• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of cellresLink to Publisher's site
Cell Res. Aug 2011; 21(8): 1248–1261.
Published online Jun 21, 2011. doi:  10.1038/cr.2011.103
PMCID: PMC3193485

The oncoprotein p28GANK establishes a positive feedback loop in β-catenin signaling

Abstract

p28GANK (also known as PSMD10 or gankyrin) is a novel oncoprotein that is highly expressed in hepatocellular carcinoma (HCC). Through its interaction with various proteins, p28GANK mediates the degradation of the tumor suppressor proteins Rb and p53. Although p53 was reported to downregulate β-catenin, whether p28GANK is involved in the regulation of β-catenin remains uncertain. Here we report that both growth factors and Ras upregulate p28GANK expression through the activation of the phosphoinositide 3-kinase-AKT pathway. Upregulation of p28GANK expression subsequently enhanced the transcription activity of β-catenin. This effect was observed in p53-deficient cells, suggesting a p53-independent mechanism for the p28GANK-mediated activation of β-catenin. p28GANK overexpression also reduced E-cadherin protein levels, leading to increased release of free β-catenin into the cytoplasm from the cadherin-bound pool. Interestingly, exogenous expression of p28GANK resulted in elevated expression of the endogenous protein. We also observed that both β-catenin and c-Myc were transcriptional activators of p28GANK, and a correlation between p28GANK overexpression and c-Myc, cyclin D1 and β-catenin activation in primary human HCC. Together, these results suggest that p28GANK expression is regulated by a positive feedback loop involving β-catenin, which may play a critical role in tumorigenesis and the progression of HCC.

Keywords: p28GANK, β-catenin, E-cadherin, positive feedback loop, hepatocellular carcinoma

Introduction

Hepatocellular carcinoma (HCC) is one of the most common cancers in the world and is a major cause of death in many countries 1. HCCs display great genomic heterogeneity and may involve at least three carcinogenesis-related pathways: the p53, Rb and Wnt/β-catenin signaling pathways 2. Three decades of basic cancer research have revealed that mutations in the components of signaling pathways that control cell growth are the basis of tumor initiation in mammals. The Ras, extracellular-signal-regulated kinase (ERK) and phosphoinositide 3-kinase (PI3K) signaling pathways form an intersecting biochemical network that, when mutated, drives cell growth in a manner unrestricted by environmental cues. These pathways drive tumorigenesis through coordinated protein phosphorylation events that directly regulate protein synthesis, cell-cycle progression and activity of transcription factors that regulate the expression of genes involved in these processes 3, 4.

Most human tumors harbor mutations that activate these master regulators or inactivate negative regulators of these regulators 3, 4, 5. Each of these proteins activates a number of downstream effectors. The activation of PI3K mediates the recruitment and activation of signaling proteins with pleckstrin homology domains including the serine-threonine kinase Akt 6. Akt phosphorylates several downstream targets, including the constitutively active glycogen synthase kinase-3 (GSK3)-β (Ser9), resulting in its inhibition 7. GSK-3β is a critical component in β-catenin signaling and provides a mechanistic link between growth factor stimulation or Ras activation and β-catenin stabilization.

The β-catenin signaling pathway plays a critical role in determining cell fate, tissue homeostasis and tumorigenesis 8. In addition to its function in the Wnt signaling pathway, β-catenin also binds to the cytoplasmic domain of type I cadherins and plays an essential role in the structural organization and function of cadherins by linking cadherins to the actin cytoskeleton via α-catenin 9. The cadherin-bound pool of β-catenin can be released and made available for nuclear signaling. Binding partners of β-catenin can modify β-catenin/TCF-dependent transcription, thus regulating the expression of target genes 10. Many cancers are initiated by inappropriate activation of the Wnt pathway, which is characterized by the accumulation of nuclear β-catenin and the constitutive transcriptional activity of the β-catenin/TCF complex 11, 12. Approximately 50%-70% of all HCCs have an abnormal accumulation of β-catenin protein in the cytoplasm and the nucleus 13, 14, 15. However, in HCC, the β-catenin mutation rate is 13%-26% 13, 15 and the axin mutation rate is 5%-10% 16, 17. Few mutations of APC have been reported so far. These facts led us to speculate that other unknown factors might be involved in regulating β-catenin in HCC.

A novel oncogene named gankyrin was cloned from HCC by cDNA subtractive hybridization 18. Its sequence is identical to that of the p28 gene, whose product is one of the non-ATPase subunits of PA700 (19S), a regulatory complex of the human 26S proteasome 19. The product of this novel gene (p28GANK) forms complexes with multiple proteins. p28GANK overexpression accelerates the hyperphosphorylation and degradation of Rb, releasing the transcription factor E2F-1 from the Rb repressor complex 18. Furthermore, the binding of p28GANK to Cdk4 prevents Cdk4 from binding to p16INK4a 20, which accelerates cell cycle progression. p28GANK also binds to the E3 ubiquitin ligase MDM2, facilitating the ubiquitylation and subsequent proteasomal degradation of p53 21. Recently, an additional target of p28GANK, C/EBPα, was identified, and the degradation of C/EBPα by the ubiquitin-proteasome system was shown to contribute to the development of liver cancer 22. In the present study, we demonstrate that activation of β-catenin signaling regulates p28GANK expression, which subsequently enhances the activation of β-catenin/TCF-mediated transcription. The oncoprotein p28GANK thus establishes a positive feedback loop that regulates β-catenin signaling and plays an important role in tumorigenesis and progression of HCC.

