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1.  Functional characterization of human oncoprotein gankyrin in Zebrafish 
Gankyrin is an oncoprotein containing seven ankyrin repeats that is overexpressed in hepatocellular carcinoma (HCC). Gankyrin binds to Mdm2, which results in accelerated ubiquitylation via degradation of p53, and it also plays an important role in cell proliferation. However, little is known about the relationships between p53 levels, cell proliferation, and gankyrin over-expression. In order to investigate the influence of gankyrin protein on p53 and Mdm2 in a zebrafish model, we injected human gankyrin (hgankyrin) containing expression vectors (pCS2-hgankyrin, pCS2-hgankyrin-EGFP) into zebrafish embryos. To measure p53 and Mdm2 expression in hgankyrin-injected embryos, RT-PCR, Northern blot and in-situ hybridization and BrdU immunostaining were used. In addition, to know the effect of hgankyrin on cell proliferation in vitro, cell viability assays such as MTT, trypan blue staining and RT-PCR following transfection of hgankyrin-containing vector into HEK 293 cell line were performed. In vivo results indicated that p53 mRNA levels decreased but those of Mdm2 were not decreased in the presence of hgankyrin. These results suggest that gankyrin downregulates p53 expression and not Mdm2 expression. In the study of cell proliferation, BrdU-positive cells were predominantly increased in the head and tail regions in hgankyrin-injected zebrafish. Additional in vitro studies using trypan blue staining and MTT assay showed that gankyrin-expressing HEK 293 cells proliferated at a faster rate, indicating that gankyrin promotes cell proliferation. Our results demonstrate that hgankyrin overexpression downregulates p53 expression and promotes cell proliferation in zebrafish. Gankyrin may play an important role in tumorigenesis via its effects on p53 and cell proliferation.
PMCID: PMC2679278  PMID: 19287195
cell proliferation; proto-oncogene proteins c-mdm2; PSMD10 protein, human; tumor suppressor protein p53; zebrafish
2.  LBH589 Inhibits proliferation and metastasis of hepatocellular carcinoma via inhibition of gankyrin/stat3/akt pathway 
Molecular Cancer  2013;12:114.
Gankyrin has shown to be overexpressed in human liver cancers and plays a complex role in hepatocarcinogenesis. Panobinostat (LBH589), a new hydroxamic acid-derived histone deacetylase inhibitor has shown promising anticancer effects recently. Here, we investigated the potential of LBH589 as a form of treatment for hepatocellular carcinoma (HCC).
Gankyrin plasmid was transfected into HCC cells, and the cells were selected for more than 4 weeks by incubation with G418 for overexpression clones. The therapeutic effects of LBH589 were evaluated in vitro and in vivo. Cell proliferation, apoptosis, cell cycle, invasive potential, and epithelial-mesenchy-mal transition (EMT) were examined.
LBH589 significantly inhibited HCC growth and metastasis in vitro and in vivo. Western blotting analysis indicated that LBH589 could decrease the expression of gankyrin and subsequently reduced serine-phosphorylated Akt and tyrosine-phosphorylated STAT3 expression although the total Akt and STAT3 were unaffected. LBH589 inhibited metastasis in vitro via down-regulation of N-cadherin, vimentin, TWIST1, VEGF and up-regulation of E-cadherin. LBH589 also induced apoptosis and G1 phase arrest in HCC cell lines. Ectopic expression of gankyrin attenuated the effects of LBH589, which indicates that gankyrin might play an important role in LBH589 mediated anticancer effects. Lastly, in vivo study indicated that LBH589 inhibited tumor growth and metastasis, without discernable adverse effects comparing to control group, with abrogating gankyrin/STAT3/Akt pathway.
Our results suggested that LBH589 could inhibit HCC growth and metastasis through down-regulating gankyrin/STAT3/Akt pathway. LBH589 may present itself as a novel therapeutic strategy for HCC.
PMCID: PMC3853770  PMID: 24093956
Hepatocellular carcinoma; LBH589; Apoptosis; Gankyrin; STAT3
3.  FXR inhibits gankyrin in mouse livers and prevents development of liver cancer 
Hepatology (Baltimore, Md.)  2013;57(3):1098-1106.
One of the early events in development of liver cancer is a neutralization of tumor suppressor proteins Rb, p53, HNF4α and C/EBPα. The elimination of these proteins is mediated by a small subunit of proteasome, gankyrin, which is activated by cancer. The aim of this study was to determine mechanisms which repress gankyrin in quiescent livers and mechanisms of activation of gankyrin in liver cancer. We found that farnesoid X receptor, FXR, inhibits expression of gankyrin in quiescent livers by silencing the gankyrin promoter through HDAC1-C/EBPβ complexes. C/EBPβ is a key transcription factor which delivers HDAC1 to gankyrin promoter and causes epigenetic silencing of the promoter. We show that down-regulation of C/EBPβ in mouse hepatoma cells and in mouse livers reduces C/EBPβ-HDAC1 complexes and activates the gankyrin promoter. Deletion of FXR signaling in mice leads to de-repression of the gankyrin promoter and to spontaneous development of liver cancer at 12 months of age. DEN-mediated liver cancer in WT mice also involves the reduction of FXR and activation of gankyrin. Examination of liver cancer in old mice and liver cancer in human patients revealed that FXR is reduced; while gankyrin is elevated during spontaneous development of liver cancer. Searching for animal models with altered levels of FXR, we found that long-lived Little mice have high levels of FXR and do not develop liver cancer with age and after DEN injections due to failure to activate gankyrin and eliminate Rb, p53, HNF4α and C/EBPα proteins.
