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Neoplasia. 2007 Nov; 9(11): 951–959.
doi: 10.1593/neo.07469
PMCID: PMC2077886
PMID: 18030363

siRNA-Mediated β-Catenin Knockdown in Human Hepatoma Cells Results in Decreased Growth and Survival1

Gang Zeng,* Udayan Apte,* Benjamin Cieply,* Sucha Singh,* and Satdarshan P S Monga*†
Author information Article notes Copyright and License information PMC Disclaimer

Abstract

β-Catenin, the chief oncogenic component of the canonical Wnt pathway, is known to be involved in a variety of cancers, including hepatocellular carcinoma (HCC). Although the mechanism of β-catenin activation in HCC is multifactorial, it is indisputably implicated at various stages of hepatocarcinogenesis, making it an attractive therapeutic target. Here we investigate the effect of small interfering RNA-mediated β-catenin knockdown on the growth and survival of human hepatoma cell lines with (HepG2) and without (Hep3B) β-catenin mutations. Transfection of HepG2 and Hep3B cells with human β-catenin (CTNNB1) small interfering RNA resulted in a significant β-catenin decrease, as confirmed by Western blot analyses and immunofluorescence, also leading to decreased expression of known target genes such as cyclin D1 and glutamine synthetase. The decrease in β-catenin activity was confirmed by TOPflash reporter luciferase assay. The functional impact of diminished β-catenin was exhibited as temporal decrease in tumor cell viability by the MTT assay. A concomitant decrease in tumor cell proliferation was also evident with [3H]thymidine incorporation and verified with soft agar assays. Thus, β-catenin is essential for the survival and growth of hepatoma cells independent of mutations in the β-catenin gene and provide a proof of principle for the significance of the therapeutic inhibition of β-catenin in HCC.

Keywords: Liver cancer, Wnt/β-catenin, development, treatment, regeneration

Introduction

The Wnt/β-catenin pathway is important for normal growth and development [1]. However, anomalous activation of Wnt signaling in adults is often associated with oncogenesis [2–4]. Aberrant signaling involving the stabilization and nuclear translocation of β-catenin has been observed in cancers of the colon, rectum, lung, shin, breast, liver, and pancreas [5–12]. Interestingly, the activation of Wnt signaling can occur due to multiple mechanisms and can result from dysregulation of upstream effectors and mutations in downstream components [13].

Hepatocellular carcinoma (HCC), which is the major primary malignant tumor of the liver, is a disease with poor prognosis due to lack of effective treatment, making it essential to identify novel therapeutic targets [14]. Aberrant activation of β-catenin has been identified in a significant subset of HCC patients with molecular bases ranging from mutations in the β-catenin gene (CTNNB1) (26–34%) or AXIN1/2 (5%) to upregulation of the frizzled-7 receptor (90%) [15–17]. Although additional known or unknown mechanisms might also contribute to β-catenin stabilization in HCC, its role in various stages of hepatocarcinogenesis ranging from hepatic adenoma to hepatoma is indisputable [18].

Based on the role of β-catenin in cellular events common to the processes of development and oncogenesis such as proliferation and survival, we initiated the current study [19–21]. We used small interfering RNA (siRNA) directed against β-catenin to examine the impact of successful β-catenin knockdown on two human HCC cell lines and to demonstrate an indispensable role of β-catenin in tumor cell survival and proliferation.

Materials and Methods

Cell Culture, Treatment, and Transient Transfection

Human HCC cell lines HepG2 and Hep3B were obtained from the American Type Culture Collection (Manassas, VA). Cells were plated in six-well plates and cultured in Eagle's minimal essential medium (EMEM) supplemented with 10% vol/vol fetal calf serum at 37°C in a humidified 5% carbon dioxide atmosphere. The cells were grown to 50% to 60% confluence, followed by serum starvation for 16 hours. For siRNA inhibition studies, the cells were transfected with validated human β-catenin (CTNNB1) siRNA or negative control siRNA 1 (Ambion, Inc., Austin, TX) at a final concentration of 100 nM in the presence of an Oligofectamine reagent (Invitrogen, Carlsbad, CA), as per the manufacturer's instructions. After transfection, the cells were harvested at 24, 48, and 72 hours for protein extraction and additional analysis. All experiments were performed in triplicate, and representative results are reported.

Protein Extraction and Western Blot Analysis

Protein extraction from cell lines and Western blot analysis were performed as previously described [5,22,23]. Briefly, the HCC cell lines from siRNA treatment were used for total cell lysate preparation. Homogenization was performed in 200 µl of RIPA buffer containing fresh protease and phosphatase inhibitors (Sigma, St. Louis, MO). The concentration of the protein in lysates was determined by bicinchoninic acid protein assay, with bovine serum albumin as standard. Aliquots of samples were stored at −80°C until use. Twenty or 50 µg of proteins was resolved by SDS-PAGE analysis using the mini-PROTEIN 3-electrophoresis module assembly (Bio-Rad, Hercules, CA) and transferred to Immobilon PVDF membranes (Bio-Rad). The primary antibodies used were against β-catenin, cyclin D1, glutamine synthetase (GS; Santa Cruz Biotechnology, Santa Cruz, CA), and β-actin (Chemicon, Temecula, CA). Horseradish peroxidase-conjugated secondary antibodies were purchased from Chemicon. The proteins were detected by Super-SignalWest Pico Chemiluminescent Substrate (Pierce, Rockford, IL) and visualized by autoradiography. Densitometric analysis on blots was performed by the NIH Imager software (NIH, Bethesda, MD), and the average integrated optical density in the β-catenin siRNA-treated group was normalized to control siRNA-treated group at the corresponding times. The differences were assessed for statistical significance with Student's t test, and P < .05 was considered significant.