Results

Growth factor stimulation and Ras activation upregulate p28GANK expression

To investigate factors that may influence p28GANK expression, we cloned a 1226 bp fragment of the 5′-flanking region of the human p28GANK gene into a pGL3 luciferase reporter vector, after analysis using the Gene2Promoter (Genomatix) online software. To determine whether p28GANK upregulation was correlated with cell cycle progression in normal and HCC cell lines, we examined the potential cell cycle dependence of p28GANK expression in HepG2 and HEK293 cells after mitogenic stimulation. Starvation has been previously shown to induce cell cycle arrest, but starved cells were able to enter S phase after stimulation with growth factors, such as epidermal growth factor (EGF) or hepatocyte growth factor (HGF) 23, 24. We thus used reporter gene assays to measure the effect of growth factor stimulation on p28GANK expression. As shown in Figure 1A and and1B,1B, p28GANK reporter gene activity was significantly increased after stimulation with different doses of EGF or HGF. Endogenous p28GANK expression was strongly induced in liver cells after 6 to 48 h of stimulation with EGF (Figure 1C, ,1D1D and Supplementary information, Figure S1A). EGF treatment also increased c-Myc protein levels (Figure 1D, Supplementary information, Figure S1A).

Figure 1
Growth factor stimulation and Ras activation upregulated p28GANK expression. Growth factor stimulation upregulated p28GANK expression in (A) HepG2 and (B) HEK293 cells. Cells were transiently transfected with p28GANK reporter plasmid and the control plasmid ...

Ras is a key signal transduction component downstream of growth factors; therefore, we aimed to determine whether Ras is involved in regulating p28GANK expression. As shown in Figure 1E, the relative luciferase activity of p28GANK reporter increased in a dose-dependent manner after Ras transfection. Quantitative RT-PCR and western blot analysis also showed that p28GANK expression was upregulated at both the mRNA and the protein levels (Figure 1F and and1G).1G). The results also demonstrated that Ras increased the levels of c-Myc protein, indicating that Ras activation regulated c-Myc and p28GANK levels simultaneously.

Growth factor stimulation and Ras activation increase p28GANK expression through the activation of PI3K signaling

The ability of growth factors and Ras to activate the PI3K-AKT and Raf-MEK-ERK kinase cascades prompted us to ask which pathway is involved in regulating p28GANK expression. LY294002, a PI3K inhibitor, significantly suppressed EGF- or HGF-induced p28GANK reporter gene activity, and conversely, the ERK inhibitors PD98059 and U0126 enhanced reporter gene activity (Figure 2A and data not shown). In Ras-transfected cells, these inhibitors evoked the same effects (Figure 2B). Moreover, inhibiting PI3K signaling in unstimulated cells also reduced p28GANK reporter expression, indicating that PI3K activity had a positive effect on p28GANK expression (Figure 2B). To evaluate the correlation between the level of expression of p28GANK and the activity levels of AKT and ERK, we assessed the expression of p28GANK and the phosphorylation of AKT and ERK in several HCC cell lines and 40 primary HCC samples. p28GANK protein levels more closely correlated with AKT activation than with ERK activation (Figure 2C, ,2D,2D, Supplementary information, Figure S1D). p28GANK expression was also examined in untreated HCC cell lines and HCC cell lines treated with EGF or PI3K inhibitors. EGF significantly activated AKT and upregulated p28GANK expression. Correspondingly, the administration of LY294002 or wortmannin, inhibitors of PI3K, inhibited AKT phosphorylation and decreased p28GANK expression (Figure 2E, Supplementary information, Figure S1B). Further, we used a constitutively active AKT construct, pCMV5-myr-AKT1 and wild-type AKT1 cDNA to investigate the effects of AKT on p28GANK expression. AKT activation upregulated p28GANK expression in L02 and HEK293 cells (Figure 2F and and2G,2G, Supplementary information, Figure S1C).

Figure 2
Growth factors and Ras increased p28GANK expression through the activation of PI3K-AKT signaling. (A) PI3K inhibition abolished growth factor-induced p28GANK upregulation. HEK293 cells were transiently transfected with a p28GANK reporter plasmid. Cells ...

p28GANK positively regulates β-catenin signaling independent of p53

Previous studies have revealed that p28GANK can promote cell proliferation. β-catenin signaling is one of the most important cascades in regulating cell survival and proliferation. Therefore, we aimed to determine whether p28GANK expression affects β-catenin/TCF-dependent transcription. The effect of p28GANK expression on β-catenin/TCF-dependent reporter activity was assessed using the TopFlash (pGL-OT and the mutant control construct pGL-OF) reporter system in HEK293 and HepG2 cells. The expression of p28GANK induced the activation of β-catenin/TCF-mediated reporter expression in a concentration-dependent manner in HEK293 cells (Figure 3A). β-catenin/TCF-dependent reporter activity was also assessed in HepG2 cells after adenovirus delivery of siRNA against p28GANK (AdSip28). Consistent with our results in HEK293 cells, pGL-OT activity was reduced by p28GANK inhibition (Figure 3B). To determine whether knockdown of endogenous β-catenin would affect p28GANK-mediated activation of the reporter, we examined reporter activation after using an shRNA to target β-catenin. As shown in Figure 3C, knockdown of β-catenin significantly suppressed p28GANK-induced activation of this reporter. Next, we analyzed the effects of p28GANK on the distribution of β-catenin in the nucleus and cytoplasm of HepG2 and HEK293 cells. We found that p28GANK not only increased β-catenin nuclear accumulation but also moderately elevated β-catenin protein levels in the cytoplasm (Figure 3D, Supplementary information, Figure S2A). To further assess the regulation of β-catenin by p28GANK, we knocked down p28GANK in HepG2 cells and detected the nuclear protein levels of β-catenin and other related proteins. As shown in Figure 3E, nuclear β-catenin, c-Myc and cyclinD1 decreased with the loss of p28GANK.

