p28GANK overexpression accelerates hepatocellular carcinoma invasiveness and metastasis via phosphoinositol 3-kinase/AKT/hypoxia-inducible factor-1α pathways

Authors

  • Jing Fu,

    1. International Cooperation Laboratory on Signal Transduction, Eastern Hepatobiliary Surgery Institute/Hospital, Shanghai, China
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    • These authors contributed equally to this work.

  • Yao Chen,

    1. International Cooperation Laboratory on Signal Transduction, Eastern Hepatobiliary Surgery Institute/Hospital, Shanghai, China
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    • These authors contributed equally to this work.

  • Jie Cao,

    1. International Cooperation Laboratory on Signal Transduction, Eastern Hepatobiliary Surgery Institute/Hospital, Shanghai, China
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    • These authors contributed equally to this work.

  • Tao Luo,

    1. International Cooperation Laboratory on Signal Transduction, Eastern Hepatobiliary Surgery Institute/Hospital, Shanghai, China
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  • You-Wen Qian,

    1. International Cooperation Laboratory on Signal Transduction, Eastern Hepatobiliary Surgery Institute/Hospital, Shanghai, China
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  • Wen Yang,

    1. International Cooperation Laboratory on Signal Transduction, Eastern Hepatobiliary Surgery Institute/Hospital, Shanghai, China
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  • Yi-Bin Ren,

    1. International Cooperation Laboratory on Signal Transduction, Eastern Hepatobiliary Surgery Institute/Hospital, Shanghai, China
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  • Bo Su,

    1. International Cooperation Laboratory on Signal Transduction, Eastern Hepatobiliary Surgery Institute/Hospital, Shanghai, China
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  • Guang-Wen Cao,

    1. Department of Epidemiology, Second Military Medical University, Shanghai, China
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  • Yuan Yang,

    1. Department of Surgery, Eastern Hepatobiliary Surgery Hospital, Shanghai, China
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  • Yi-Qun Yan,

    1. Department of Surgery, Eastern Hepatobiliary Surgery Hospital, Shanghai, China
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  • Feng Shen,

    1. Department of Surgery, Eastern Hepatobiliary Surgery Hospital, Shanghai, China
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  • Meng-Chao Wu,

    1. Department of Surgery, Eastern Hepatobiliary Surgery Hospital, Shanghai, China
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  • Gen-Sheng Feng,

    1. International Cooperation Laboratory on Signal Transduction, Eastern Hepatobiliary Surgery Institute/Hospital, Shanghai, China
    2. Department of Pathology, School of Medicine, and Section of Molecular Biology, Division of Biological Sciences, University of California San Diego, La Jolla, CA
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  • Hong-Yang Wang

    Corresponding author
    1. International Cooperation Laboratory on Signal Transduction, Eastern Hepatobiliary Surgery Institute/Hospital, Shanghai, China
    2. State Key Laboratory of Oncogenes and Related Genes, Cancer Institute of Shanghai Jiao Tong University, Shanghai, China
    • Laboratory of Signal Transduction, EasternHepatobiliarySurgery Institute/Hospital, 225 Changhai Road, Shanghai 200438, China
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    • fax: 86 21 65566851


  • Potential conflict of interest: Nothing to report.

Abstract

The overall survival of patients with hepatocellular carcinoma (HCC) remains poor, and the molecular mechanisms underlying HCC progression and aggressiveness are unclear. Here, we report that increased expression of p28GANK (Gankyrin, PSMD10, or p28) in human HCC predicts poor survival and disease recurrence after surgery. Patients with HCC who have large tumors, with vascular invasion and intrahepatic or distant metastasis, expressed high levels of p28GANK. Invasive tumors overexpressing p28GANK were featured by active epithelial-mesenchymal transition (EMT), and exhibited increased angiogenesis associated with vascular endothelial growth factor overexpression, whereas silencing p28GANK expression attenuated EMT and motility/invasion of tumor cells. The p28GANK activates phosphoinositide 3-kinase (PI3K)–V-akt Murine Thymoma Viral Oncogene Homolog (AKT)–hypoxia-inducible factor 1α (HIF-1α) signaling to promote TWIST1, vascular endothelial growth factor, and metalloproteinase 2 expression. Suppression of the PI3K–AKT–HIF-1α pathway interfered with p28GANK-mediated EMT and invasion. Consistently, we detected a significant correlation between p28GANK expression and p-AKT levels in a cohort of HCC biopsies, and the combination of these two parameters is a more powerful predictor of poor prognosis. Conclusion: These results present novel mechanistic insight into a critical role of p28GANK in HCC progression and metastasis. (HEPATOLOGY 2011)