Immunofluorescence Microscopy

Cells were grown to 50% confluence on glass cover slips in 24-well plates. After β-catenin siRNA transfection for 48 hours, the cover slips were washed once with phosphate-buffered saline (PBS) and fixed in 100% methanol for 3 minutes at −20°C. Staining was performed as described elsewhere [24]. The secondary antibody was Cy3, which was conjugated and obtained from Jackson Immunoresearch (West Grove, PA). Nuclei were counterstained with 40,6-diamidino-2-phenylindole. The cover slips were then placed on slides with a drop of gelvatol and viewed on a Nikon Eclipse epifluorescence microscope (Nikon), and images were obtained with a Sony CCD camera (Sony).

β-Catenin/Tcf Transcription Reporter Assay

β-catenin/Tcf transcriptional reporter activity was performed as previously described [23]. Briefly, after β-catenin siRNA transfection for 24 hours, the cells were transiently transfected with the reporter construct TOPflash or FOPflash (Upstate, Lake Placid, NY). TOPflash has three copies of the Tcf/Lef sites upstream of a thymidine kinase (TK) promoter and the firefly luciferase gene. FOPflash has mutated copies of Tcf/Lef sites and is used as a control for measuring nonspecific activation of the reporter. All transfections were performed using 1.8 µg of TOPflash or FOPflash plasmids and FuGene HD reagent (Roche, Indianapolis, IN). To normalize transfection efficiency in reporter assays, the cells were cotransfected with 0.2 µg of internal control reporter Renilla reniformis luciferase driven under the TK promoter (pRL-TK; Promega, Madison, WI). Twenty hours after TOPflash or FOPflash transfection, luciferase assay was performed, using the Dual Luciferase Assay System kit, in accordance with the manufacturer's protocols (Promega). Relative luciferase activity (in arbitrary units) was reported as fold induction after normalization for transfection efficiency. The normalized luciferase activity from experiments performed in triplicate was compared between the experimental and control groups for statistical significance with unpaired Student's t test. P < .01 was considered significant.

Cell Viability Assay

Cell viability assay was performed as previously described [23]. All experiments were performed in triplicate. Briefly, at 24, 48, and 72 hours after β-catenin siRNA transfection, 10% vol/vol of 5 mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma) diluted in PBS was added to HepG2 and Hep3B cultures. After 30 minutes of incubation, the medium was aspirated and washed with PBS. Isopropanol (600 ml) was added and shaken gently for 5 minutes. Two hundred microliters of this solution was transferred to a 96-well plate, and absorbance was measured at 570 nM. The mean (±SEM) absorbance units obtained from three experiments for each group were compared for statistical significance with Student's t test, and P < .01 was considered highly significant. The data were normalized to their respective controls and are presented as a bar graph.

Cell Proliferation Assay

[3H]Thymidine incorporation analyses were performed as previously described [23]. Briefly, 4 hours after β-catenin or control siRNA transfection in Hep3B and HepG2 cells, [3H]thymidine (2.5 µCi/ml) was added to culture media. These cells were cultured for [3H]thymidine incorporation for an additional 24, 48, or 72 hours. Alternatively, an additional set of HepG2 cells was transfected with siRNA against β-catenin a second time, at 24 hours after the initial transfection. This was followed 4 hours later by the addition of [3H]thymidine (2.5 µCi/ml), and the cells were cultured for 24 or 48 hours. Next, the cells were washed with 0.9% NaCl and lysed in 0.2% SDS, 1× SSC (150 mM NaCl and 15 mM Na-citrate, pH 7.0), and 5 mM EDTA, and aliquots were used to determine [3H]thymidine incorporation on a Beckman Scintillation counter (Beckman), and total DNA using Hoechst dye H33258 and a fluorescence meter (DyNA Quant 200; Hoefer). Counts from the experiments performed in triplicate from each group were compared for statistical significance with Student's t test. P < .01 was considered highly significant, and P < .05 was considered significant. The analysis from sequential (2×) siRNA-treated cells was normalized to their respective controls and is presented as a bar graph.