FXR prevents liver cancer by inhibiting the gankyrin promoter via C/EBPβ-HDAC1 complexes leading to subsequent protection of tumor suppressor proteins from degradation.
PMCID: PMC3649861  PMID: 23172628
Cancer; liver; gankyrin; C/EBP; HDAC1
4.  Evidence that Proteasome-Dependent Degradation of the Retinoblastoma Protein in Cells Lacking A-Type Lamins Occurs Independently of Gankyrin and MDM2 
PLoS ONE  2007;2(9):e963.
A-type lamins, predominantly lamins A and C, are nuclear intermediate filaments believed to act as scaffolds for assembly of transcription factors. Lamin A/C is necessary for the retinoblastoma protein (pRB) stabilization through unknown mechanism(s). Two oncoproteins, gankyrin and MDM2, are known to promote pRB degradation in other contexts. Consequently, we tested the hypothesis that gankyrin and/or MDM2 are required for enhanced pRB degradation in Lmna−/− fibroblasts. Principal Findings. To determine if gankyrin promotes pRB destabilization in the absence of lamin A/C, we first analyzed its protein levels in Lmna−/− fibroblasts. Both gankyrin mRNA levels and protein levels are increased in these cells, leading us to further investigate its role in pRB degradation. Consistent with prior reports, overexpression of gankyrin in Lmna+/+ cells destabilizes pRB. This decrease is functionally significant, since gankyrin overexpressing cells are resistant to p16ink4a-mediated cell cycle arrest. These findings suggest that lamin A-mediated degradation of pRB would be gankyrin-dependent. However, effective RNAi-enforced reduction of gankyrin expression in Lmna−/− cells was insufficient to restore pRB stability. To test the importance of MDM2, we disrupted the MDM2-pRB interaction by transfecting Lmna−/− cells with p14arf. p14arf expression was also insufficient to stabilize pRB or confer cell cycle arrest, suggesting that MDM2 also does not mediate pRB degradation in Lmna−/− cells.
Our findings suggest that pRB degradation in Lmna−/− cells occurs by gankyrin and MDM2-independent mechanisms, leading us to propose the existence of a third proteasome-dependent pathway for pRB degradation. Two findings from this study also increase the likelihood that lamin A/C functions as a tumor suppressor. First, protein levels of the oncoprotein gankyrin are elevated in Lmna−/− fibroblasts. Second, Lmna−/− cells are refractory to p14arf-mediated cell cycle arrest, as was previously shown with p16ink4a. Potential roles of lamin A/C in the suppression of tumorigenesis are discussed.
PMCID: PMC1978514  PMID: 17896003
5.  Gankyrin Is Frequently Overexpressed in Cervical High Grade Disease and Is Associated with Cervical Carcinogenesis and Metastasis 
PLoS ONE  2014;9(4):e95043.
Our previous studies have showed that Gankyrin expression is correlated with a malignant phenotype in endometrial carcinoma. Here, we investigated the possible role of Gankyrin in cervical disease. The increasing protein level of Gankyrin was observed in high-grade cervical intraepithelial neoplasia and carcinoma compared with benign cervical tissues and low-grade cervical intraepithelial neoplasia. In para-carcinoma tissues, it was found interestingly that there was no lymph node metastasis when nuclei Gankyrin was positively expressed, but lymph node metastasis rate was 30% (6/20) when nuclei Gankyrin was negatively expressed. In vitro, the transfection of Gankyrin resulted in markedly up-regulating of Vimentin, β-catenin and Twist2, as well as down-regulating of E-cadherin in cervical carcinoma cells. Our results suggested that Gankyrin may be functional in cervical carcinogenesis and metastasis.
PMCID: PMC3994022  PMID: 24751719
6.  The oncoprotein p28GANK establishes a positive feedback loop in β-catenin signaling 
Cell Research  2011;21(8):1248-1261.
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.
PMCID: PMC3193485  PMID: 21691299
p28GANK; β-catenin; E-cadherin; positive feedback loop; hepatocellular carcinoma
7.  Effects of Ligand Binding on the Mechanical Properties of Ankyrin Repeat Protein Gankyrin 
PLoS Computational Biology  2013;9(1):e1002864.