Hepatocellular carcinoma (HCC) is the third leading cause of cancer death worldwide, and the second in China.1, 2 HCCs that grow rapidly with early vascular invasion are highly resistant to chemotherapy.3-5 The extremely poor prognosis of patients with HCC is largely due to the high frequency of tumor recurrence or distant metastasis after surgical resection.6 Extensive epidemiological studies have identified major risk factors of HCC, and significant advances have been made in understanding the pathogenesis.7 However, little is known about molecular mechanisms underlying recurrence or metastasis. Therefore, a most critical issue is to search for molecular markers related to metastasis, which will provide new targets for intervention of metastatic recurrence of HCC.

p28GANK (also known as gankyrin, PSMD10, or p28) was identified as an oncoprotein that is frequently overexpressed in human liver cancers.8, 9 Up-regulation of p28GANK correlates well with cell cycle progression in human hepatocytes.10, 11 Gankyrin overexpression confers tumorigenicity to NIH3T3 cells and inhibits apoptosis in cultured human tumor cells exposed to chemotherapeutic agents.8, 12 The tumorigenic effect of p28GANK might be associated with its antiapoptotic property,11–14 and down-regulation of p28GANK-induced apoptosis inhibits tumor growth.11, 12, 14 The antiapoptotic activity is attributable, at least in part, to increased degradation of p53, resulting in reduced expression of p53-dependent proapoptotic genes.12 Additionally, p28GANK was shown to bind v-rel reticuloendotheliosis viral oncogene homolog A (RelA)/nuclear factor κB (NF-κB) and suppress NF-κB activity.15, 16 Therefore, p28GANK may play a complex role in hepatocarcinogenesis, which is yet to be elucidated.

In this study, we extensively investigated p28GANK expression pattern and determined its contribution to HCC invasion and metastasis. We also dissected the molecular mechanisms by which p28GANK mediates tumor metastasis. Results presented here suggest that p28GANK overexpression promotes HCC aggression via modulation of phosphoinositide 3-kinase (PI3K)–v-akt murine thymoma viral oncogene homolog 1 (AKT)–hypoxia-inducible factor-1α (HIF-1α) signaling. We propose that combination of p28GANK/p-AKT is a new powerful predictor for HCC recurrence and metastasis and a new potential target for adjuvant treatment of aggressive HCCs after surgical resection.

Abbreviations

CRC, colorectal cancer; DFS, disease-free survival; EMT, epithelial mesenchymal transition; ESCC, esophageal squamous cell carcinoma; GFP, green fluorescent protein; HCC, hepatocellular carcinoma; MMP, matrix metalloproteinase; NF-κB, nuclear factor κB; OS, overall survival; PVTT, portal vein tumor thrombus; qRT-PCR, quantitative reverse transcription polymerase chain reaction; SEM, standard error of the mean; siRNA, small interfering RNA; TMA, tissue microarray; TNM, tumor-node-metastasis; VEGF, vascular endothelial growth factor.

Patients and Methods

Detailed description of Patients and Methods can be found in the online Supporting Information.

Results

Elevated Expression of P28GANK in HCC Cell Lines With Metastatic Potential and in Invasive HCC Specimens.

We first examined the p28GANK protein amounts in several HCC cell lines, with varying metastatic capability.17, 18 The p28GANK protein levels increased progressively from normal liver cells (HL-7702 and QSG-7701), low metastatic SMMC-7721 and MHCC-97L cells, to highly metastatic HepG2 and HCC-LM3 cells (Fig. 1A; Supporting Information Fig. 1A). Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis showed that p28GANK expression was significantly higher in invasive HCC samples than in normal liver tissue or noninvasive HCC tumors (28.4-fold and 9.3-fold, respectively; Supporting Information Fig. 1B). Immunoblotting of protein extracts from the same set of patients' samples confirmed the association of p28GANK overexpression with features of tumor metastasis (Fig. 1B; Supporting Information Fig. 1C), suggesting p28GANK involvement in HCC aggressiveness.

Figure 1.

Increased levels of p28GANK indicate worsening prognosis and recurrence/metastasis of HCC. (A) p28GANK expression was evaluated in the indicated cell lines. (B) Protein levels of p28GANK were determined in 10 normal liver (Norm), 12 noninvasive HCC (Non-inv, without vascular invasion), and 15 invasive HCC (Inv, with vascular invasion) samples. Box plot graph shows the statistical analysis of p28GANK expression in all samples. The expression of p28GANK was normalized against β-actin. (C) Protein levels of p28GANK in 10 pairs of HCC tissue samples (Normal, normal liver; Primary, primary HCC; PVTT, portal vein tumor thrombus). Box plot graph shows the quantitative results of p28GANK expression. (D,E) HCC samples in a tissue microarray were immunostained with a monoclonal anti-gankyrin antibody. Representative low-p28GANK (L: below the median value) and high-p28GANK expression (H: above the median value) samples are shown (×200). The disease-free and overall survival rates of 201 patients with HCC were compared between the low-p28GANK and high-p28GANK groups.