Soft Agar Assay

A 1.5-ml layer of 0.5% agar (wt/vol) in EMEM with 10% FBS was poured in 35-mm Petri dishes. HepG2 and Hep3B cells were treated with control or β-catenin siRNA for 24 hours at 100 nM in the presence of Oligofectamine reagent (Invitrogen) and resuspended in 0.35%agar (wt/vol) in EMEM with 5% FBS at a density of 5000 cells/1.5 ml. Cell suspensions (1.5 ml) were poured on the top of the base layer, allowed to solidify, and incubated at 37°C in the presence of 5% CO2 for 14 days (HepG2 cells) and 21 days (Hep3B cells). The colonies were stained with 0.005% crystal violet for 1 hour. Colonies containing > 10 cells were counted under an Olympus microscope (Olympus). Absolute counts were compared for statistical significance with Student's t test and are presented as a bar graph.

Results

siRNA Inhibits β-Catenin Expression in HCC Cell Lines

The human hepatoma cell lines Hep3B and HepG2 were grown to around 50% confluence, followed by transfection with either human β-catenin (CTNNB1) or control siRNA (referred to as control), as indicated in the Materials and Methods section. Protein lysates obtained from the tumor cells at different time points after CTNNB1 siRNA transfection showed a temporal decrease in total β-catenin protein in both HepG2 and Hep3B cells compared to controls (Figure 1A). HepG2 cells harbor two species of β-catenin: wild-type or the 96-kDa form, and a more prominent truncated or stable 75-kDa form (tβ-catenin) due to partial exon 3 deletion that contains phoshorylation sites critical for eventual degradation of β-catenin. siRNA transfection led to a decrease in both species of β-catenin in HepG2 cells, albeit the wild-type form showed an immediate and robust knockdown compared to tβ-catenin. Although control siRNA-transfected HepG2 cells showed a time-dependent increase in tβ-catenin protein levels, β-catenin siRNA-treated cells failed to show this increase, thus suggesting that de novo synthesis of β-catenin might also contribute to the truncated species of β-catenin. Densitometric analysis of Western immunoblots identified a dramatic and highly significant (P < .01) decrease in the full-length β-catenin protein in both HepG2 and Hep3B cells within 24 hours, although a more prominent effect was evident after 48 hours (Figure 1, B and C). In addition, a modest but significant (P < .01) decrease in tβ-catenin in HepG2 cells was observed after 48 hours, with a more dramatic difference at 72 hours after a single transfection of CTNNB1 siRNA (Figure 1B).

An external file that holds a picture, illustration, etc. Object name is neo0911_0951_fig001.jpg

Effect of CTNNB1 siRNA on β-catenin, GS, and cyclin D1 in human hepatoma cell lines (Hep3B and HepG2). All results for densitometry are presented as the mean ± SEM of three experiments and normalized to their respective controls. (A) Western blot analysis shows a decrease in total β-catenin, GS, and cyclin D1 in HepG2 (left panel) and Hep3B (right panel) cells. A lower-molecular-weight (75 kDa) truncated form of β-catenin (tβ-catenin) in HepG2 cells showed only a modest decrease, especially at 48 and 72 hours. β-Actin confirmed equal loading. (B) Densitometric analysis of HepG2 Western immunoblots revealed a significant decrease in full-length β-catenin protein (left panel) at 24 hours (P < .01), and in tβ-catenin (right panel) at 48 hours (P < .05), with a 40% additional loss at 72 hours (P < .01). (C) Densitometric analysis of Hep3B immunoblots shows a significant decrease in β-catenin protein at 24 hours, with a 50% additional decrease at 48 hours and a 70% additional decrease at 72 hours. (D) A significant decrease in cyclin D1 at 48 and 72 hours, and in GS at 72 hours (P < .05), after β-catenin siRNA transfection has been confirmed with densitometry in HepG2 cells. (E) A significant decrease in cyclin D1 and GS at 24, 48, and 72 hours (P < .01) after β-catenin siRNA transfection has been confirmed by densitometry in Hep3B cells.

siRNA Inhibits β-Catenin Activation as Observed by a Downregulation of Known Target Genes Cyclin D1 and GS

Next, to confirm whether the decrease in total β-catenin protein in both HepG2 and Hep3B cells following siRNA treatment also affected the transcriptional regulatory function of β-catenin, the protein lysates were examined for the protein expression of two known targets: cyclin D1 and GS. Although the former is regulated by additional factors as well, GS is a more selective Wnt/β-catenin target in the liver [21]. After CTNNB1 siRNA transfection, a decrease in cyclin D1 and GS was detectable in both cell lines, supporting a functional decrease in the β-catenin protein in both cell lines (Figure 1A). This decrease was more dramatic in Hep3B cells than in HepG2 cells, although it was significant in both cells following β-catenin siRNA transfections (Figure 1, D and E).

Decrease in β-Catenin and Its Nuclear Localization Are Also Detectable By Immunofluorescence in Hep3B and HepG2 Cells after siRNA Transfection

To confirm a decrease in total β-catenin, as well as any changes in its localization following CTNNB1 or control siRNA transfection, Hep3B and HepG2 cells were examined for β-catenin with immunofluorescence, as discussed in the Materials and Methods section. Although Hep3B cells transfected with control siRNA for 48 hours showed membranous and nuclear localization, a vivid decrease in β-catenin was evident in CTNNB1 siRNA-transfected cultures at the same time, which displayed occasional cells with membranous or, even rarely, nuclear β-catenin (Figure 2, A and B). In addition, although control HepG2 cells showed extensive membranous and nuclear β-catenin, the CTNNB1 siRNA-transfected cells showed a similar and pronounced β-catenin decrease at 48 hours as well (Figure 2, C and D).