Figure 3
p28GANK positively regulated β-catenin signaling. (A) p28GANK increased β-catenin/TCF signaling activity. HEK293 cells were transferred to a 48-well plate and co-transfected with pGL-OT or pGL-OF in combination with increasing amounts ...

It is known that overexpression of wild-type p53 downregulates β-catenin in a variety of cell types, and this effect is dependent on the integrity and functionality of p53 25, 26. Previous reports have shown that the binding of p28GANK to MDM2 facilitates p53-MDM2 interactions and increases the ubiquitylation and degradation of p53 24. We therefore asked whether p28GANK might regulate β-catenin through p53. We examined the activity of the pGL-OT reporter in p53-null Hep3B cells. As shown in Figure 3F, the reporter activity decreased with decreasing expression of p28GANK. The real-time PCR assay also indicated that a decrease in p28GANK resulted in the downregulation of c-Myc and cyclin D1 expression (Figure 3G). Our observations suggest a novel role for p28GANK in positively regulating β-catenin/TCF-mediated transcription.

Recently, a study revealed that p28GANK can activate AKT 27, which phosphorylates several downstream targets including GSK-3β (Ser9), resulting in its inhibition 7. Given that GSK-3β provides a mechanistic link between growth factor stimulation or Ras activation and β-catenin stabilization, we examined the ability of p28GANK to regulate AKT and GSK-3β phosphorylation. In HepG2, L02 and Huh7 cells, the expression of p28GANK significantly increased p-AKT levels, but had no effect on p-GSK-3β levels (Supplementary information, Figure S2B-S2D). Similar results were observed when p28GANK was stably knocked down in Huh7 and LM3 cells (Supplementary information, Figure S2E). Taken together, these data indicate that the GSK-3β pathway may not be the main mechanism through which p28GANK plays its regulatory role.

Expression of p28GANK decreased E-cadherin levels and increased free cytoplasmic β-catenin

To determine whether the induction of β-catenin signaling was due to β-catenin being released from the membrane-associated E-cadherin/β-catenin complex pool, levels of E-cadherin from HEK293 cells overexpressing p28GANK and HepG2 cells were analyzed by immunoblotting. As shown in Figure 4A, overexpression of p28GANK significantly downregulated E-cadherin expression in HEK293 cells. The use of adenovirus-delivered siRNA to suppress p28GANK expression in HepG2 cells decreased levels of p28GANK and increased E-cadherin protein levels (Figure 4B). Further, in Huh7 and QSG7701 cells, adenovirus-mediated overexpression of p28GANK markedly decreased E-cadherin protein levels (Figure 4C and and4D).4D). Next, we performed co-IP assays in liver cancer cells by precipitating either p28GANK or E-cadherin. Even though p28GANK and E-cadherin can both form a complex with MDM2, we could not detect a p28/E-cadherin complex (Supplementary information, Figure 3A and 3B). These data are consistent with previous studies that identified E-cadherin as an MDM2-binding protein and confirmed that E-cadherin was a substrate for the MDM2 E3 ubiquitin ligase 28.

Figure 4
Expression of p28GANK led to decreased E-cadherin and increased free cytoplasmic β-catenin. (A) Expression of p28GANK led to decreased E-cadherin in HEK293 cells. HEK293 cells were seeded in a 12-well plate and transfected with increasing amounts ...

To further characterize the effect of p28GANK, we evaluated the level of the E-cadherin/β-catenin complex by immunoprecipitation. We observed that the level of E-cadherin/β-catenin complex decreased upon expression of p28GANK (data not shown). To further confirm this result, we loaded lysates containing equal amounts of E-cadherin protein to repeat the immunoprecipitation assay. As shown in Figure 4E, the levels of associated β-catenin protein were moderately decreased upon p28GANK expression. Cell fractionation with Triton X-100 was used to evaluate the attachment of the E-cadherin/catenins complex to the cytoskeleton. As seen in Figure 4F, the Triton X-100-insoluble fraction of p28GANK-expressing cells contained significantly less E-cadherin/β-catenin complex than that of control cells, indicating that p28GANK attenuated the attachment of the E-cadherin/β-catenin complex to the cytoskeleton. Notably, β-catenin levels were higher in the Triton X-100 soluble fraction of p28GANK-expressing cells than in that of control cells (Figure 4F, Supplementary information, Figure 3C). These data support the hypothesis that the expression of p28GANK is correlated with reduced membrane localization of the E-cadherin/β-catenin complex, which results in increased levels of cytoplasmic β-catenin.