Portal vein tumor thrombus (PVTT) from HCC notably deteriorates hepatic function and serves as a poor prognostic factor associated with frequent recurrence and intrahepatic metastasis.19 By analyzing an additional 10 pairs of HCC samples, we found that p28GANK protein levels were low in normal liver tissue, relatively higher in primary HCCs by 2.1-fold, and further increased in PVTT by 3.3-fold (Fig. 1C; Supporting Information Fig. 1D), further suggesting its potential role not only at the origin but also in invasive progression of HCCs.

p28GANK Overexpression in HCCs Predicts a Poor Prognosis.

We next sought to determine whether p28GANK expression in HCC is associated with disease recurrence and poor survival. Tissue microarrays from 201 patients with HCC who underwent liver resection (Supporting Information Table 1) were examined by immunostaining with p28GANK monoclonal antibody. The average expression level of p28GANK protein was significantly higher in HCC tissues than in peritumoral tissues (Supporting Information Table 2). Intriguingly, the p28GANK expression levels were found to be significantly higher in HCCs with increased tumor size (P = 0.002), vascular invasion (P = 0.0366), and intrahepatic (P = 0.0429) and distant metastasis (P < 0.0001; Table 1). Based on the immunohistochemical result, all 201 patients with HCC were divided into two groups: the high-expression (n = 100) and low-expression group (n = 101). Patients in the high expression group had either shorter disease-free survival (DFS, median DFS time were 11 and 32 months, respectively, difference = 21 month, P = 0.002) or worse overall survival (OS, median OS time were 28 and >49 months, respectively, difference >21 month, P = 0.007) (Fig. 1D,E; Supporting Information Table 3) than the low-expression group. Consistently, the 1-year, 3-year, and 5-year OS and DFS after surgery were much worse for p28GANK-high than p28GANK-low expression group (Table 2). Thus, p28GANK expression is a valuable predicting factor for recurrence and survival in patients with HCC.

Table 1. Relationship Between Intratumoral p28GANK Expression and Clinicopathologic Features
Variablep28GANK DensityP Value
Low(n = 101)High(n = 100)
  • Patients with HCC were divided into p28GANK “High” group (whose final density was higher than the median) and “Low” group (whose final density was lower than the median). The patient and disease profiles in each group were compared.

  • *

    Fisher's exact test.

  • AFP, serum alpha-fetoprotein; anti-HCV, anti-hepatitis C virus antibody; HBsAg, hepatitis B surface antigen; NS, not significant between any groups; TNM, tumor-node-metastasis.

Sex  NS
 Male9287 
 Female913 
Age(years)  NS
 ≤ 505054 
 > 505146 
Tumor size(cm)  0.002
 ≤ 55730 
 > 54470 
AFP  NS
  ≤ 20 U/L2528 
 >20 U/L7672 
 ≤ 1000 U/L6657 
 >1000 U/L3543 
HBsAg  NS
 Positive8389 
 Negative1811 
Anti-HCV  NS*
 Positive52 
 Negative9698 
Liver cirrhosis  NS
 Yes6351 
 No3849 
Vascular invasion  0.0366
 Yes5266 
 No4934 
Intrahepatic metastasis  0.0429
 Yes1830 
 No8370 
Capsular formation  NS
 Yes7059 
 No3141 
Capsular invasion  NS
 Yes2524 
 No7676 
Distant metastasis  <0.0001
 Yes1541 
 No8659 
TNM stage  NS
 I-II8172 
 III-IV2028 
Edmondson  NS
 I-II118 
 III-IV9092 
Table 2. Relationship Between Intratumoral p28GANK Expression and Survival Rate
Survival Measurementp28GANK DensityP Value*
LowHigh
  • Disease-free survival is defined as a period without recurrence, the diagnosis of which is based on the typical feature presented in a computed tomography/magnetic resonance imaging scan and an elevated serum alpha-fetoprotein level.

  • *

    Log-rank test.

1-year overall survival (%)83.2 ± 3.770.0 ± 4.60.007
3-year overall survival (%)65.2 ± 4.844.8 ± 5.0
5-year overall survival (%)53.7 ± 5.739.2 ± 5.6
1-year disease-free survival (%)69.3 ± 4.646.0 ± 5.00.002
3-year disease-free survival (%)45.8 ± 5.027.8 ± 4.5
5-year disease-free survival (%)39.0 ± 6.124.6 ± 4.6

p28GANK Enhances Invasive Potential of HCC Cells In Vitro.

To determine the significance of the above clinical data, we generated lentiviral constructs expressing p28GANK (LV-p28GANK) to infect low-invading MHCC-97L cells. p28GANK overexpression significantly enhanced their invasive capacity by 2.2-fold, as compared with lentiviral–green fluorescent protein (LV-GFP)-treated cells (Fig. 2A), and enhanced adhesion to several cell matrix proteins (Supporting Information Fig. 2A). In contrast, silencing endogenous p28GANK with lentivirus-mediated microRNA (LV-mip28GANK) in HCC-LM3 cells significantly reduced cell invasion by 58% (Supporting Information Fig. 2B), and resulted in inhibition of adhesion to cell matrix proteins (Supporting Information Fig. 2C). Evidently, p28GANK acts to promote the invasive property of HCC cells in vitro.