An external file that holds a picture, illustration, etc. Object name is neo0911_0951_fig002.jpg

Changes in β-catenin expression and localization after siRNA treatment in Hep3B and HepG2 cells by immunofluorescence. Left panel: Original magnification, ×600. Right panel: Original magnification, ×1000. (A) Membranous and nuclear (arrowhead) β-catenin is observed in Hep3B cells at 48 hours after control siRNA transfection. (B) Decreased expression and nuclear localization of β-catenin at 48 hours after β-CTNNB1 siRNA transfection in Hep3B cells. (C) Membranous and nuclear (arrowhead) β-catenin is observed in HepG2 cells at 48 hours after control siRNA transfection. (D) Decreased expression and nuclear localization of β-catenin at 48 hours after β-CTNNB1 siRNA transfection in HepG2 cells. A small subset of cells continued to show nuclear β-catenin (arrowheads).

β-Catenin/Tcf Reporter Assay Confirms siRNA-Mediated Loss of β-Catenin Activity in Hep3B and HepG2 Cells

Next, we examined the effect of β-catenin knockdown on its activity with the TOPflash reporter assay, which is a direct and reliable measure of the β-catenin/Tcf-dependent transcriptional activity. TOPflash and FOPflash activities were measured following β-catenin siRNA transfection for 48 hours, as described in the Materials and Methods section. A significant downregulation in TOPflash reporter activity, without any effect on FOPflash activity, was apparent after CTNNB1 siRNA transfection in Hep3B and HepG2 tumor cells compared to control siRNA-transfected cells (Figure 3). The activity was decreased around threefold to fivefold in HepG2 cells, and by fivefold to eightfold in Hep3B cells, clearly identifying a pronounced loss of β-catenin function in both tumor cells.

An external file that holds a picture, illustration, etc. Object name is neo0911_0951_fig003.jpg

TOPflash reporter assay demonstrates a significant decrease in β-catenin transcriptional activity in Hep3B and HepG2 cells following β-catenin siRNA transfection for 48 hours (P < .01). Luciferase activity in FOPflash remained unaffected, confirming a lack of nonspecific activation of the reporter system. A vector containing Renilla luciferase was used as an internal control for transfection efficiency, and the results are expressed as relative firefly/Renilla luciferase activity. The results presented are the mean ± SEM for three experiments.

Effect of β-Catenin Knockdown on the Survival of Hepatoma Cells

To address any biologic relevance of the loss of β-catenin function, we investigated the impact of β-catenin siRNA on the survival of Hep3B and HepG2 cells. CTNNB1 siRNA-transfected tumor cells were examined for viability by MTT assay, as described in the Materials and Methods section. Although a consistently small but insignificant effect on the survival of HepG2 cells was apparent after 24 hours of transfection, a more robust (∼ 35%) and significant (P < .01) decrease was observed at 48 and 72 hours (Figure 4A).

An external file that holds a picture, illustration, etc. Object name is neo0911_0951_fig004.jpg

Loss of β-catenin affects Hep3B and HepG2 cell viability by MTT assay. The results represent the mean ± SEM of three experiments and are presented as a bar graph after normalizing to the respective controls. (A) A significant decrease in the viability of HepG2 cells was observed after 48 and 72 hours of β-catenin siRNA transfection. (B) A significant decrease in the viability of Hep3B cells was observed at 24, 48, and 72 hours after β-catenin siRNA transfection.

A significant decrease in Hep3B cell viability was observed as early as 24 hours after CTNNB1 siRNA transfection (P < .01), which was further augmented to around a 40% decrease in viability by 48 to 72 hours (Figure 4B). These results demonstrate an important role in tumor cell viability.

Effect of β-Catenin siRNA on the Proliferation of HCC Cells

Next, we examined the effect of β-catenin loss on tumor cell proliferation. Thymidine incorporation was employed as a measure of DNA synthesis after CTNNB1 or control siRNA transfection, as described in the Materials and Methods section. A significant decrease in thymidine incorporation in β-catenin siRNA-transfected HepG2 cells (P < .05), which ranged from around 20% to 35%, was identified after 48 and 72 hours (Figure 5A). Although a 20% and significant decrease in β-catenin siRNA-transfected Hep3B cells was observed at 48 hours (P < .05), a pronounced (> 50%) and highly significant (P < .01) decrease was readily identifiable at 72 hours (Figure 5B).