β-catenin and c-Myc are transcriptional activators of p28GANK

β-catenin and c-Myc are two transcription factors that play important roles in regulating cell growth and cell cycle progression; therefore, we asked whether there was a relationship between p28GANK and these two factors. Reporter gene assays were performed in HEK293 cells that expressed exogenous β-catenin and c-Myc. To our surprise, both β-catenin and c-Myc significantly increased p28GANK promoter activity (Figure 5A and and5B).5B). The quantitative PCR results further confirmed the positive roles of β-catenin and c-Myc in regulating the expression of p28GANK (Figure 5C and and5D).5D). In HEK293 cells, overexpression of wild-type β-catenin by transient transfection increased p28GANK protein levels (Figure 5E, Supplementary information, Figure S4A). Overexpression of c-Myc also induced p28GANK protein levels (Figure 5F, Supplementary information, Figure S4B). Using shRNA to inhibit β-catenin expression in L02 and Huh7 cells, we found that p28GANK expression decreased with decreasing levels of β-catenin (Figure 5G and and5H5H).

Figure 5
β-catenin and c-Myc are transcriptional activators of p28GANK. (A, B) p28GANK promoter activity was upregulated by β-catenin and c-Myc. HEK293 cells were transiently transfected with pGL3-p28 and increasing amounts of β-catenin ...

To assess the role of β-catenin and c-Myc in the transcriptional activation of p28GANK, we analyzed the p28GANK promoter using MatInspector (Genomatix) and found putative DNA-binding sites for E2F, NFAT, STAT1, SP1, v-Myb, HIF-1, GATA and other transcription factors. Interestingly, putative binding sites for TCF/LEF and Myc/Max were also predicted. To determine whether β-catenin and Myc transcription complexes functioned through these two predicted sites, we mutated the predicted sites in the reporter construct (Supplementary information, Figure S4C). The mutation of these two predicted sites did not eliminate the activation of the reporter upon the overexpression of β-catenin or c-Myc (Supplementary information, Figure S4D and S4E). Furthermore, no direct binding of β-catenin/TCF or Myc/Max complexes to the putative transcriptional regulation sites was detected by ChIP assay (data not shown).

p28GANK expression correlates with β-catenin activation and establishes a positive feedback loop to regulate β-catenin signaling in HCC

Although previous studies have reported varying expression levels and localization of wild-type and mutant β-catenin proteins, most reports indicate that the nuclear localization of β-catenin is correlated with tumor progression and tumor cell proliferation 29, 30, 31. c-Myc and cyclin D1 are major regulators of cell cycle progression and cell proliferation. Increased β-catenin levels may promote neoplastic conversion by triggering the expression of c-Myc and cyclin D1, resulting in uncontrolled cell cycle progression. Thus, we assessed the correlation between β-catenin signaling and the expression of p28GANK in human primary hepatic tumors and found a correlation between the activation of β-catenin signaling and the expression of p28GANK, c-Myc and cyclin D1 (Figure 6A, Supplementary information, Figure S5A). We previously reported that β-catenin signaling was significantly activated in tumorigenic OV6-positive liver progenitor cells 32. We thus detected the expression of c-Myc, cyclin D1 and p28GANK in sorted OV6+ SMMC7721 liver carcinoma cells and found that the levels of these gene products were higher in OV6+ cells than in OV6- cells (Figure 6B). These data indicate that β-catenin signaling-induced p28GANK expression may be an important mechanism for promoting cell cycle activation in human liver cancers.

Figure 6
p28GANK expression correlated with β-catenin and established a positive feedback loop in β-catenin signaling. (A) Immunoblot analysis of proteins prepared from primary human hepatic tumors (T) and apparently normal liver tissues (N). ...

We further examined the effect of p28GANK expression on the activity of its own promoter in HEK293 and Hep3B cells. p28GANK significantly increased the expression of a reporter driven by the p28GANK promoter in HEK293 cells (Figure 6C) but had no effect on the activity of a pGL3-luc control construct (Supplementary information, Figure S5B). Furthermore, knocking down endogenous p28GANK in Hep3B cells resulted in a decrease in p28GANK promoter activity (Figure 6D). Conversely, transient transfection of HEK293 cells with a GFP-tagged p28GANK expression vector or HepG2 cells with myc-p28GANK resulted in increased levels of endogenous p28GANK (Figure 6E, ,6F6F and Supplementary information, Figure S5C and S5D).

Discussion

This study demonstrates that growth factor stimulation and Ras activation upregulate p28GANK expression through the activation of β-catenin signaling. Elevated expression of β-catenin, which results in the constitutive activation of numerous β-catenin/TCF target genes, has been detected in various types of cancers, including HCC. In the present study, we found a correlation between the presence of activated β-catenin and increased p28GANK expression. Interestingly, elevated p28GANK expression positively regulated β-catenin/TCF transcriptional activity. Growth factors and Ras activation activated β-catenin and c-Myc through the Raf-ERK and PI3K-AKT pathways; our results indicate that the PI3K pathway positively controlled p28GANK expression. Akt is a general mediator of growth factor-induced survival and in a number of cell types it has been shown to suppress apoptosis induced by growth factor withdrawal, cell-cycle discordance, loss of cell adhesion and DNA damage 6, 33. Thus, a signaling pathway has been defined in which growth factor receptor activation leads to the sequential activation of PI3K and Akt, which then promotes cell survival and inhibits apoptosis. Our studies and others have revealed that p28GANK can increase resistance to DNA damage-induced apoptosis 24, 34.