Ankyrin repeat proteins are elastic materials that unfold and refold sequentially, repeat by repeat, under force. Herein we use atomistic molecular dynamics to compare the mechanical properties of the 7-ankyrin-repeat oncoprotein Gankyrin in isolation and in complex with its binding partner S6-C. We show that the bound S6-C greatly increases the resistance of Gankyrin to mechanical stress. The effect is specific to those repeats of Gankyrin directly in contact with S6-C, and the mechanical ‘hot spots’ of the interaction map to the same repeats as the thermodynamic hot spots. A consequence of stepwise nature of unfolding and the localized nature of ligand binding is that it impacts on all aspects of the protein's mechanical behavior, including the order of repeat unfolding, the diversity of unfolding pathways accessed, the nature of partially unfolded intermediates, the forces required and the work transferred to the system to unfold the whole protein and its parts. Stepwise unfolding thus provides the means to buffer repeat proteins and their binding partners from mechanical stress in the cell. Our results illustrate how ligand binding can control the mechanical response of proteins. The data also point to a cellular mechano-switching mechanism whereby binding between two partner macromolecules is regulated by mechanical stress.
Author Summary
Here we use molecular dynamics simulation to compare the mechanical properties of the 7-ankyrin-repeat oncoprotein Gankyrin in isolation and in complex with binding partner S6-C. Tandem repeat proteins like Gankyrin comprise tandem arrays of small structural motifs that pack linearly to produce elongated architectures. They are elastic, mechanically weak molecules and they unfold and refold repeat by repeat under force. We show that S6-C binding greatly increases the resistance of Gankyrin to mechanical stress. The enhanced mechanical stability is specific to those ankyrin repeats in contact with S6-C, and the localized nature of the effect results in fundamental changes in the way the protein responds to force. Thus, the forced unfolding of isolated Gankryin involves a diverse set of pathways with a preference for a C- to N-terminus unfolding mechanism whereas this diversity is reduced upon complex formation with the central repeats, which are those most tightly bound to the ligand, tending to unfold last. Our study shows how stepwise unfolding can buffer repeat proteins and their binding partners from mechanical stress in the cell. It also points to a mechano-switching mechanism whereby binding between two partner macromolecules is regulated by mechanical stress.
PMCID: PMC3547791  PMID: 23341763
8.  Intracellular Signaling and Hepatocellular Carcinoma 
Seminars in cancer biology  2010;21(1):28-34.
Liver cancer is the fifth most common cancer and the third most common cause of cancer related death in the world. The recent development of new techniques for the investigations of global change in the gene expression, signaling pathways and wide genome binding has provided novel information for the mechanisms underlying liver cancer progression. Although these studies identified gene expression signatures in hepatocelluar carcinoma, the early steps of the development of hepatocellular carcinomas (HCC) are not well understood. The development of HCC is a multistep process which includes the progressive alterations of gene expression leading to the increased proliferation and to liver cancer. This review summarizes recent progress in the identification of the key steps of the development of HCC with the focus on early events of carcinogenesis and on the role of translational and epigenetic alterations in the development of HCC. Quiescent stage of the liver is supported by several tumor suppressor proteins including p53, Rb and C/EBPα. Studies with chemical models of liver carcinogenesis and with human HCC have shown that the elevation of gankyrin is responsible for the elimination of these three proteins at early steps of carcinogenesis. Later stages of progression of the liver cancer are associated with alterations in many signaling pathways including translation which leads to epigenetic silencing/activation of many genes. Particularly, recent reports suggest a critical role of histone deacetylase 1, HDAC1, in the development of HCC through the interactions with transcription factors such as C/EBP family proteins.
PMCID: PMC3228644  PMID: 20850540
liver cancer; C/EBPα; p53; Rb; gankyrin
9.  Dissection of Protein-Protein Interaction and CDK4 Inhibition in the Oncogenic versus Tumor Suppressing Functions of Gankyrin and P16† 
Journal of molecular biology  2007;373(4):990-1005.
Protein-protein interactions usually involve a large number of residues; thus it is difficult to elucidate functional and structural roles of specific residues located in the interface. This problem is particularly challenging for ankyrin repeat proteins (ARs), which consist of linear arrays of small repeating units and play critical roles in almost every life process via protein-protein interactions, because the residues involved are discontinuously dispersed in both the ARs and their partners. Our previous studies showed that while both P16 and gankyrin bind to CDK4 in similar fashion, only P16 inhibits the kinase activity of CDK4. While this could explain why P16 is a tumor suppressor and gankyrin is oncogenic, the structural basis of these contrasting properties was unknown. In this study we show that a double mutant of gankyrin, L62H/I79D, inhibits the kinase activity of CDK4, similar to P16, and such CDK4-inhibtory activity is associated with the I79D but not L62H mutation. In addition, mutations at I79 and L62 bring about moderate decrease in the stability of gankyrin. Further structural and biophysical analyses suggest that the substitution of Ile 79 with Asp leads to local conformational changes in loops I–III of gankyrin. Taken together, our results allow the dissection of the “protein-protein binding” and “CDK4 inhibition” functions of P16, show that the difference between tumor suppressing and oncogenic functions of P16 and gankyrin, respectively, mainly resides in a single residue, and provide structural insight to the contrasting biological functions of the two AR proteins.
PMCID: PMC2693045  PMID: 17881001
10.  p38α inhibits liver fibrogenesis and consequent hepatocarcinogenesis by curtailing accumulation of reactive oxygen species 
Cancer research  2012;73(1):215-224.