An external file that holds a picture, illustration, etc. Object name is neo0911_0951_fig005.jpg

Loss of β-catenin compromises tumor cell proliferation by thymidine incorporation. The results represent the mean ± SEM of three experiments. (A) A significant decrease in thymidine incorporation in HepG2 cells observed at 48 and 72 hours after β-catenin siRNA transfection (P < .05). (B) A significant decrease in thymidine incorporation in Hep3B cells observed after 48 and 72 hours of β-catenin siRNA transfection (P < .01). (C) Bar graph shows a significant decrease in thymidine incorporation (P < .001) by 40% and 70% in HepG2 cells at 24 and 48 hours, respectively, after two sequential siRNA transfections performed 24 hours apart. Data presented were normalized to control siRNA for each time point and treatment. (D) A representative photograph shows a noteworthy decrease in the numbers of soft agar colonies formed by HepG2 cells at 14 days, and by Hep3B cells at 21 days, after the initial 24-hour transfection with β-catenin siRNA compared to the controls. (E) Histogram shows a significant decrease in the number of colonies of both HepG2 (top) and Hep3B (bottom) in soft agar in the β-catenin siRNA-treated group compared to the respective controls at the same time (P < .01). The experiment was performed in triplicate, and average (±SD) data were presented.

Because HepG2 cells harbor a β-catenin deletion rendering a more stable protein, we examined the efficacy of employing serial CTNNB1 siRNA transfections in HepG2 cells, as detailed in the Materials and Methods section. An additional transfection of CTNNB1 siRNA led to a dramatic and extremely significant (P < .001) decrease in thymidine incorporation after 24 hours (40%) and 48 hours (> 70%) in HepG2 cells (Figure 5C). Thus, the above results demonstrate a critical role of β-catenin in tumor cell proliferation.

Effect of β-Catenin siRNA on the Soft Agar Growth of Hepatoma Cells

Next, we examined the effect of β-catenin loss on the growth of hepatoma cells in soft agar. Twenty-four hours after the control or β-catenin siRNA transfection of HepG2 and Hep3B cells, these were cultured in soft agar for 14 and 21 days, respectively. A dramatic decrease in crystal violet-stained colonies was evident in the β-catenin siRNA-transfected HepG2 and Hep3B cells, as shown in representative cultures (Figure 5D). This difference was statistically significant, with the numbers of colonies in the β-catenin siRNA-transfected cells being around 30% of the control siRNA-treated group (Figure 5E).

Discussion

HCC is the commonest primary malignant tumor of the liver that has been observed with increasing incidence due to an increased prevalence of risk factors, including nonalcoholic and alcoholic fatty liver disease and infections such as hepatitis B and C [14,25]. In addition, the lack of optimal treatment adds to the overall grim prognosis of the disease, making it prudent to elucidate novel molecular mechanisms, which in turn might reveal new therapeutic targets [25]. β-Catenin, which is the chief oncogenic component of the canonical Wnt pathway, has been identified to be active in a significant subset of HCC, which, based on mechanisms, ranges from 15% to 90% [12,17,26]. Furthermore, β-catenin activation and cytoplasmic/nuclear localization have been associated with increased proliferation and survival of hepatocytes in normal physiology and tumor cell survival/proliferation in HCC [2,20,21,26–28]. This implicates β-catenin as an attractive therapeutic target in HCC. The current study provides a proof of principle that β-catenin inhibition is feasible, is of clinical relevance, and has functional consequences in HCC cells, which show β-catenin overexpression and/or activation.

We examined the effect of the inhibition of β-catenin by siRNA in human HCC cell lines. In our current study, we used two hepatoma cell lines that have appreciable β-catenin expression but have distinct bases of β-catenin activation. HepG2 cells have a heterozygous deletion of 348 nucleotides in exon 3 of the β-catenin gene, which encode a total of 116 amino acids from trytophan-25 to asparagine-141 [29]. Critical amino acids in this region have been shown to be necessary for GSK3β or casein kinase-driven β-catenin phosphorylation and eventual degradation [30–32]. Hep3B cells, however, were derived from hepatitis B-infected liver tumor, and although they do not contain any mutations or deletions in the β-catenin gene, these cells show high levels of β-catenin protein [33,34]. One of the mechanisms of such observation may be the presence of elevated levels of inactive GSK3β (GSK3β Ser9-phosphorylated) in these cells [34]. In our current study, we used these two cell lines to examine the efficacy of β-catenin knockdown by siRNA against CTNNB1 along with its functional consequences, including effects on proliferation and survival.

A dramatic decrease in β-catenin expression, nuclear localization, and activation was evident within 24 to 48 hours in Hep3B cells, which exhibit elevated wild-type β-catenin. A concomitant decrease in tumor cell proliferation, growth in soft agar, and survival was also evident at the corresponding times. The extent of the decrease in these biologic events was fairly robust in these cells. A similar effect of CTNNB1 siRNA on the wild-type species of β-catenin in HepG2 cells was evident within 24 to 48 hours. However, the effect of siRNA on the truncated form, which is the predominant functional form of β-catenin in HepG2 cells, was less dramatic and mainly evident after 48 hours. There was a modest but significant decrease in tumor cell viability and proliferation after 48 hours of CTNNB1 siRNA in this case, which might be the consequence of the decrease in both wild-type and truncated β-catenin species. In addition, the soft agar growth of HepG2 was equally affected by siRNA against β-catenin and was comparable to Hep3B cells, suggesting that suppression of β-catenin transcription was equally efficacious in long-term studies, irrespective of the mutational status of β-catenin. Alternatively, tβ-catenin was dramatically decreased in HepG2 cells after two consecutive 24-hour transfections with CTNNB1 siRNA, with a more robust decrease in tumor cell proliferation. These findings clearly indicate the clinical relevance of the transcriptional downregulation of β-catenin in HCC.