Previous studies have found that the activation of β-catenin signaling is mediated by multiple target genes, most of which are direct β-catenin/TCF-dependent transcriptional targets 11, 35. We demonstrated that p28GANK is regulated by β-catenin signaling and that the elevated expression of p28GANK positively regulates β-catenin/TCF transcriptional activity. Earlier studies have shown that high levels of functional p53 can downregulate the amount and transcriptional activity of β-catenin 25, 26. This effect was eliminated by blocking the activities of components that regulate the turnover of β-catenin, such as GSK-3β and the proteasomal system. In addition, Higashitsuji et al. demonstrated that p28GANK can bind to MDM2 and increase the ubiquitination and degradation of p53 21. These reports led us to hypothesize that p28GANK might affect β-catenin through the function of p53. By performing experiments using the p53-negative cell line Hep3B, we showed that the effect of p28GANK on β-catenin was independent of p53.

Man et al. demonstrated that p28GANK plays an essential role in Ras-induced tumorigenesis and promotes activation of AKT 27. Given that GSK-3β provides a mechanistic link between growth factor stimulation or Ras activation and β-catenin stabilization, we examined whether p28GANK regulates AKT and GSK-3β phosphorylation. We found that p28GANK significantly enhanced the phosphorylation of AKT, but showed no effect on GSK-3β. We were unable to demonstrate a direct protein-protein interaction between p28GANK and β-catenin or GSK3β, suggesting that additional proteins are likely to play roles in regulating β-catenin. p28GANK is a component of the 26S proteasome, which is capable of directly or indirectly regulating turnover of most proteins. Yang et al. reported that MDM2 associates with E-cadherin and regulates its degradation 28. This interaction suggests a potential role for the association of p28GANK with MDM2 in regulating E-cadherin stability. Our results demonstrate that overexpression of p28GANK decreased the levels of E-cadherin, which, in turn, decreased the levels of membrane-bound β-catenin, resulting in a corresponding increase in cytoplasmic β-catenin. Furthermore, we investigated whether p28GANK, MDM2 and E-cadherin form a complex and found that even though p28GANK and E-cadherin both formed a complex with MDM2, we were unable to detect an interaction between p28GNAK and E-cadherin. Therefore, the exact mechanism by which p28GANK downregulates E-cadherin remains to be elucidated. The role of p28GANK in regulating the distribution of β-catenin between the membrane and the cytoplasm may partially explain the mechanism by which p28GANK regulates β-catenin signaling.

In HCC, accumulation of β-catenin presents at an early stage. Most authors have reported that the prevalent type of HCC is of a moderately differentiated to well-differentiated grade with β-catenin expression at the membrane and in the cytoplasm 14, 29, 30. These conclusions are consistent with recent results showing that p28GANK plays an oncogenic role primarily at the early stages of human hepatocarcinogenesis 36. β-catenin can enter the nucleus and regulate the transcription of target genes such as cyclin D1 and c-Myc 10, 30. Both proteins play a role in the G1-S checkpoint of the cell cycle by affecting the activity of retinoblastoma tumor-suppressor (Rb) 37. p28GANK can accelerate the degradation of Rb and is coordinately expressed with β-catenin, c-Myc and cyclin D1, further indicating that p28GANK plays a role similar to that of c-Myc and cyclin D1 in HCC progression. Interestingly, more than 50% of murine HCCs induced by transgenic expression of c-Myc contained mutations in the β-catenin regulatory sequence 13. Similar mutations were found in HCCs of H-ras transgenic mice. These results indicate that the Ras and c-Myc cancer pathways in this tissue operate in parallel with mutant β-catenin. The function of p28GANK in tumorigenesis coincides with Ras and c-Myc. β-catenin signaling contributes to the activation of tumorigenic OV6+ liver progenitor cells 32 and we detected p28GANK expression after sorting OV6+ cells. Surprisingly, much higher p28GANK mRNA levels were present in OV6+ liver cells than in OV6- cells (Figure 6B), indicating a potential role of p28GANK in regulating stem cell-like self-renewal.

Cancer cells of different origins often use the mechanisms of normal embryonic development to achieve their malignant status. The β-catenin/TCF pathway is a classic example. Similarly, p28GANK was previously shown to be highly expressed during normal liver regeneration as well as in HCC 38. This oncofetal pattern of p28GANK expression could potentially be a result of β-catenin/TCF activation during tumorigenesis. Transcriptional upregulation of p28GANK by β-catenin/TCF may therefore contribute to the pleiotropic effects of the β-catenin/TCF signaling pathway and could result in a significant function for p28GANK in β-catenin-mediated oncogenesis. Future studies will define the role of p28GANK in β-catenin/TCF-induced HCC tumorigenesis. Our findings here outline a positive feedback loop involving β-catenin signaling and p28GANK in which excess β-catenin induces the accumulation of p28GANK and high p28GANK levels upregulate β-catenin/TCF transcriptional activation. This feedback loop may therefore underlie the high expression of p28GANK observed in HCC and disruption of the feedback loop through knockdown of p28GANK may affect tumorigenesis.