Most hepatocellular carcinomas (HCCs) develop in the context of severe liver fibrosis and cirrhosis caused by chronic liver inflammation, which also results in accumulation of reactive oxygen species (ROS). In this study, we examined whether the stress activated protein kinase p38α (Mapk14) controls ROS metabolism and development of fibrosis and cancer in mice given thioacetamide (TAA) to induce chronic liver injury. Liver-specific p38α ablation was found to enhance ROS accumulation, which appears to be exerted through the reduced expression of anti-oxidant protein heat shock protein (HSP) 25 (Hspb1), a mouse homologue of HSP27. Its re-expression in p38α-deficient liver prevents ROS accumulation and TAA-induced fibrosis. p38α-deficiency increased expression of SOX2, a marker for cancer stem cells, and the liver oncoproteins c-Jun (Jun) and Gankyrin (Psmd10) and led to enhanced TAA-induced hepatocarcinogenesis. The up-regulation of SOX2 and c-Jun was prevented by administration of the antioxidant butylated hydroxyanisole. Intriguingly, the risk of human HCC recurrence is positively correlated with ROS accumulation in liver. Thus, p38α and its target HSP25/HSP27 appear to play a conserved and critical hepatoprotective function by curtailing ROS accumulation in liver parenchymal cells engaged in oxidative metabolism of exogenous chemicals. Augmented oxidative stress of liver parenchymal cells may explain the close relationship between liver fibrosis and hepatocarcinogenesis.
PMCID: PMC3605785  PMID: 23271722
HSP27; liver fibrosis; ROS; SOX2; Gankyrin
11.  Purification, crystallization and preliminary X-ray diffraction analysis of the non-ATPase subunit Nas6 in complex with the ATPase subunit Rpt3 of the 26S proteasome from Saccharomyces cerevisiae  
The complex of the non-ATPase subunit Nas6 with the C-terminal domain of the ATPase subunit Rpt3 of the 26S proteasome from S. cerevisiae was co-expressed in E. coli and purified to homogeneity. The crystals obtained from the protein complex diffracted to a resolution of 2.2 Å.
The non-ATPase subunit Nas6, which is the human orthologue of gankyrin, was co-expressed with the C-terminal domain of the ATPase subunit Rpt3 of the yeast 26S proteasome in Escherichia coli, purified to near-homogeneity and crystallized using the hanging-drop vapour-diffusion method. The protein crystallized in space group P21, with unit-cell parameters a = 60.38, b = 100.22, c = 72.20 Å, β = 94.70° and with three Nas6–Rpt3C molecules per asymmetric unit. The crystal diffracted to beyond 2.2 Å resolution using synchrotron radiation.
PMCID: PMC2330193  PMID: 17329811
Nas6; gankyrin; oncoproteins; Rpt3; proteasome; protein degradation; ubiquitin
12.  Upregulator of Cell Proliferation Predicts Poor Prognosis in Hepatocellular Carcinoma and Contributes to Hepatocarcinogenesis by Downregulating FOXO3a 
PLoS ONE  2012;7(7):e40607.
The goal of the present study was to investigate the potential correlation between the expression level of upregulator of cell proliferation (URGCP/URG4) and the prognosis of hepatocellular carcinoma (HCC), and to examine the biological function of URGCP/URG4 in the progression of HCC, to better understand its underlying molecular mechanism in hepatic tumorigenesis.
URGCP/URG4 expression was analyzed in 15 HCC cell lines, in 278 archived paraffin-embedded HCC sections, and in 10 pairs of fresh HCC tumor and para-tumor non-cancerous tissues using immunohistochemistry (IHC) and Western blotting analysis (WB). The effect of URGCP/URG4 on cell proliferation and tumorigenesis was examined in vitro and in vivo. WB and luciferase reporter analyses were performed to identify the effects of URGCP/URG4-overexpression or -knockdown on expression of cell cycle regulators and transcriptional activity of FOXO3a.
IHC results revealed an upregulation of URGCP/URG4 in all HCC cell lines and fresh HCC samples as compared with normal liver cells and para-tumor tissues, respectively. URGCP/URG4 was also expressed at a high level in 122 of the 278 (43.8%) archived HCC specimens. The expression level of URGCP/URG4 was significantly correlated with clinical staging and poor patient survival of HCC in the study cohort, and in various clinical subgroups. Strikingly, ectopic expression of URGCP/URG4 induced proliferation and anchorage-independent growth of HCC cells, while silencing of URGCP/URG4 had the opposite effect. Furthermore, URGCP/URG4 overexpression in HCC cells increased cellular entry into the G1/S transitional phase, associated with downregulation of p27Kip1 and p21Cip1 and upregulation of cyclin D1. These effects were accompanied by enhanced Akt activity and reduced FOXO3a transcriptional activity.
URGCP/URG4 plays an important role in promoting proliferation and tumorigenesis of HCC and may represent a novel prognostic biomarker and therapeutic target for this disease.