Figure 2.

Overexpression of p28GANK in HCC cells promotes invasiveness in vitro and accelerates proliferation and metastasis in vivo. (A) The number of invasive MHCC-97L cells infected with lentiviral-delivering GFP (LV-GFP) or p28GANK (LV-p28GANK) was calculated with crystal violet staining as described in Patients and Methods, and represented the average count of six random microscopic fields (magnification, ×100). Bar graphs show mean ± standard error of the mean (SEM) performed in triplicate compared with mock cells. p28GANK expression was analyzed by immunoblot. (B) Photomicrographs were taken for liver tumors and lung metastasis in nude mice, 4 weeks after orthotopic xenograft transplantation of mock, LV-GFP–infected or LV-p28GANK–infected MHCC-97L cells. Representative images of a mouse in each group were presented. Arrowheads indicate the metastatic nodes. Tumor volumes from each group were measured as described in Patients and Methods. (C) (Upper) Representative lung tissue sections from each group were shown (hematoxylin and eosin stain; magnification, ×100). Black arrows indicate lung metastatic tumors. (Lower) The number of lung metastatic foci in each group was calculated. Classification of metastatic foci was based on the cell number in the metastatic lesion. (D) Immunostaining of p28GANK and CD31 was performed, and the microvessel density (MVD) was calculated in tumors from each group. Results display the mean ± SEM from triplicate experiments. *P < 0.05, **P < 0.001 versus mock and control.

p28GANK Promotes Localized Growth, Angiogenesis, and Metastasis of HCC In Vivo.

We further examined the effect of p28GANK on HCC growth and pulmonary metastasis by establishing an orthotopic liver tumor model in nude mice. MHCC-97L cells having low metastatic potential were infected with LV-GFP or LV-p28GANK, and used for orthotopic model studies. Compared to mock control or LV-GFP groups, p28GANK overexpression resulted in significant increase of tumor size, the number of pulmonary metastatic foci, increased average size of pulmonary metastatic lesions, and enlarged average microvessel density (Fig. 2B-D). Furthermore, the subcutaneous xenograft model based on low-metastatic potential SMMC-7721 cells also showed that p28GANK overexpression promoted tumor cell proliferation and lung metastasis, while suppressing retinoblastoma 1 (RB) expression (Supporting Information Fig. 3A-D), consistent with previous reports on p28GANK promotion of RB phosphorylation and degradation.9, 14 On the contrary, down-regulation of p28GANK expression by LV-mip28GANK retarded growth of HepG2-derived or HCCLM3 cell–derived tumors and reduced pulmonary metastases dramatically (Supporting Information Fig. 3E,F). Together, these results reveal functional significance of increased p28GANK expression in metastatic HCC and in aggressive tumors with high propensity to develop recurrence after surgery.

p28GANK Promotes Tumor Metastasis by Enhancing EMT.

Given that p28GANK promotes HCC metastasis, we investigated the effect of p28GANK on EMT, a critical event in tumor invasion. p28GANK overexpression by LV-p28GANK resulted in morphologic changes from tightly packed colonies to scattered growth structure (Fig. 3A), suggesting induction of EMT. We performed qRT-PCR for molecular markers of EMT. The epithelial markers such as E-cadherin, cytokeratin-8, cytokeratin-17, cytokeratin-18, claudin-1, and claudin-8 were lower in the LV-p28GANK group than that in the LV-GFP group, whereas the mesenchymal markers such as N-cadherin, vimentin, HEY1 (hairy/enhancer-of-split related with YRPW motif 1), HEY2, Jagged 1, Jagged 2, Goosecoid, and the EMT major regulator TWIST1 were increased (Fig. 3B). Immunoblotting also detected lower expression of E-cadherin in MHCC-97L-LV-p28GANK cells but an increase in HCC-LM3-LV-mip28GANK cells. In contrast, the expression of N-cadherin, vimentin, and TWIST1 increased in MHCC-97L-LV-p28GANK cells but decreased in HCC-LM3-LV-mip28GANK cells (Fig. 3C). However, no alteration in other EMT inducers, such as Snail, Slug, and SIP1 (survival of motor neuron protein interacting protein 1), was observed in MHCC-97L-LV-p28GANK or HCC-LM3-LV-mip28GANK cells (Supporting Information Fig. 4A). We next investigated the occurrence of EMT in vivo. LV-p28GANK tumors exhibited the typical EMT phenotype, including focal loss of the epithelial marker E-cadherin, translocation of β-catenin (dissociation of membranous β-catenin and translocation into the nucleus), and concurrent gain of the mesenchymal marker vimentin and N-cadherin (Fig. 3D). Thus, p28GANK overexpression induced oncogenic EMT in HCC in vivo.

