transfection of C/EBPα-saRNA in HepG2 cells regulates hepatocyte function and affects cell proliferation. (A) Methylation assay of the CpG islands at the promoter regions of C/EBPA and (B) DBP demonstrated reduction in methylation when compared to control. Data represent mean ± SD. (C) An ELISA specific for human albumin detected a significant increase of albumin secretion following transfection of 20 nM CEPBA-saRNA. Data represent mean ± SD. (D) Expression of the gene encoding ornithine cycle enzyme OTC increased in C/EBPα-saRNA transfected cells suggesting an improved ability of urea production. (E) Decreased expression of the gene encoding AFP suggested improved regulation of cell differentiation. Data represent mean ± SD. (F) A WST-1 cell proliferation assay on HepG2 cells over 96 hours transfected with increasing amounts of C/EBPα-saRNA. Results show a dose-dependent reduction in cell proliferation. Data represent mean ± SD.

To determine the biological relevance of increased albumin mRNA transcripts in C/EBPα-saRNA-transfected HepG2 cells, a human albumin specific ELISA was performed. Secreted albumin peptide was detected in the culture media of the transfected cells (Fig. 2C).

To establish if enhanced albumin secretion in HepG2 cells by C/EBPα-saRNA also affected other hepatocyte-specific functions and maintenance of hepatocyte differentiation, we measured expression levels of the ornithine cycle enzyme ornithine transcarbamylase (OTC) and alpha-fetoprotein (AFP). C/EBPα-saRNA caused an increase in OTC levels (Fig. 2D), suggesting an improved ability of urea production. The expression level of AFP decreased (Fig. 2E), indicative of the negative regulation typically observed with normal hepatocytes.[26] In addition to the observed gene changes described, we also observed that C/EBPα-saRNA caused a marked down-regulation of HepG2 cell proliferation (Fig. 2F). This observation confirms the known antiproliferative effects of C/EBPα.[14, 27]

Intravenous Injection of C/EBPα-saRNA in Male Wistar Rats Bearing Liver Cirrhosis/HCC Promoted Increased Circulating Levels of Albumin, Amelioration of Liver Function, and Reduced Tumor Burden

The stability of C/EBPα-saRNA was initially tested in circulating serum by performing a nuclease activity assay using blood samples from C/EBPα-saRNA-treated rats. We observed a significant reduction in the stability of C/EBPα-saRNA duplex by 48 hours (Fig. 3A,B). We thus injected cirrhotic rats over a period of 1 week with repeat doses of C/EBPα-saRNA-dendrimer. Measurement of circulating albumin showed a significant increase of over 30% after three doses of C/EBPα-saRNA-dendrimer injection when compared to PBS control or scramble-saRNA-dendrimer control groups (Fig. 3C). Further blood analysis demonstrated that the worsening of bilirubin levels was significantly less in the C/EBPα-saRNA-dendrimer-treated group by at least 17% when compared to both control groups (Fig. 3D). There was also a significant drop in levels of the liver enzymes aspartate aminotransferase (AST) and alanine aminotransferase (ALT) by at least 10% and 30%, respectively, in the C/EBPα-saRNA-dendrimer-treated group when compared to both control groups (Fig. 3E,F). Histological examination of the liver showed a significant reduction in tumor nodules from C/EBPα-saRNA-dendrimer-injected rats when compared to both control groups (Fig. 4A,B). These results were consistent with immunohistology studies of tissue sections from C/EBPα-saRNA-treated rat liver stained for placenta-form of glutathione S-transferase (GST-p). Independent conclusions by two pathologists suggested that there was evidence of reduced carcinogenesis by treatment of C/EBPα-saRNA-dendrimer when compared to the PBS control or scramble-saRNA-dendrimer control groups. Furthermore, there were no differences in liver fibrosis between the PBS control, scramble-saRNA-dendrimer, or C/EBPα-saRNA-dendrimer-treated groups (Fig. 4C). The average density of positive staining for GST-p from control groups was 70 (±5.0%), and that from C/EBPα-saRNA-dendrimer injected rats was 32 (±6.5%). Since overexpression of GST-p is observed during rat liver preneoplastic state and neoplastic transformation,[28, 29] these data suggest that C/EBPα-saRNA-dendrimer treatment may reduce this process.