PMCID: PMC3398045  PMID: 22815774
13.  Quantitative proteomic analysis in HCV-induced HCC reveals sets of proteins with potential significance for racial disparity 
The incidence and mortality of hepatitis C virus (HCV)-induced hepatocellular carcinoma (HCC) is higher in African Americans (AA) than other racial/ethnic groups in the U.S., but the reasons for this disparity are unknown. There is an urgent need for the discovery of novel molecular signatures for HCV disease progression to understand the underlying biological basis for this cancer rate disparity to improve the clinical outcome.
We performed differential proteomics with isobaric labeling tags for relative and absolute quantitation (iTRAQ) and MS/MS analysis to identify proteins differentially expressed in cirrhotic (CIR) and HCC as compared to normal tissues of Caucasian American (CA) patients. The raw data were analyzed using the ProteinPilot v3.0. Searches were performed against all known sequences populating the Swiss-Prot, Refseq, and TrEMBL databases. Quality control analyses were accomplished using pairwise correlation plots, boxplots, principal component analysis, and unsupervised hierarchical clustering. Supervised analysis was carried out to identify differentially expressed proteins. Candidates were validated in independent cohorts of CA and AA tissues by qRT-PCR or Western blotting.
A total of 238 unique proteins were identified. Of those, around 15% were differentially expressed between normal, CIR & HCC groups. Target validation demonstrates racially distinct alteration in the expression of certain proteins. For example, the mRNA expression levels of transferrin (TF) were 2 and18-fold higher in CIR and HCC in AA as compared to CA. Similarly; the expression of Apolipoprotein A1 (APOA1) was 7-fold higher in HCC of AA. This increase was mirrored in the protein expression levels. Interestingly, the level of hepatocyte nuclear factor4α (HNF4α) protein was down regulated in AA, whereas repression of transcription is seen more in CA compared to AA. These data suggest that racial disparities in HCC could be a consequence of differential dysregulation of HNF4α transcriptional activity.
This study identifies novel molecular signatures in HCV-induced HCC using iTRAQ-based tissue proteomics. The proteins identified will further enhance a molecular explanation to the biochemical mechanism(s) that may play a role in HCC racial disparities.
PMCID: PMC3850534  PMID: 24283668
Hepatocellular carcinoma; Hepatitis C; Tissue proteomics; Isobaric tags for relative and absolute quantification (iTRAQ); Cancer racial disparity
14.  Dynamic Expression Patterns of Differential Proteins during Early Invasion of Hepatocellular Carcinoma 
PLoS ONE  2014;9(3):e88543.
Tumor cell invasion into the surrounding matrix has been well documented as an early event of metastasis occurrence. However, the dynamic expression patterns of proteins during early invasion of hepatocellular carcinoma (HCC) are largely unknown. Using a three-dimensional HCC invasion culture model established previously, we investigated the dynamic expression patterns of identified proteins during early invasion of HCC.
Materials and Methods
Highly metastatic MHCC97H cells and a liver tissue fragment were long-term co-cultured in a rotating wall vessel (RWV) bioreactor to simulate different pathological states of HCC invasion. The established spherical co-cultures were collected on days 0, 5, 10, and 15 for dynamic expression pattern analysis. Significantly different proteins among spheroids at different time points were screened and identified using quantitative proteomics of iTRAQ labeling coupled with LC–MS/MS. Dynamic expression patterns of differential proteins were further categorized by K-means clustering. The expression modes of several differentially expressed proteins were confirmed by Western blot and qRT–PCR.
Time course analysis of invasion/metastasis gene expressions (MMP2, MMP7, MMP9, CD44, SPP1, CXCR4, CXCL12, and CDH1) showed remarkable, dynamic alterations during the invasion process of HCC. A total of 1,028 proteins were identified in spherical co-cultures collected at different time points by quantitative proteomics. Among these proteins, 529 common differential proteins related to HCC invasion were clustered into 25 types of expression patterns. Some proteins displayed significant dynamic alterations during the early invasion process of HCC, such as upregulation at the early invasion stage and downregulation at the late invasion stage (e.g., MAPRE1, PHB2, cathepsin D, etc.) or continuous upregulation during the entire invasion process (e.g., vitronectin, Met, clusterin, ICAM1, GSN, etc.).
Dynamic expression patterns of candidate proteins during the early invasion process of HCC facilitate the discovery of new molecular targets for early intervention to prevent HCC invasion and metastasis.
PMCID: PMC3948617  PMID: 24614035
15.  Coronin-1C is a novel biomarker for hepatocellular carcinoma invasive progression identified by proteomics analysis and clinical validation 
To better search for potential markers for hepatocellular carcinoma (HCC) invasion and metastasis, proteomic approach was applied to identify potential metastasis biomarkers associated with HCC.
Membrane proteins were extracted from MHCC97L and HCCLM9 cells, with a similar genetic background and remarkably different metastasis potential, and compared by SDS-PAGE and identified by ESI-MS/MS. The results were further validated by western blot analysis, immunohistochemistry (IHC) of tumor tissues from HCCLM9- and MHCC97L-nude mice, and clinical specimens.