Figure 3.

Overexpression of p28GANK enhances EMT in HCC cells. (A) Morphology of SMMC-7721 and MHCC-97L infected with either LV-GFP or LV-p28GANK is shown by phase contrast microscopy (magnification, ×200). (B) qRT-PCR was performed to assess messenger RNA levels of epithelial markers E-cadherin, cytokeratin-8, cytokeratin-17, cytokeratin-18, claudin-1, and claudin-8 and mesenchymal markers N-cadherin, vimentin, HEY1, HEY2, Jagged1, Jagged2, Goosecoid, and an EMT regulator TWIST1 in LV-p28GANK–infected versus LV-GFP–infected MHCC-97L cells. Results, normalized against β-actin, are presented as mean ± SEM from three independent experiments. (C) Expression of indicated molecules in MHCC-97L cells infected with LV-GFP or LV-p28GANK and in HCC-LM3 cells infected with LV-miNon or LV-mip28GANK were detected by western blot. (D) Tumors from MHCC-97L mock, LV-GFP, or LV-p28GANK groups were immunostained for indicated molecules. The yellow arrows indicate membranous expression of E-cadherin, N-cadherin, or β-catenin; blue arrows indicate the cytoplasmic translocation of β-catenin; and red arrows indicate the nuclear expression of β-catenin. Pictures are representative of three independent experiments. (E) Immunoblots were performed to detect expression of indicated molecules in SMCC-7721 LV-p28GANK or MHCC-97L LV-p28GANK cells transfected with siRNA targeting TWIST1 (TWIST1 siRNA) or scrambled siRNA (Con siRNA). *P < 0.05, **P < 0.001.

We asked whether TWIST1 is involved in p28GANK-induced E-cadherin down-regulation. E-cadherin expression could be rescued by silencing TWIST1 in SMMC-7721-LV-p28GANK or MHCC-97L-LV-p28GANK cells (Fig. 3E), suggesting that TWIST1 is required for p28GANK-driven EMT.

HIF-1α Is Essential for p28GANK-Mediated Vascular Endothelial Growth Factor and Matrix Metalloproteinase 2 Expression.

Given that orthotopic intrahepatic implantation of MHCC97L-LV-p28GANK cells generated aggressive and highly vascularized tumors, we examined the expression levels of vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMPs). Compared with vector control, the protein amounts of both VEGF and MMP2, but not MMP9, were significantly increased in LV-p28GANK groups but decreased in LV-mip28GANK group (Fig. 4A, upper). Gelatin zymography assay showed that MMP2 activity increased in MHCC-97L-LV-p28GANK cells but decreased in HCC-LM3-LV-mip28GANK cells (Fig. 4A, lower).

Figure 4.

p28GANK activates VEGF and MMP2 correlated with HIF-1α. (A) (Upper) Protein contents of VEGF and p28GANK were examined by immunoblot in LV-p28GANK–infected versus LV-GFP–infected MHCC-97L cells, and LV-mip28GANK–infected versus LV-miCon–infected HCC-LM3 cells. (Lower) The above-mentioned cells were serum-starved for 18 hours. The supernatants were immunoblotted by MMP2 or MMP9 antibodies, and MMP2 activity was analyzed by zymography. Equal amounts of total proteins were loaded in each lane. (B) Luciferase activity was measured in the indicated cells in triplicate. Firefly luciferase activity was normalized by Renilla luciferase activity, and the value of the empty pGL3-basic vector was used as control. Values are given as the mean ± SEM of three independent experiments. **P < 0.001. (C) The effect of p28GANK on HIF-1α expression was analyzed by immunoblot in indicated cells. (D) Representative immunohistochemistry of VEGF, MMP2, and HIF-1α in MHCC-97L mock, MHCC-97L LV-GFP, or MHCC-97L LV-p28GANK induced tumors. The yellow arrows indicate mesenchymal expression of MMP2, blue arrows indicate the cytoplasmic translocation of MMP2, and red arrows indicate nuclear expression of HIF-1α. Pictures are representative of three independent experiments. (E) Expression of indicated molecules in SMCC-7721 LV-p28GANK or MHCC-97L LV-p28GANK cells transfected with HIF-1α siRNA or scrambled siRNA (Con siRNA). MMP2 expression in cell supernatants was detected as above.