Based on the above findings, there appears to be an obvious merit of β-catenin suppression in β-catenin-overexpressing liver tumors independent of any mutations in CTNNB1. However, the caveat is that β-catenin suppression would need to be prolonged in the event of mutations in the β-catenin gene itself, as the truncated protein is stable over prolonged time. Nonetheless, sustained β-catenin suppression in overexpressing tumors would have promising biologic consequences on tumor cell viability and proliferation.

Antisense modalities are being tested preclinically and clinically, and are a part of a bigger “antigene” strategy for cancer prevention and treatment [35]. Antisense therapies for treatment in a clinical setup are still in the stage of infancy. The current clinical trials are mainly aimed at chemosensitization of the tumors and similar applications [36–38]. Although no clinical studies are available, preclinical β-catenin suppression using antisense modalities has been shown to have relevance in colorectal cancer cells and other tumor cells such as melanoma and sarcoma [39–41]. It remains to be seen whether successful β-catenin suppression through antisense techniques, including classic antisense oligodeoxynucleotide technology, antisense RNA technology, or additional means, will have translational significance. Successful β-catenin suppression in vivo has been achieved by phosphomorpholino oligonucleotides during liver regeneration [42].

A yet another prospective way of β-catenin suppression in the clinical setting might be the use of agents that inhibit β-catenin gene and protein expression, along with its activity. PPAR-γ agonists suppress β-catenin transcriptionally and posttranslationally [43,44]. We have recently identified a role for R-Etodolac in inhibiting β-catenin expression and activation as well [23]. Such agents would therefore have strong clinical relevance in inhibiting β-catenin activity in liver cancers exhibiting β-catenin overexpression independent of mutations in the β-catenin gene.

Footnotes

1This report was funded, in part, by the American Cancer Society (grant RSG-03-141-01-CNE to S.P.S.M.) and the National Institutes of Health (grants 1RO1DK62277 and 1R01CA124414 to S.P.S.M.) Rango's Fund for Enhancement of Pathology Research.