Membrane proteins were extracted from MHCC97L and HCCLM9 cell and compared by SDS-PAGE analyses. A total of 14 differentially expressed proteins were identified by ESI-MS/MS. Coronin-1C, a promising candidate, was found to be overexpressed in HCCLM9 cells as compared with MHCC97L cells, and validated by western blot and IHC from both nude mice tumor tissues and clinical specimens. Coronin-1C level showed an abrupt upsurge when pulmonary metastasis occurred. Increasing coronin-1C expression was found in liver cancer tissues of HCCLM9-nude mice with spontaneous pulmonary metastasis. IHC study on human HCC specimens revealed that more patients in the higher coronin-1C group had overt larger tumor and more advanced stage.
Coronin-1C could be a candidate biomarker to predict HCC invasive behavior.
PMCID: PMC2845108  PMID: 20181269
16.  Large isoform of MRJ (DNAJB6) reduces malignant activity of breast cancer 
Mammalian relative of DnaJ (MRJ [DNAJB6]), a novel member of the human DnaJ family, has two isoforms. The smaller isoform, MRJ(S), is studied mainly for its possible role in Huntington's disease. There are no reports of any biologic activity of the longer isoform, MRJ(L). We investigated whether this molecule plays any role in breast cancer. Our studies were prompted by interesting observations we made regarding the expression of MRJ in breast cancer cell lines and breast cancer tissue microarrays, as described below.
Expression of MRJ(L) from several breast cancer cell lines was evaluated using real-time PCR. Relative levels of the small and large isoforms in breast cancer cell lines were studied using Western blot analysis. A breast cancer progression tissue microarray was probed using anti-MRJ antibody. MRJ(L) was ectopically expressed in two breast cancer cell lines. These cell lines were evaluated for their in vitro correlates of tumor aggressiveness, such as invasion, migration, and anchorage independence. The cell lines were also evaluated for in vivo tumor growth and metastasis. The secreted proteome of the MRJ(L) expressors was analyzed to elucidate the biochemical changes brought about by re-expression of MRJ(L).
We found that MRJ(L) is expressed at a significantly lower level in aggressive breast cancer cell lines compared with normal breast. Furthermore, in clinical cases of breast cancer expression of MRJ is lost as the grade of infiltrating ductal carcinoma advances. Importantly, MRJ staining is lost in those cases that also had lymph node metastasis. We report that MRJ(L) is a protein with a functional nuclear localization sequence. Expression of MRJ(L) via an exogenous promoter in breast cancer cell line MDA-MB-231 and in MDA-MB-435 (a cell line that metastasizes from the mammary fat pad) decreases their migration and invasion, reduces their motility, and significantly reduces orthotopic tumor growth in nude mice. Moreover, the secreted proteome of the MRJ(L)-expressing cells exhibited reduced levels of tumor progression and metastasis promoting secreted proteins, such as SPP1 (osteopontin), AZGP1 (zinc binding α2-glycoprotein 1), SPARC (osteonectin), NPM1 (nucleophosmin) and VGF (VGF nerve growth factor inducible). On the other hand, levels of the secreted metastasis-suppressor KiSS1 (melanoma metastasis suppressor) were increased in the secreted proteome of the MRJ(L)-expressing cells. We confirmed by quantitative RT-PCR analysis that the secreted profile reflected altered transcription of the respective genes.
Collectively, our data indicate an important role for a totally uncharacterized isoform of DNAJB6 in breast cancer. We show that MRJ(L) is a nuclear protein that is lost in breast cancer, that regulates several key players in tumor formation and metastasis, and that is functionally able to retard tumor growth.
PMCID: PMC2397520  PMID: 18328103
17.  Quantitative proteomics analysis of early recurrence/metastasis of huge hepatocellular carcinoma following radical resection 
Proteome Science  2014;12:22.
Hepatic resection is the preferred treatment for huge hepatocellular carcinoma (>10 cm in diameter; H-HCC). However, the patients with H-HCC suffer from poor prognosis due to the early recurrence/metastasis. The underlying mechanism of H-HCC’s early recurrence/metastasis is currently not well understood.
Here, we describe an Isobaric Tags for relative and absolute quantification (iTRAQ)-based quantitative proteomics approach to analyze the early recurrence/metastasis related proteins of H-HCC after radical resection through multidimensional chromatography coupled with tandem mass spectrometry (2DLC-MS/MS). The different protein expression profiles between the early recurrence/metastasis within 6 months(R/M≤6months) and late recurrence/metastasis within 6–12 months after surgery (R/M6-12months) were confirmed and might reveal different underlying molecular mechanisms. We identified 44 and 49 significantly differentially expressed proteins in the R/M≤6months group and the R/M6-12months group compared to the group who had no recurrence within 2 years post surgery (the NR/M group), respectively. Moreover, among those proteins, S100A12 and AMACR were down regulated in the R/M≤6months group but up-regulated in the R/M6-12months group; and this regulation was further confirmed in mRNA and protein level by Q-PCR, Western-Blot and Immunohistochemistry (IHC).
This current study presents the first proteomic profile of the early recurrence/metastasis of H-HCC. The results suggest that S100A12 and AMACR might be potential prognostic markers for predicting the early recurrence/metastasis of H-HCC after hepatectomy.