Figure 3.

Intravenous injection of C/EBPα-saRNA-dendrimer in male Wistar rats with liver cirrhosis and HCC shows improved liver function. (A) C/EBPα-saRNA-dendrimer was tested for nuclease sensitivity in rat serum for the indicated times. RNA stability was visualized on a 2% denaturing agarose gel and (B) quantified by densitometry analysis (data represent mean ± SD, n = 3). (C) C/EBPα-saRNA injected rats showed a significant change in circulating levels of albumin when compared to PBS control (Control) or scramble-saRNA-dendrimer control groups. (D) Changes in bilirubin levels suggested that C/EBPα-saRNA-dendrimer injected rats had at least a 10% improvement when compared to both control groups. Changes in (E) AST and (F) ALT demonstrated at least a 10% and 30% improvement in values when compared to the control groups. Data represent mean ± SD.

Figure 4.

Intravenous injection of C/EBPα-saRNA-dendrimer in male Wistar rats with liver cirrhosis and HCC shows reduced tumor burden. (A) Liver tumor nodules were visibly reduced in C/EBPα-saRNA injected rats when compared to both PBS control and Scramble-saRNA control groups.(B) Tumor burden was assessed by the volume of all tumor nodules with a diameter in excess of 3 mm. C/EBPα-saRNA-injected rats had significantly reduced tumor burden after 2 weeks of treatment when compared to both control groups. Data represent mean ± SD. (C) 2 μm liver sections from PBS control, scramble-saRNA-dendrimer control, and C/EBPα-saRNA-dendrimer-injected rats were immunostained for expression of placenta-form glutathione-S-transferase (GST-p). PBS control rats showed 70% (± 5.0%) of positive staining for the preneoplastic marker, scramble-saRNA-dendrimer-injected rats showed 64% (± 10.0%) of positive staining, while C/EBPα-saRNA-dendrimer-injected rats only showed 32% (± 6.5%) of positive staining.

Total RNA extracted from liver biopsies of seven animals from each group were screened for transcript levels of albumin (Fig. 5A), C/EBPα (Fig. 5B), hepatocyte nuclear factor 4-alpha (HNF4α) (Fig. 5C), and hepatocyte nuclear factor 1-alpha (HNF1α) (Fig. 5D). A significant increase in mRNA level was observed for all the factors, consistent with the role of HNF4α in hepatocyte differentiation together with C/EBPα and HNF1α in promoting expression of albumin. Taken together, lower mRNA levels of hepatocyte growth factor (HGF) (Fig. 5E) and increased levels of 4-hydroxyphenylpyruvic acid dioxygenase (HPD1) (Fig. 5F) and plasminogen (Fig. 5G) are suggestive of improved liver function in these cirrhotic rats treated with C/EBPα-saRNA-dendrimer.[30]

Figure 5.

Intravenous injection of C/EBPα-saRNA-dendrimer in male Wistar rats with liver cirrhosis and HCC positively regulates expression of factors for liver function (A) Total RNA extracts from 7 control rats versus 7 C/EBPα-saRNA injected rats were analyzed for albumin gene expression, (B)C/EBPα gene expression, (C) HFN4α gene expression, and (D) HNF1α gene expression showed an increase in these factors. (E) Decreased mRNA levels encoding HGF and (F) increased levels of hydroxyphenylpyruvic acid dioxygenase (HPD1) and (G) plasminogen indicated positive regulation of cell proliferation and improved liver function. Data represent mean ± SD.

Pathway Gene Microarray Analysis Suggests That C/EBPα-saRNA Contributes to Up-Regulation of Tumor Suppressor Genes and Down-Regulation of Genes Involved in Liver Cancer

To investigate other liver-specific factors that might be affected in response to C/EBPα-saRNA;, we analyzed the gene expression profile of a panel of 84 liver cancer-specific genes (Qiagen/SABiosciences Human Liver Cancer RT2 Profiler) in C/EBPα-saRNA-transfected HepG2 cells (Fig. 6). Of particular interest was the observed up-regulation of 20 genes (Supporting Table 1), 18 of which are known tumor suppressor genes in HCC (Supporting Table 3) including RB. The most significantly up-regulated (over-3 fold) included the death agonist gene BH3-interacting domain (BID), and tumor protein 53 gene (TP53), encoding p53. BID interacts with BCl2-associated X protein (BAX) which in turn is up-regulated by wild-type p53 to regulate cell cycle arrest and apoptosis in response to DNA damage.[31, 32]

Figure 6.