HIF-1α plays a pivotal role in promoting angiogenesis, through regulation of target genes including VEGF and MMPs.20–22 Thus, we asked whether p28GANK regulates HIF-1α activity in HCC cells. As shown in Fig. 4B, overexpression of p28GANK in MHCC-97L cells resulted in up-regulation of HIF-1α–responsive luciferase reporter which contains hypoxia response element–binding sites (9-fold), whereas down-regulation of p28GANK in HCC-LM3 cells led to a decrease in the HIF-1α reporter level (79.5%). Consistently, HIF-1α protein level was higher in MHCC-97L-LV-p28GANK cells than in LV-GFP cells, whereas silencing p28GANK suppressed HIF-1α expression in HCC-LM3 cells (Fig. 4C). Increased level of HIF-1α accompanied with VEGF and MMP2 enhancement was also observed in p28GANK-overexpressing tumors (Fig. 4D).

HIF-1α small interfering RNA (siRNA) significantly down-regulated HIF-1α protein levels but not p28GANK, whereas p28GANK miRNA inhibited both p28GANK and HIF-1α expression, indicating that HIF-1α is downstream of p28GANK (Fig. 4C,E). Moreover, p28GANK-mediated VEGF and MMP2 production was counteracted by silencing HIF-1α in SMMC-7721 or MHCC-97L cells (Fig. 4E). In addition, HIF-1α suppression reduced p28GANK-induced TWIST1 but restored p28GANK-reduced E-cadherin (Fig. 4E). These results suggest that p28GANK may promote EMT response and tumor cell invasion through HIF-1α.

The PI3K/AKT Pathway Plays a Critical Role in Mediating p28GANK Function.

Signaling pathways activated by p28GANK were analyzed by expression of phosphorylated forms of AKT, p38 mitogen-activated protein kinase, extracellular signal-regulated kinase (ERK), and JNK by immunoblot. Only the p-AKT signal was observed to be significantly higher in MHCC-97L-p28GANK cells, whereas there was a decrease in HCC-LM3-LV-mip28GANK cells (Fig. 5A; Supporting Information Fig. 4B). Silencing AKT expression by siRNA suppressed both proliferation of LV-GFP and LV-p28GANK cells. Interestingly, compared with LV-GFP, LV-p28GANK still significantly promoted proliferation of MHCC-97L cells even when AKT was down-regulated (Supporting Information Fig. 4C), suggesting that AKT does not play a key role in p28GANK-mediated cell proliferation. However, suppression of AKT profoundly blocked p28GANK-induced matrigel invasion in MHCC-97L cells, and ectopic expression of AKT restored the invasiveness of LV-mip28GANK–treated HCC-LM3 cells (Fig. 5B). LV-p28GANK–enhanced adhesion to cell matrix proteins was reduced in the absence of AKT (Supporting Information Fig. 4D). Consistently, PI3K-AKT inhibitors (LY294002 and rapamycin), rather than Ras-ERK (PD98059) or p38–mitogen-activated protein kinase (SB203580), could markedly block p28GANK-induced invasion (Supporting Information Fig. 4E). Taken together, these data show requirement for the PI3K/AKT pathway in p28GANK-mediated invasion and adhesion, but not proliferation.

Figure 5.

PI3K-AKT pathway is involved in mediating p28GANK action. (A) p-AKT was evaluated by immunoblot analysis with AKT protein and β-actin as controls. (B) Matrigel invasion assay was done for the indicated cells following treatment with AKT siRNA or ectopic AKT expression. (C) SMCC-7721 LV-p28GANK or MHCC-97L LV-p28GANK cells were transfected with AKT siRNA or Con siRNA, and were subjected to immunoblot with the indicated antibodies. (D) The MHCC-97L vector control or p28GANK overexpressing cells were injected into right flanks of 10 nude mice. Fourteen days after injection, mice were randomly assigned into two groups and set for the rapamycin sensitivity assay. Pulmonary metastasis lesions were analyzed at 30 days later. (E) Immunoblot analysis of the interaction between p28GANK, RhoGDIα, and RhoA in lysates of MHCC-97L LV-GFP or MHCC-97L LV-p28GANK cells after immunoprecipitation with anti-p28GANK and whole-cell lysates (WCL). IgL, immunoglobin light chain.

Either knockdown or small AKT inhibitors (LY294002 and rapamycin), rather than ERK inhibitor (PD98059) or p38 inhibitor (SB203580), blocked p28GANK-activated HIF-1α activation, whereas mip28GANK-diminished HIF-1α reporter was reversed by ectopic expression of AKT, indicating PI3K-AKT involvement in induction of HIF-1α by p28GANK (Supporting Information Fig. 5A,B). Moreover, AKT knockdown repressed p-AKT signal and HIF-1α expression in LV-p28GANK groups (Fig. 5C). More importantly, inhibition of PI3K-AKT pathway in vivo by rapamycin dramatically impeded the pulmonary metastasis of p28GANK-overexpressing cells (Fig. 5D). Collectively, these findings support a critical role of AKT during p28GANK-induced invasiveness and metastasis in cancer cells.