PMCID: PMC4023177  PMID: 24839399
Huge hepatocellular carcinoma (H-HCC); Early recurrence/metastasis; Quantitative proteomics; iTRAQ; Potential prognostic biomarker
18.  Redox Proteomics in Selected Neurodegenerative Disorders: From Its Infancy to Future Applications 
Antioxidants & Redox Signaling  2012;17(11):1610-1655.
Several studies demonstrated that oxidative damage is a characteristic feature of many neurodegenerative diseases. The accumulation of oxidatively modified proteins may disrupt cellular functions by affecting protein expression, protein turnover, cell signaling, and induction of apoptosis and necrosis, suggesting that protein oxidation could have both physiological and pathological significance. For nearly two decades, our laboratory focused particular attention on studying oxidative damage of proteins and how their chemical modifications induced by reactive oxygen species/reactive nitrogen species correlate with pathology, biochemical alterations, and clinical presentations of Alzheimer's disease. This comprehensive article outlines basic knowledge of oxidative modification of proteins and lipids, followed by the principles of redox proteomics analysis, which also involve recent advances of mass spectrometry technology, and its application to selected age-related neurodegenerative diseases. Redox proteomics results obtained in different diseases and animal models thereof may provide new insights into the main mechanisms involved in the pathogenesis and progression of oxidative-stress-related neurodegenerative disorders. Redox proteomics can be considered a multifaceted approach that has the potential to provide insights into the molecular mechanisms of a disease, to find disease markers, as well as to identify potential targets for drug therapy. Considering the importance of a better understanding of the cause/effect of protein dysfunction in the pathogenesis and progression of neurodegenerative disorders, this article provides an overview of the intrinsic power of the redox proteomics approach together with the most significant results obtained by our laboratory and others during almost 10 years of research on neurodegenerative disorders since we initiated the field of redox proteomics. Antioxid. Redox Signal. 17, 1610–1655.
I. Introduction
II. Protein (/Lipid) Oxidation and Protein Dysfunction
A. Protein carbonyls
B. Protein nitration
1. Peroxynitrite (ONOO−)
2. Nitrogen dioxide (NO2)
C. HNE adduction to proteins
D. Importance of clearance and detoxification systems
1. The proteasome, parkin, ubiquitin carboxy-terminal hydrolase-L1, and HSPs
2. Superoxide dismutase
3. Catalase
4. Peroxiredoxins
5. Trx and Trx reductase
6. Glutathione reductase
7. Vitamins in neurodegeneration
8. Involvement of iron in neurodegeneration
E. Role of iron in neurodegeneration
1. Fe homeostasis in AD
2. Fe homeostasis in PD
3. Fe homeostasis in ALS
4. Fe homeostasis in HD
F. Some known consequences of protein oxidation
III. Overview of Redox Proteomics
A. Global, gel-based approaches
B. Targeted, gel-free approach
1. Enrichment of PCO modified proteins
2. Enrichment of HNE modified proteins
3. Enrichment of 3-NT modified proteins
IV. Application of Redox Proteomics to Selected Neurodegenerative Disorders
A. Alzheimer's disease
1. PCO in AD
2. Identification of carbonylated proteins in brain of subjects with AD
a. Sample: the brain
b. Energy dysfunction
c. Excitotoxicity
d. Proteosomal dysfunction
e. Neuritic abnormalities
f. APP regulation, tau hyperphosphorylation, and cell cycle regulation
g. Synaptic abnormalities and LTP
h. pH maintenance
i. Mitochondrial abnormalities
3. Carbonylated proteins in brain of subjects with amnestic MCI
4. EAD carbonylated proteins
5. PCAD vs. amnestic MCI protein carbonylation in brain
6. Protein-bound HNE in brain and progression of Alzheimer's disease
7. Protein-bound 3-NT in brain and progression of Alzheimer's disease
8. Nitrated brain proteins in MCI
9. Nitrated proteins in EAD
B. Parkinson disease
1. Redox proteomics in PD
C. Amyotrophic lateral sclerosis
1. Redox proteomics studies in ALS transgenic mice
D. Huntington disease
1. Redox proteomics-transgenic mouse model of HD
2. Proteomics of HD brain
E. Down syndrome
1. Redox proteomics in DS transgenic mice
V. Conclusions and Future Directions
PMCID: PMC3448942  PMID: 22115501
19.  Proteomic Consequences of a Single Gene Mutation in a Colorectal Cancer Model 
Journal of Proteome Research  2011;11(2):1184-1195.
The proteomic effects of specific cancer-related mutations have not been well characterized. In colorectal cancer (CRC), a relatively small number of mutations in key signaling pathways appear to drive tumorigenesis. Mutations in adenomatous polyposis coli (APC), a negative regulator of Wnt signaling, occur in up to 60% of CRC tumors. Here we examine the proteomic consequences of a single gene mutation by using an isogenic CRC cell culture model in which wildtype APC expression has been ectopically restored. Using LC–MS/MS label free shotgun proteomics, over 5000 proteins were identified in SW480Null (mutant APC) and SW480APC (APC restored). We observed 155 significantly differentially expressed proteins between the two cell lines, with 26 proteins showing opposite expression trends relative to gene expression measurements. Protein changes corresponded to previously characterized features of the APCNull phenotype: loss of cell adhesion proteins, increase in cell cycle regulators, alteration in Wnt signaling related proteins, and redistribution of β-catenin. Increased expression of RNA processing and isoprenoid biosynthetic proteins occurred in SW480Null cells. Therefore, shotgun proteomics reveals proteomic differences associated with a single gene change, including many novel differences that fall outside known target pathways.