Materials and Methods

Antibodies and reagents

p28GANK-(sc-8991), c-Myc-(sc-40), β-catenin-(sc-7963) and GSK-3β-(sc-9166) specific antibodies were obtained from Santa Cruz Biotechnology. The HA-tag-, cyclin D1- and histone H3-specific antibodies were obtained from Cell Signaling Technology (Beverly, MA). The MDM2 (F-414) antibodies were obtained from Bioworld technology. The β-actin- and GAPDH-specific antibodies were obtained from Kangcheng Biosystem. EGF was purchased from BD Biosciences (San Diego, CA) and HGF was obtained from PeproTech (Rocky Hill, NJ). Inhibitors PD98059 and LY294002 were purchased from Sigma (St. Louis, MO) and used at a final concentration of 15 μM.

Plasmids

The human p28GANK reporter vector was constructed by cloning a 1 226 bp putative p28GANK promoter region into a pGL3-basic reporter vector containing the promoter driving the firefly luciferase gene (Promega). The proximal promoter region was first amplified by PCR from the human genome using primers based on the genomic DNA sequence of the p28GANK gene. The forward primer (5′-ACAGCCGTTAGAGCTTCACCAATCAC-3′) contained an Xho I restriction enzyme site at the 5′ end and the reverse primer (5′-ACAATAGGGAACACTAATTCTGAGCC-3′) carried a Hind III site. The PCR product was then cloned into the pGL3-Basic reporter vector with the same sites. PSG5-PI3Kp85 was a kind gift from Dr Claudia Cosentino (Genome Stability Cancer Research Institute, London, UK); PCMV5-myr-AKT1 and pCMV5-wt-AKT1 were provided by Dr Jin Q Cheng (Department of Pathology, University of South Florida, USA); and pGL-β-catenin-OT was kindly provided by Daru Lu (Fudan University, Shanghai, China). The expression vectors containing Ras, pCMV-c-Myc and pcDNA3-β-catenin were obtained from the Chinese National Human Genome Center (Shanghai, China).

Cell lines, transfections, adenoviral infection and luciferase assays

The cell lines were obtained from the American Type Culture Collection (Manassas, VA) or the Cancer Institute of Shanghai Jiao Tong University (Shanghai, China). Cells were grown in DMEM or RPMI 1640 (Gibco BRL, Life Technologies) supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin. Cells were maintained in a humidified 37 °C incubator with 5% CO2. Adenovirus was produced as previously described 34. Viral infection was performed in a minimal volume of serum-free DMEM for 1.5 h. Mock cultures that did not contain virus were used as controls. After infection for 1.5 h, 2 ml of fresh growth medium was added and cells were placed in the incubator. Cells were co-transfected with a mixture of the indicated luciferase reporter plasmids, the renilla luciferase control reporter vector pRL-TK and the indicated amounts of constructs by PEI (Polyplus, AFAQ). After treatment, the cells were lysed, and luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer's recommendations.

Reverse transcription polymerase chain reaction and real-time qPCR

Total RNA was isolated with TRIzol reagent (Invitrogen) and reverse transcribed with SuperScript III reverse transcriptase (Invitrogen) according to the manufacturer's instructions. PCR amplification was performed in a 50 μl reaction mixture containing 2 μl cDNA, 1× PCR buffer, 1.5 mmol/L MgCl2, 0.8 mmol/L deoxynucleotide triphosphatase, 0.2 μmol/L each primer and 1 unit Taq DNA polymerase (Roche Molecular Systems, Inc., Pleasanton, CA). For real-time PCR, we used the specific SYBR-Fluo from TaKaRa Biotechnology Co., Ltd. PCR was performed with the following primers: p28-F, 5′-TGGAGGGGTGTGTGTCTAAC-3′ and p28-R, 5′-ACTTGCAGGGGTGTCTTTTC-3′ c-Myc-F, 5′-TCCAGCTTGTACCTGCAGGATCTGA-3′ and c-Myc-R, 5′-CCTCCAGCAGAAGGTGATCCAGACT-3′ cyclin D1-F, 5′-CCGATGCCAACCTCCTCAAC-3′ and cyclin D1-R, 5′-GCGGGCCAGGTTCCACTTGAG-3′ β-actin-F, 5′-GGA CTC CTA TGT GGG TGG CGA GG-3′ and β-actin-R, 5′-GGGAGAGCATGCCCTCGTAGAT-3′.

Primary human tissue samples

Protein extracts were prepared from tissue samples obtained from patients with primary hepatic tumors in the course of direct surgery at the Eastern Hepatobiliary Surgery Institute. Specimens obtained from 2006 to 2009 were from patients in the Eastern Hepatobiliary Surgery Institute in Shanghai, China. Informed consent was obtained from all patients for subsequent use of their resected tissues. From each patient, tissue samples were taken from non-neoplastic mucosa at the proximal surgical margin and from the primary tumor.