Microarray analysis of 84 liver cancer pathway genes. Negative regulation of genes (in green) or positive regulation of genes (in red) following transfection of 20 nM C/EBPα-saRNA in HepG2 cells. Control (CONTROL-1 to 4) and C/EBPA-saRNA transfected (C/EBPA-1 to 4) are shown as four repeats.

Growth arrest and DNA-damage-inducible, 45 beta (GADD45B), also up-regulated, is a member of the growth arrest DNA damage inducible gene family associated with cell growth control, which together with p53 induces hepatoprotection in HepG2 cells.[33] Deleted in Liver Cancer 1 (DLC1) gene is a reported tumor suppressor for human liver cancer inhibiting cell growth and proliferation, as well as inducing apoptosis.[34] Our data suggest that DLC1 is up-regulated in C/EBPα-saRNA-transfected HepG2 cells (Supporting Table 3).

Runt-related transcription factor-3 (RUNX3) is a member of the runt domain family of transcription factor and has been frequently been observed in HCC, where its expression is significantly lower than in surrounding normal tissue.[35] Since ectopic expression of RUNX3 reverses epithelial-mesenchymal transition (EMT) in HCC cells,[36] we also observed, in the C/EBPα-saRNA-transfected HepG2 cells, an up-regulation of RUNX3 (Supporting Table 3) and down-regulation of four genes involved in EMT. These included CTNB1 (encoding β-catenin), hepatocyte growth factor (HGF), small body size mothers against decapentaplegic homolog 7 (SMAD7), and transforming factor beta 1 (TGFB1) (Supporting Table 4).

Suppression of cytokine signaling 3 (SOCS3) was also detected. SOCS3 is a member of the STAT-induced STAT inhibitor (SSI) which function as negative regulators of cytokine signaling. Decreased expression of SOCS3 is correlated with increased phosphorylation of STAT3 in HCC.[37] SOCS3 furthermore has been implicated in negatively regulating cyclin D1 (CCND1), and antiapoptotic genes including XIAP, survivin (BIRC5), and myeloid leukemia cell differentiation protein (MCL1).[38] Here we observed a significant increase in expression of SOCS3 (Supporting Table 3) and a significant decrease in STAT3, CCND1, XIAP, BIRC5, and MCL1 expression (Supporting Table 4). Similar to the in vivo observations of reduction in GST-p (Fig. 2D), the array data also confirmed down-regulation in expression of GSTP1 (Supporting Table 4).

Overall, the down-regulated genes were strongly enriched for functions related to negative regulation of apoptosis and cell death (gene ontology (GO) terms GO:0043066 and GO:0060548; P 2 × 10−9 and 2 × 10−9, respectively), whereas the up-regulated genes were enriched for functions related to positive regulation of cell differentiation (GO:0045597; P = 5 × 10−3).

Transfection of C/EBPα-saRNA in HepG2 Suppresses STAT3, IL6R, and cMyc in HepG2 Cells

Previously published reports demonstrate that IL6R promotes hepatic oncogenesis by directly activating STAT3 and in turn up-regulating expression of c-Myc.[39] Since a ChIP-Seq analysis of these three genes show the presence of C/EBPα binding sites within their promoter regions (Fig. 7A-C), we assessed whether transfection of C/EBPα-saRNA in HepG2 cells would affect expression levels of these three factors. We observed a significant reduction in mRNA levels of STAT3 (Fig. 7D), cMyc (Fig. 7E), and IL6R (Fig. 7F) when compared to untransfected cells. This trend in gene reduction was also observed for MYC and STAT in our previously described gene expression array (Supporting Table 2, in bold). When the methylation status of the CpG islands at the promoter regions of STAT3 (Fig. 8A), MYC (Fig. 8B), and IL6R (Fig. 8C) were assessed using the EpiTect Methyl II PCR assay (Qiagen), an increase in methylation state at the promoters of all three genes was detected. A western blot also confirmed a reduction in the phosphorylation status of STAT3 and in the protein level of IL6R (Fig. 8D). Collectively, we show that in vivo delivery of C/EBPα might have a positive effect in assisting liver function and decreasing aberrant cell proliferation in a cirrhotic/HCC setting.