References

1. Peifer M, Polakis P. Wnt signaling in oncogenesis and embryogenesis—a look outside the nucleus. Science. 2000;287(5458):1606–1609. [PubMed] [Google Scholar]
2. Morin PJ. Beta-catenin signaling and cancer. Bioessays. 1999;21(12):1021–1030. [PubMed] [Google Scholar]
3. He TC, Sparks AB, Rago C, Hermeking H, Zawel L, da Costa LT, Morin PJ, Vogelstein B, Kinzler KW. Identification of c-MYC as a target of the APC pathway. Science. 1998;281(5382):1509–1512. [PubMed] [Google Scholar]
4. Pennisi E. How a growth control path takes a wrong turn to cancer. Science. 1998;281(5382):1438–1439. (1441 [news; comment; published erratum appears in Science281 (5384), 1809, 1998]) [PubMed] [Google Scholar]
5. Zeng G, Germinaro M, Micsenyi A, Monga NK, Bell A, Sood A, Malhotra V, Sood N, Midda V, Monga DK, et al. Aberrant Wnt/beta-catenin signaling in pancreatic adenocarcinoma. Neoplasia. 2006;8(4):279–289. [PMC free article] [PubMed] [Google Scholar]
6. Smalley M, Leiper K, Tootle R, McCloskey P, O'Hare MJ, Hodgson H. Immortalization of human hepatocytes by temperature-sensitive SV40 large-T antigen. In Vitro Cell Dev Biol Anim. 2001;37(3):166–168. [PubMed] [Google Scholar]
7. Rimm DL, Caca K, Hu G, Harrison FB, Fearon ER. Frequent nuclear/cytoplasmic localization of beta-catenin without exon 3 mutations in malignant melanoma. Am J Pathol. 1999;154(2):325–329. [PMC free article] [PubMed] [Google Scholar]
8. Jaiswal AS, Kennedy CH, Narayan S. A correlation of APC and c-myc mRNA levels in lung cancer cell lines. Oncol Rep. 1999;6(6):1253–1256. [PubMed] [Google Scholar]
9. de La Coste A, Romagnolo B, Billuart P, Renard CA, Buendia MA, Soubrane O, Fabre M, Chelly J, Beldjord C, Kahn A, et al. Somatic mutations of the beta-catenin gene are frequent in mouse and human hepatocellular carcinomas. Proc Natl Acad Sci USA. 1998;95(15):8847–8851. [PMC free article] [PubMed] [Google Scholar]
10. Hugh TJ, Dillon SA, O'Dowd G, Getty B, Pignatelli M, Poston GJ, Kinsella AR. Beta-catenin expression in primary and metastatic colorectal carcinoma. Int J Cancer. 1999;82(4):504–511. [PubMed] [Google Scholar]
11. Buendia MA. Genetics of hepatocellular carcinoma. Semin Cancer Biol. 2000;10(3):185–200. [PubMed] [Google Scholar]
12. Laurent-Puig P, Legoix P, Bluteau O, Belghiti J, Franco D, Binot F, Monges G, Thomas G, Bioulac-Sage P, Zucman-Rossi J. Genetic alterations associated with hepatocellular carcinomas define distinct pathways of hepatocarcinogenesis. Gastroenterology. 2001;120(7):1763–1773. [PubMed] [Google Scholar]
13. Bafico A, Liu G, Goldin L, Harris V, Aaronson SA. An autocrine mechanism for constitutive Wnt pathway activation in human cancer cells. Cancer Cell. 2004;6(5):497–506. [PubMed] [Google Scholar]
14. Muller C. Hepatocellular carcinoma—rising incidence, changing therapeutic strategies. Wien Med Wochenschr. 2006;156(13—14):404–409. [PubMed] [Google Scholar]
15. Satoh S, Daigo Y, Furukawa Y, Kato T, Miwa N, Nishiwaki T, Kawasoe T, Ishiguro H, Fujita M, Tokino T, et al. AXIN1 mutations in hepatocellular carcinomas, and growth suppression in cancer cells by virus-mediated transfer of AXIN1. Nat Genet. 2000;24(3):245–250. [PubMed] [Google Scholar]
16. Miyoshi Y, Iwao K, Nagasawa Y, Aihara T, Sasaki Y, Imaoka S, Murata M, Shimano T, Nakamura Y. Activation of the beta-catenin gene in primary hepatocellular carcinomas by somatic alterations involving exon 3. Cancer Res. 1998;58(12):2524–2527. [PubMed] [Google Scholar]
17. Merle P, de la Monte S, Kim M, Herrmann M, Tanaka S, Von Dem Bussche A, Kew MC, Trepo C, Wands JR. Functional consequences of frizzled-7 receptor overexpression in human hepatocellular carcinoma. Gastroenterology. 2004;127(4):1110–1122. [PubMed] [Google Scholar]
18. Zucman-Rossi J, Jeannot E, Nhieu JT, Scoazec JY, Guettier C, Rebouissou S, Bacq Y, Leteurtre E, Paradis V, Michalak S, et al. Genotype-phenotype correlation in hepatocellular adenoma: new classification and relationship with HCC. Hepatology. 2006;43(3):515–524. [PubMed] [Google Scholar]
19. Monga SP, Monga HK, Tan X, Mule K, Pediaditakis P, Michalopoulos GK. Beta-catenin antisense studies in embryonic liver cultures: role in proliferation, apoptosis, and lineage specification. Gastroenterology. 2003;124(1):202–216. [PubMed] [Google Scholar]
20. Nhieu JT, Renard CA, Wei Y, Cherqui D, Zafrani ES, Buendia MA. Nuclear accumulation of mutated beta-catenin in hepatocellular carcinoma is associated with increased cell proliferation. Am J Pathol. 1999;155(3):703–710. [PMC free article] [PubMed] [Google Scholar]
21. Tan X, Behari J, Cieply B, Michalopoulos GK, Monga SP. Conditional deletion of beta-catenin reveals its role in liver growth and regeneration. Gastroenterology. 2006;131(5):1561–1572. [PubMed] [Google Scholar]
22. Monga SP, Mars WM, Pediaditakis P, Bell A, Mulé K, Bowen WC, Wang X, Zarnegar R, Michalopoulos GK. Hepatocyte growth factor induces Wnt-independent nuclear translocation of beta-catenin after Met-beta-catenin dissociation in hepatocytes. Cancer Res. 2002;62(7):2064–2071. [PubMed] [Google Scholar]