Cell fractionation

Cytosolic and nuclear proteins were prepared from cells using a Nuclear and Cytoplasmic Extraction Reagent Kit (Pierce, USA) as described before [39]. To obtain Triton X-100-soluble and -insoluble fractions, cells were incubated with Triton buffer (1% Triton X-100, 0.3 M sucrose, 25 mM HEPES, pH 7.4, 100 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgCl2, 10 μg/ml aprotinin, 10 μg/ml leupeptin and 1 mM phenylmethylsulfonyl fluoride) for 15 min on a rocking platform. After centrifugation, the supernatant (Triton X-100-soluble fraction) was collected. The cell pellet was resuspended in SDS lysis buffer (20 mM Tris, pH 7.5, 2.5 mM EDTA, 1% SDS and 1 mM dithiothreitol) and subjected to standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analysis.

Immunoblotting and immunoprecipitation

Cells were lysed in RIPA buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 5 mM EDTA, pH 8.0, 30 mM NaF, 1 mM Na3VO4, 40 mM β-glycerophosphate, 0.1 mM PMSF, protease inhibitors, 10% glycerol and 1% Nonidet-P40) and equal volumes of cell lysates from each condition were resolved by 8%-16% SDS-PAGE. Protein phosphorylation was visualized by western blotting using antibodies directed against the indicated antigens. For coimmunoprecipitation studies, whole-cell lysates from HEK293 cells were prepared in 1 ml RIPA buffer, incubated overnight with 2 μg of the indicated antibody, and then incubated with 40 μl of a 1:1 slurry of protein A and protein G sepharose beads (Santa Cruz Biotechnology) for 3 h at 4 °C. The beads were washed four times with lysis buffer and resuspended in equal volumes of RIPA buffer.

Statistical analysis

Results are expressed as the mean±SEM. Student's t-tests were used for statistical analysis and statistical significance was defined as P < 0.05.

Acknowledgments

We thank Dr Claudia Cosentino and Dr Jin Q Cheng for kindly providing some of the plasmids. We are also grateful to Dr David Blake and Dr Xiaoni Kong (School of Public Health, Johns Hopkins University, Baltimore, MD) for helpful discussion. Research was supported by grants from the Funds for Creative Research Groups of China (30921006), the State Key Project for Liver Cancer (2008ZX10002), the State Key Laboratory of Oncogenes and Related Genes (91-10-02, 91-10-03), the National Natural Science Foundation of China (81001075, 81071778).

Footnotes

(Supplementary information is linked to the online version of the paper on the Cell Research website.)