Figure 7.

Transfection of C/EBPα-saRNA in HepG2 cells results in negative regulation of cell proliferating factors. STAT3, c-Myc (MYC), and IL6R have one or more C/EBPα binding sites. The panels show the genomic region 2000 nucleotides upstream and downstream of (A) STAT3, (B) MYC, and (C) IL6R. Figure panels shown are as described in (Fig. 1E-G). C/EBPα-saRNA transfected HepG2 cells show negative regulation in mRNA expression levels of genes encoding (D) STAT3, (E) c-Myc, and (F) IL6 receptor (IL6R). Data represent mean ± SD.

Figure 8.

Transfection of C/EBPα-saRNA in HepG2 cells targets STAT3, c-Myc, and IL6R signaling. A methylation assay of the CpG islands at the promoter regions of (A) STAT3, (B) MYC, and (C) IL6R demonstrated hypermethylation when compared to control. Data represent mean ± SD. (D) A western blot analysis showed decreased phosphorylation of STAT3 at residues 705 and 727 and down-regulation of IL6R in cells transfected with C/EBPα-saRNA.

Discussion

HCC develops in most patients from a background of liver cirrhosis and accounts for 90% of all liver cancers. Although much progress has been made in targeting therapy to HCC, few of these treatments have had much impact on patient outcome.

The initial aim of this investigation was to study the therapeutic potential of using saRNAs to help ameliorate liver function in a clinically relevant rat model of liver cirrhosis with HCC. By enhancing expression of the gene encoding C/EPBα, a liver enriched transcription factor that enhances albumin and confers antimitotic activity, we primarily sought to increase circulating albumin in these rats. Using our previously published concept of designing saRNA oligonucleotide to increase the expression of a target gene,[7, 11] C/EPBα-saRNA was generated. This was initially tested in the HCC line (HepG2) where introduction of the saRNA oligonucleotide led to increased transcript levels of C/EPBα and albumin. Both genes furthermore contained the recognition motif of C/EPBα, CGAAT within their promoter regions. It was therefore unsurprising to detect a loss in methylation status at their CpG islands following transfection of C/EPBα-saRNA.

The biological significance of increasing albumin transcript levels in C/EPBα-saRNA-transfected cells corresponded well with the increased secretion of albumin. Interestingly, we found that the maximum albumin gene expression was achieved at 5 nM of C/EPBα-saRNA with no further increase at higher saRNA levels. In addition to the albumin gene, we also found increased gene expression in other important biological markers such as ornithine cycle enzyme OTC and AFP.[40]

To test the potential therapeutic value of the C/EPBα-saRNA, we subsequently performed an in vivo study using an HCC rat model. For targeted delivery of C/EPBα-saRNA we linked the duplex RNA molecule to cationic PAMAM dendrimers. These nanoparticle have previously been evaluated where biodistribution studies demonstrate that they preferentially accumulate in peripheral blood mononuclear cells and the liver with no discernible toxicity.[25] Intravenous injection of C/EPBα-saRNA-dendrimers over a course of 1 week showed a significant improvement by 30% in the circulating levels of albumin where compared to PBS control or scramble-saRNA-dendrimer control groups. Changes in bilirubin levels showed a 10% improvement in the C/EBPα-saRNA group when compared to the control groups. Additionally, a 10% improvement in AST levels and 30% improvement in ALT levels were observed in the C/EBPα-saRNA-treated group when compared to the control groups. More significant was the reduction in tumor burden and the inhibition of preneoplastic lesions as detected by a 40% reduction in GST-p staining in the liver sections from the C/EBPα-saRNA-treated group. From a clinical perspective, this represents a very attractive therapeutic avenue since the expression level of C/EBPα in matched tumor tissues and nontumor tissues of HCC patients is down-regulated in the majority of tumor specimens. Moreover, patients with tumor samples showing higher levels of C/EBPα have a longer survival rate than those patients with tumor samples in which the expression of the C/EBPα is lower.[41] Our data support this evidence, suggesting that up-regulation of C/EBPα provides a strong antiproliferative role in hepatocytes.[14, 42]