We did not observe any change in expression of AKT signaling regulators (PI3K, pyruvate dehydrogenase kinase 1 [PDK1], phosphatase and tensin homolog [PTEN], SH-2 containing inositd phosphatase-1 (SHIP-1), and SH-2 containing inositd phosphatase-2 (SHIP-2)), nor any change in PI3K, PDK1 kinase activity by p28GANK. Moreover, no association of p28GANK with those regulators was observed by immunoprecipitation in MHCC-97L cells (data not shown). Through coimmunoprecipitation, we detected a complex consisting of p28GANK, RhoGDIα (RhoGDIa (Rho GDP dissociation inhibitor (GDI) α), and RhoA (ras homolog gene family, member A) proteins (Fig. 5E), which results in repression of ROCK2 (Rho-associated, coiled-coil containing protein kinase 2) activity (Supporting Information Fig. 6). This result suggests a mechanism in which p28GANK activates p-AKT signaling through control of the RhoGDIα/RhoA/ROCK2 pathway. Consistent with this, Man et al. reported recently that p28GANK promoted RhoGDIα interaction with RhoA, leading to inhibition of RhoA/ROCK2 activity, and prolonged AKT activation in NIH3T3 cells, human embryonic kidney 293 cells, and lung cancer cells.23

Combination of p28GANK and p-AKT Levels Has Better Prognostic Value for HCC.

We further analyzed the expression levels of p28GANK, p-AKT, E-cadherin, TWIST1, HIF-1α, RB, and p53 in clinical HCC samples. Tissue microarray analysis of 130 patient specimens revealed a strong correlation of p28GANK expression with p-AKT levels (r = 0.2505, P = 0.0004) (Fig. 6A). Moreover, patients whose tumors expressed above-average levels of p28GANK or p-AKT exhibited significantly decreased trend in any of the prognostic indicators, including time to DFS and OS due to HCC-related death (Supporting Information Fig. 7A,B). For patients whose tumors had above-average levels of both p28GANK and p-AKT, adverse outcomes were exacerbated (Fig. 6B). Using the combination of these two parameters increased the prognostic value, as compared to p28GANK or p-AKT overexpression alone (Fig. 6B; Supporting Information Fig. 7A,B). In conclusion, evaluation of both p28GANK expression and p-AKT signal is a powerful predictor of poor prognosis, further supporting a model of p28GANK activation of PI3K–AKT–HIF-1α signaling, resulting in EMT occurrence, VEGF and MMP2 production, and thus metastases of HCC cells (Fig. 6C).

Figure 6.

Combination of elevated p28GANK expression and p-AKT signal is a powerful predictor of poor clinical outcome in HCCs. (A) Correlation between p28GANK expression and p-AKT level was examined in tumor tissues derived from 130 patients, r = 0.2505, P = 0.0004. Representative immunostaining for p28GANK and p-AKT is shown for three patient samples. (B) Combination of p28GANK and p-AKT enhanced correlation to clinical parameters and the significance for poor prognosis. The median value for p28GANK and p-AKT expression in HCCs was used to divide the patients into high (above median) and low (below median) p28GANK and p-AKT groups. (C) A model for p28GANK action in HCC progression and metastasis is shown, with p28GANK acting as a critical driving factor in metastatic carcinoma cells counteracting with various barriers.

Discussion

Efforts to elucidate the molecular mechanism underlying HCC tumorigenicity, invasion, and metastasis of HCC are warranted in order to identify biomarkers for prediction and intervention. In this study, we determined the significance and underlying mechanism for p28GANK overexpression in HCC progression and metastasis. The p28GANK content was low in normal hepatocytes, increased in noninvasive and primary HCC cells, and reached the highest level in invasive HCC and PVTT cells. This progressively increased expression profile paralleled with deterioration of the disease, suggesting a role of p28GANK in progression of HCC.

Analyzing the association of p28GANK expression with pathological characteristics in 201 patients with HCC by tissue microarray revealed a significant correlation of p28GANK expression with tumor size, vein invasion, intrahepatic metastasis, and distant metastasis, which are all hallmarks for poor prognosis of HCC.24, 25 Indeed, the Kaplan-Meier analysis shows that patients with HCC who had high p28GANK expression in general had worse prognosis than those with low expression. We believe that p28GANK is an attractive candidate gene for risk prognostication and therapy of HCC. However, our data is apparently at odds with a recent report suggesting that the cumulative survival rate of patients with gankyrin-positive HCC was significantly higher than those patients with gankyrin-negative HCC.26 The discrepancy may be due to different backgrounds of specimens used, including the proportion of hepatitis C virus and hepatitis B virus infection, sex of patients, and the classification/criteria of tumor-node-metastasis (TNM) staging. Recently, Ortiz and Tang reported that gankyrin messenger RNA and protein increased in human esophageal squamous cell carcinoma (ESCC) or colorectal cancer (CRC), and its overexpression is poor prognosis of ESCC or CRC due to its significant correlation with TNM stages and metastasis of these tumors, respectively.27, 28 Therefore, p28GANK overexpression may be involved in development of human digestive malignancies such as HCC, ESCC, and CRC.