Supplementary Information

Supplementary information, Figure S1

Supplementary information, Figure S2

Supplementary information, Figure S3

Supplementary information, Figure S4

Supplementary information, Figure S5

References

  • Befeler AS, Di Bisceglie AM. Hepatocellular carcinoma: diagnosis and treatment. Gastroenterology. 2002;122:1609–1619. [PubMed]
  • Laurent-Puig P, Zucman-Rossi J. Genetics of hepatocellular tumors. Oncogene. 2006;25:3778–3786. [PubMed]
  • Luo J, Manning BD, Cantley LC. Targeting the PI3K-Akt pathway in human cancer: rationale and promise. Cancer Cell. 2003;4:257–262. [PubMed]
  • McCormick F. Signalling networks that cause cancer. Trends Cell Biol. 1999;9:M53–M56. [PubMed]
  • Bader AG, Kang S, Zhao L, Vogt PK. Oncogenic PI3K deregulates transcription and translation. Nat Rev Cancer. 2005;5:921–929. [PubMed]
  • Fresno Vara JA, Casado E, de CJ, Cejas P, Belda-Iniesta C, Gonzalez-Baron M. PI3K/Akt signalling pathway and cancer. Cancer Treat Rev. 2004;30:193–204. [PubMed]
  • Frame S, Cohen P. GSK3 takes centre stage more than 20 years after its discovery. Biochem J. 2001;359:1–16. [PMC free article] [PubMed]
  • Reya T, Clevers H. Wnt signalling in stem cells and cancer. Nature. 2005;434:843–850. [PubMed]
  • Jamora C, Fuchs E. Intercellular adhesion, signalling and the cytoskeleton. Nat Cell Biol. 2002;4:E101–E108. [PubMed]
  • Giles RH, van Es JH, Clevers H. Caught up in a Wnt storm: Wnt signaling in cancer. Biochim Biophys Acta. 2003;1653:1–24. [PubMed]
  • Polakis P. The oncogenic activation of beta-catenin. Curr Opin Genet Dev. 1999;9:15–21. [PubMed]
  • Taipale J, Beachy PA. The Hedgehog and Wnt signalling pathways in cancer. Nature. 2001;411:349–354. [PubMed]
  • de La CA, Romagnolo B, Billuart P, et al. Somatic mutations of the beta-catenin gene are frequent in mouse and human hepatocellular carcinomas. Proc Natl Acad Sci USA. 1998;95:8847–8851. [PMC free article] [PubMed]
  • Suzuki T, Yano H, Nakashima Y, Nakashima O, Kojiro M. Beta-catenin expression in hepatocellular carcinoma: a possible participation of beta-catenin in the dedifferentiation process. J Gastroenterol Hepatol. 2002;17:994–1000. [PubMed]
  • Wong CM, Fan ST, Ng IO. beta-Catenin mutation and overexpression in hepatocellular carcinoma: clinicopathologic and prognostic significance. Cancer. 2001;92:136–145. [PubMed]
  • Taniguchi K, Roberts LR, Aderca IN, et al. Mutational spectrum of beta-catenin, AXIN1, and AXIN2 in hepatocellular carcinomas and hepatoblastomas. Oncogene. 2002;21:4863–4871. [PubMed]
  • Satoh S, Daigo Y, Furukawa Y, et al. AXIN1 mutations in hepatocellular carcinomas, and growth suppression in cancer cells by virus-mediated transfer of AXIN1. Nat Genet. 2000;24:245–250. [PubMed]
  • Higashitsuji H, Itoh K, Nagao T, et al. Reduced stability of retinoblastoma protein by gankyrin, an oncogenic ankyrin-repeat protein overexpressed in hepatomas. Nat Med. 2000;6:96–99. [PubMed]
  • Hori T, Kato S, Saeki M, et al. cDNA cloning and functional analysis of p28 (Nas6p) and p40.5 (Nas7p), two novel regulatory subunits of the 26S proteasome. Gene. 1998;216:113–122. [PubMed]
  • Dawson S, Apcher S, Mee M, et al. Gankyrin is an ankyrin-repeat oncoprotein that interacts with CDK4 kinase and the S6 ATPase of the 26 S proteasome. J Biol Chem. 2002;277:10893–10902. [PubMed]
  • Higashitsuji H, Higashitsuji H, Itoh K, et al. The oncoprotein gankyrin binds to MDM2/HDM2, enhancing ubiquitylation and degradation of p53. Cancer Cell. 2005;8:75–87. [PubMed]
  • Wang GL, Shi X, Haefliger S, et al. Elimination of C/EBPα through the ubiquitin-proteasome system promotes the development of liver cancer in mice. J Clin Invest. 2010;120:2549–2562. [PMC free article] [PubMed]
  • Richman RA, Claus TH, Pilkis SJ, Friedman DL. Hormonal stimulation of DNA synthesis in primary cultures of adult rat hepatocytes. Proc Natl Acad Sci USA. 1976;73:3589–3593. [PMC free article] [PubMed]
  • Marker AJ, Galloway E, Palmer S, et al. Role of the adenylate cyclase, phosphoinositidase C and receptor tyrosyl kinase systems in the control of hepatocyte proliferation by hepatocyte growth factor. Biochem Pharmacol. 1992;44:1037–1043. [PubMed]
  • Levina E, Oren M, Ben-Ze'ev A. Downregulation of beta-catenin by p53 involves changes in the rate of beta-catenin phosphorylation and axin dynamics. Oncogene. 2004;23:4444–4453. [PubMed]
  • Sadot E, Geiger B, Oren M, Ben-Ze'ev A. Down-regulation of beta-catenin by activated p53. Mol Cell Biol. 2001;21:6768–6781. [PMC free article] [PubMed]
  • Man JH, Liang B, Gu YX, et al. Gankyrin plays an essential role in Ras-induced tumorigenesis through regulation of the RhoA/ROCK pathway in mammalian cells. J Clin Invest. 2010;120:2829–2841. [PMC free article] [PubMed]
  • Yang JY, Zong CS, Xia W, et al. MDM2 promotes cell motility and invasiveness by regulating E-cadherin degradation. Mol Cell Biol. 2006;26:7269–7282. [PMC free article] [PubMed]
  • Buendia MA. Genetics of hepatocellular carcinoma. Semin Cancer Biol. 2000;10:185–200. [PubMed]
  • Inagawa S, Itabashi M, Adachi S, et al. Expression and prognostic roles of beta-catenin in hepatocellular carcinoma: correlation with tumor progression and postoperative survival. Clin Cancer Res. 2002;8:450–456. [PubMed]
  • Kondo Y, Kanai Y, Sakamoto M, et al. Beta-catenin accumulation and mutation of exon 3 of the beta-catenin gene in hepatocellular carcinoma. Jpn J Cancer Res. 1999;90:1301–1309. [PubMed]
  • Yang W, Yan HX, Chen L, et al. Wnt/beta-catenin signaling contributes to activation of normal and tumorigenic liver progenitor cells. Cancer Res. 2008;68:4287–4295. [PubMed]
  • Datta SR, Dudek H, Tao X, et al. Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery. Cell. 1997;91:231–241. [PubMed]
  • Li H, Fu X, Chen Y, et al. Use of adenovirus-delivered siRNA to target oncoprotein p28GANK in hepatocellular carcinoma. Gastroenterology. 2005;128:2029–2041. [PubMed]
  • Eastman Q, Grosschedl R. Regulation of LEF-1/TCF transcription factors by Wnt and other signals. Curr Opin Cell Biol. 1999;11:233–240. [PubMed]
  • Umemura A, Itoh Y, Itoh K, et al. Association of gankyrin protein expression with early clinical stages and insulin-like growth factor-binding protein 5 expression in human hepatocellular carcinoma. Hepatology. 2007;47:493–502. [PubMed]
  • Levy L, Renard CA, Wei Y, Buendia MA. Genetic alterations and oncogenic pathways in hepatocellular carcinoma. Ann N Y Acad Sci. 2002;963:21–36. [PubMed]
  • Qin JM, Fu XY, Li SJ, et al. Gene and protein expressions of p28GANK in rat with liver regeneration. World J Gastroenterol. 2003;9:2523–2527. [PubMed]

Articles from Cell Research are provided here courtesy of Nature Publishing Group
PubReader format: click here to try

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...