To better understand the global molecular effect of C/EPBα-saRNA more specific to liver cancer, we performed a liver cancer pathway gene expression profile analysis. Such analysis of whole tumors is frequently confounded by the presence of cell types other than those with a transformed phenotype.[43] Therefore, we profiled the gene expression changes brought about by C/EPBα-saRNA in HepG2 cells.

The expression pattern of the liver cancer genes varied greatly between untransfected and C/EPBα-saRNA-transfected HepG2 cells. After normalization and cluster analysis, several important genes were significantly altered in expression. From the list of 20 genes that were up-regulated, 18 were known tumor suppressor genes. Of note was the up-regulation of RB, TP53, BID, and BAX to regulate cell cycle and apoptosis. The down-regulation of key genes were also noted, in particular ADAM17, a metalloproteinase reported as being a pathological feature of HCC.[44] ADAM17 is known to cause the shedding of receptor ligands such as epidermal growth factor (EGF) and tumor necrosis factor alpha (TNFα),[45, 46] thus preventing regulation of key signaling events for normal cell signaling.

Upon further analysis of the tumor suppressor genes, we noticed a pathway-defined trend where key effector genes of the tumor suppressors were down-regulated. Examples of this included repression of RHOA following up-regulation of the tumor suppressor DLC1, or up-regulation of RUNX3 to reverse expression of the oncogenes involved in EMT. Here we observed down-regulation of CTNB1 (β-catenin), HGF, SMAD7, and TGFB1. We also observed increased expression of the tumor suppressor SOC3, a known regulator of apoptosis and cell adhesion. Concomitantly, we also observed down-regulation of the associated SOC3 oncogenes including STAT3, cyclin-D1 (CCND1), XIAP, BIRC5, and MCL1. STAT3 activation together with IL6R is known to enhance hepatic oncogenesis as part of a feedback loop,[47] and moreover perturbation in any of the components from this network is sufficient to suppress HCC.[39] Here we demonstrated by gene expression analysis and detection of hypermethylation within the gene promoters that both STAT3 and IL6R were down-regulated following C/EBPα-saRNA transfection. In addition to the well-characterized antimitotic activity of C/EPBα involving retinoblastoma, p21, and the cyclin dependent proteins, our data here suggest that C/EPBα may regulate other liver-specific oncogenic pathways including c-Myc (MYC).[48] Our observed reduction in the EMT factors, the positive regulation of apoptosis and down-regulation of IL6R, STAT3 and MYC, and the presence of numerous C/EBPα binding motifs within the promoter regions of these three genes provide a novel landscape to further study the role of C/EPBα in improving the function of hepatocytes in a cirrhotic/HCC setting.

In summary, we initially designed saRNAs targeting the liver enriched transcription factor C/EBPα with the aim of addressing hypoalbuminemia. This was successfully done in vitro and in vivo. In the course of this work we also confirmed the well known antiproliferative effects of C/EPBα in a clinically relevant cirrhotic/HCC model. In addition to regulating known targets of C/EPBα that controls cell proliferation, we demonstrated using a liver cancer-specific gene array analysis that C/EPBα potentially targets numerous other oncogenes and tumor suppressor genes which must be further investigated. C/EPBα-saRNAs therefore may have a profound effect at the transcriptional level for liver cancer. Currently, most therapeutic disciplines such as surgery, chemotherapy, radiotherapy, and biologics are associated with variable decrease of liver dysfunction.[49, 50] The data presented here offer a new approach to targeting liver cancer cells.

Acknowledgment

We are sincerely grateful to Dr. Albert Deisseroth and Professor Farzin Farzaneh for their valuable input to the construction of this manuscript.

Ancillary