The effect of p28GANK on tumor invasion and metastasis was directly demonstrated in our in vitro and in vivo studies. In both subcutaneous and orthotopic xenografts, overexpression of p28GANK generated larger primary tumors and more lung metastasis foci, and higher levels of vascularization and angiogenesis, indicating their more aggressive and metastatic properties. Moreover, down-regulation of p28GANK led to severe suppression of tumor growth and lung metastasis of HCC in mice. To our knowledge, this is the first report that p28GANK expression is critical for HCC metastasis, in addition to tumor proliferation and growth.

In this study, we found that TWIST1 is indeed involved in p28GANK-driven EMT. Moreover, p28GANK modulated HIF-1α hyperactivation and expression correlated with TWIST up-regulation and E-cadherin down-regulation. Thus, our data suggest a requirement for HIF-1α in p28GANK-driven EMT. We also observed a role of HIF-1α in p28GANK-regulated VEGF and MMP2 expression, consistent with previous reports that HIF-1α up-regulates VEGF, promoting angiogenesis and invasion of HCC.29–31 Taken together, this study clearly demonstrates a crucial role for p28GANK in induction of EMT and angiogenesis through regulation of HIF-1α, VEGF, and MMP2 expression.

An increase in AKT signal is a key tumor survival mechanism, and promotes tumor metastatic processes including EMT, resistance to apoptosis, and angiogenesis.32–34 Previous studies have demonstrated that activated AKT plays a critical role in hematogenous intrahepatic metastasis in an orthotopic implantation model of HCC.35 Our group previously showed a protective role of p28GANK in HCC cells against endoplasmic reticulum stress-induced apoptosis, partially through enhancing AKT phosphorylation.14 Our current in vitro and in vivo studies suggest that p-AKT is responsible for p28GANK-mediated invasion/metastasis. Furthermore, we found that p28GANK interacted with RhoGDIα and RhoA, resulting in inhibition of ROCK2 activity in HCC cells, an observation supported by a recent report that p28GANK negatively regulates the RhoA/ROCK2/PTEN pathway for activation of AKT.23 Given complex p-AKT pathways, whether other upstream regulators are involved in p28GANK-promoting p-AKT signal remains to be further determined. Remarkably, the predictive range of p28GANK expression levels combined with p-AKT signal was more sensitive than that of p28GANK alone for OS and cumulative recurrence, strongly suggesting that the concerted activities of p28GANK and p-AKT detected in our experiments are recapitulated in clinical patients with HCC. Identification of tumor p28GANK alone or combined evaluation of p28GANK/p-AKT levels as a new prognostic marker in patients with HCC is important because they provide not only a new criterion for prognosis, but also a potential therapeutic target.

The most interesting part of the results shown here is the remarkable function of p28GANK in transforming noninvasive HCC cells into highly aggressive cells that generate tumors similar to those in patient-derived samples (Fig. 6C). p28GANK is a cytoplasmic protein that contains seven ankyrin repeats to mediate protein-protein interactions, and acts as a chaperone for the assembly of the 19S structure of the 26S proteasome.36 However, neither 26S proteasome activity nor the overall levels of polyubiquitinated proteins were changed in p28GANK-overexpressed or knockdown cells (Supporting Information Fig. 8A,B), indicating that the proteasome system is not involved in p28GANK-mediated invasion/metastasis. Previous studies showed that p28GANK plays its oncogenic role by controlling the activities of pRb and p53.8, 12 Intriguingly, even in both Rb and p53-deficient Hep3B cells, p28GANK overexpression still promoted their invasion (Supporting Information Fig. 8C). Combined with no evident correlation of pRb or p53 with p28GANK in clinical HCC samples (data not shown), it is likely that p28GANK-induced invasion/metastasis is independent of Rb and/or p53 status.

In conclusion, we have identified p28GANK as a key regulator that controls multiple facets essential for HCC development and metastasis. In particular, the data has led us to propose that p28GANK or combination of p28GANK with p-AKT is a novel marker in the prognosis of HCC and a potential therapeutic target. Because p28GANK is also overexpressed in other types of cancers, including lung,23 esophageal,27 colon,28 gastric carcinoma, rectal, bladder, breast, ovary, and uterus endometrium cancers (Fu and Chen, unpublished observations), we believe that this oncoprotein may be widely involved in tumorigenesis in human cancers.

Acknowledgment: We thank Dangsheng Li (Shanghai Institutes for Biological Sciences, China) for critical review of the manuscript and helpful suggestions. We also thank Guoqiang Chen (Shanghai Jiao Tong University, Shanghai, China) for the gift of pGL3–HIF-1α plasmid.

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