23. Behari J, Zeng G, Otruba W, Thompson MD, Muller P, Micsenyi A, Sekhon SS, Leoni L, Monga SP. R-Etodolac decreases beta-catenin levels along with survival and proliferation of hepatoma cells. J Hepatol. 2006;46(5):849–857. [PMC free article] [PubMed] [Google Scholar]
24. Monga SP, Pediaditakis P, Mule K, Stolz DB, Michalopoulos GK. Changes in WNT/beta-catenin pathway during regulated growth in rat liver regeneration. Hepatology. 2001;33(5):1098–1109. [PMC free article] [PubMed] [Google Scholar]
25. McKillop IH, Moran DM, Jin X, Koniaris LG. Molecular pathogenesis of hepatocellular carcinoma. J Surg Res. 2006;136(1):125–135. [PubMed] [Google Scholar]
26. Schmitt-Graeff A, Ertelt-Heitzmann V, Allgaier HP, Olschewski M, Nitschke R, Haxelmans S, Koelble K, Behrens J, Blum HE. Coordinated expression of cyclin D1 and LEF-1/TCF transcription factor is restricted to a subset of hepatocellular carcinoma. Liver Int. 2005;25(4):839–847. [PubMed] [Google Scholar]
27. Apte U, Zeng G, Thompson M, Muller P, Micsenyi A, Cieply B, Kaestner KH, Monga SP. Beta-catenin is critical for early postnatal liver growth. Am J Physiol Gastrointest Liver Physiol. 2007;292(6):G1578–G1585. [PubMed] [Google Scholar]
28. Micsenyi A, Tan X, Sneddon T, Luo JH, Michalopoulos GK, Monga SP. Beta-catenin is temporally regulated during normal liver development. Gastroenterology. 2004;126(4):1134–1146. [PubMed] [Google Scholar]
29. Carruba G, Cervello M, Miceli MD, Farruggio R, Notarbartolo M, Virruso L, Giannitrapani L, Gambino R, Montalto G, Castagnetta L. Truncated form of beta-catenin and reduced expression of wild-type catenins feature HepG2 human liver cancer cells. Ann NY Acad Sci. 1999;886:212–216. [PubMed] [Google Scholar]
30. Amit S, Hatzubai A, Birman Y, Andersen JS, Ben-Shushan E, Mann M, Ben-Neriah Y, Alkalay I. Axin-mediated CKI phosphorylation of beta-catenin at Ser 45: a molecular switch for the Wnt pathway. Genes Dev. 2002;16(9):1066–1076. [PMC free article] [PubMed] [Google Scholar]
31. Liu X, Rubin JS, Kimmel AR. Rapid, Wnt-induced changes in GSK3beta associations that regulate beta-catenin stabilization are mediated by Galpha proteins. Curr Biol. 2005;15(22):1989–1997. [PubMed] [Google Scholar]
32. Wu G, He X. Threonine 41 in beta-catenin serves as a key phosphorylation relay residue in beta-catenin degradation. Biochemistry. 2006;45(16):5319–5323. [PubMed] [Google Scholar]
33. Cha MY, Kim CM, Park YM, Ryu WS. Hepatitis B virus X protein is essential for the activation of Wnt/beta-catenin signaling in hepatoma cells. Hepatology. 2004;39(6):1683–1693. [PubMed] [Google Scholar]
34. Desbois-Mouthon C, Blivet-Van Eggelpoel MJ, Beurel E, Boissan M, Delélo R, Cadoret A, Capeau J. Dysregulation of glycogen synthase kinase-3beta signaling in hepatocellular carcinoma cells. Hepatology. 2002;36(6):1528–1536. [PubMed] [Google Scholar]
35. Pillay V, Dass CR, Choong PF. The urokinase plasminogen activator receptor as a gene therapy target for cancer. Trends Biotechnol. 2007;25(1):33–39. [PubMed] [Google Scholar]
36. Gleave M, Chi KN. Knock-down of the cytoprotective gene, clusterin, to enhance hormone and chemosensitivity in prostate and other cancers. Ann NY Acad Sci. 2005;1058:1–15. [PubMed] [Google Scholar]
37. Schmitz G. Drug evaluation: OGX-011, a clusterin-inhibiting anti-sense oligonucleotide. Curr Opin Mol Ther. 2006;8(6):547–554. [PubMed] [Google Scholar]
38. Stein CA, Benimetskaya L, Mani S. Antisense strategies for oncogene inactivation. Semin Oncol. 2005;32(6):563–572. [PubMed] [Google Scholar]
39. Mikami I, You L, He B, Xu Z, Batra S, Lee AY, Mazieres J, Reguart N, Uematsu K, Koizumi K, et al. Efficacy of Wnt-1 monoclonal anti-body in sarcoma cells. BMC Cancer. 2005;5(1):53. [PMC free article] [PubMed] [Google Scholar]
40. Verma UN, Surabhi RM, Schmaltieg A, Becerra C, Gaynor RB. Small interfering RNAs directed against beta-catenin inhibit the in vitro and in vivo growth of colon cancer cells. Clin Cancer Res. 2003;9(4):1291–1300. [PubMed] [Google Scholar]
41. You L, He B, Xu Z, Uematsu K, Mazieres J, Fujii N, Mikami I, Reguart N, McIntosh JK, Kashani-Sabet M, et al. An anti-Wnt-2 monoclonal antibody induces apoptosis in malignant melanoma cells inhibits tumor growth. Cancer Res. 2004;64(15):5385–5389. [PubMed] [Google Scholar]
42. Sodhi D, Micsenyi A, Bowen WC, Monga DK, Talavera JC, Monga SP. Morpholino oligonucleotide-triggered beta-catenin knockdown compromises normal liver regeneration. J Hepatol. 2005;43(1):132–141. [PubMed] [Google Scholar]
43. Hedvat M, Jain A, Carson DA, Leoni LM, Huang G, Holden S, Lu D, Corr Fox W, Agus DB. Inhibition of HER-kinase activation prevents ERK-mediated degradation of PPARgamma. Cancer Cell. 2004;5(6):565–574. [PubMed] [Google Scholar]
44. Liu J, Wang H, Zuo Y, Farmer SR. Functional interaction between peroxisome proliferator-activated receptor gamma and beta-catenin. Mol Cell Biol. 2006;26(15):5827–5837. [PMC free article] [PubMed] [Google Scholar]
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