The proteomic effects of specific cancer-related mutations have not been well characterized. In colorectal cancer (CRC), a relatively small number of mutations in key signaling pathways appear to drive tumorigenesis. Mutations in adenomatous polyposis coli (APC), a negative regulator of Wnt signaling, occur in up to 60% of CRC tumors. Here we examine the proteomic consequences of a single gene mutation. Shotgun proteomics reveals proteomic differences associated with a single gene change, including many novel differences that fall outside known target pathways.
PMCID: PMC3271737  PMID: 22103262
shotgun proteomics; label-free quantitation; multiple reaction monitoring (MRM); LC−MS/MS; colorectal cancer; APC
20.  Cell Surface Glycan Alterations in Epithelial Mesenchymal Transition Process of Huh7 Hepatocellular Carcinoma Cell 
PLoS ONE  2013;8(8):e71273.
Background and Objective
Due to recurrence and metastasis, the mortality of Hepatocellular carcinoma (HCC) is high. It is well known that the epithelial mesenchymal transition (EMT) and glycan of cell surface glycoproteins play pivotal roles in tumor metastasis. The goal of this study was to identify HCC metastasis related differential glycan pattern and their enzymatic basis using a HGF induced EMT model.
HGF was used to induce HCC EMT model. Lectin microarray was used to detect the expression of cell surface glycan and the difference was validated by lectin blot and fluorescence cell lectin-immunochemistry. The mRNA expression levels of glycotransferases were determined by qRT-PCR.
After HGF treatment, the Huh7 cell lost epithelial characteristics and obtained mesenchymal markers. These changes demonstrated that HGF could induce a typical cell model of EMT. Lectin microarray analysis identified a decreased affinity in seven lectins ACL, BPL, JAC, MPL, PHA-E, SNA, and SBA to the glycan of cell surface glycoproteins. This implied that glycan containing T/Tn-antigen, NA2 and bisecting GlcNAc, Siaα2-6Gal/GalNAc, terminal α or βGalNAc structures were reduced. The binding ability of thirteen lectins, AAL, LCA, LTL, ConA, NML, NPL, DBA, HAL, PTL II, WFL, ECL, GSL II and PHA-L to glycan were elevated, and a definite indication that glycan containing terminal αFuc and ± Sia-Le, core fucose, α-man, gal-β(α) GalNAc, β1,6 GlcNAc branching and tetraantennary complex oligosaccharides structures were increased. These results were further validated by lectin blot and fluorescence cell lectin-immunochemistry. Furthermore, the mRNA expression level of Mgat3 decreased while that of Mgat5, FucT8 and β3GalT5 increased. Therefore, cell surface glycan alterations in the EMT process may coincide with the expression of glycosyltransferase.
The findings of this study systematically clarify the alterations of cell surface glycan in cancer EMT, and may provide novel insight for HCC metastasis.
PMCID: PMC3748092  PMID: 23977005
21.  Promoting Colonization in Metastatic HCC Cells by Modulation of Autophagy 
PLoS ONE  2013;8(9):e74407.
Autophagy is an important adaptive survival mechanism, which has been postulated to be involved in cancer metastasis. The purpose of this study was to investigate autophagy in metastasis of hepatocellular carcinoma (HCC).
Immunohistochemical analysis of autophagic activity in metastatic and paired primary HCC tissues using LC3 as autophagosome marker was performed in samples from 216 HCC patients diagnosed with metastasis (including 158 intravascular, 42 intrabiliary, 8 lymph node, 4 bone and 4 lung metastases). Then a mouse model of pulmonary metastasis was established using a highly metastatic HCC cell line (HCCLM3). Autophagy in pulmonary metastases and paired primary tumors were analyzed by LC3 immunohistochemistry, transmission electron microscopy (TEM) and western blot analysis. Further, mouse model of pulmonary metastasis and in vitro cell migration, invasion and detachment models were established using a stable GFP-LC3-expressing HCCLM3 cell line (HCCLM3-GFP-LC3). Autophagic alterations during metastatic colonization, migration, invasion and detachment were determined by GFP-LC3 analysis and western blot analysis.
LC3 immunohistochemistry of metastases and primary tumors from HCC patients revealed significantly higher LC3 expression in metastases than primary HCC, which suggested a higher level of autophagy in HCC metastases. Further immunohistochemical, TEM, western blot and in vivo GFP-LC3 analyses of lung metastases and primary tumors in mouse model of pulmonary metastasis confirmed that metastatic colonies displayed higher level of autophagy than primary tumors and the early metastatic colonies displayed highest level. The dynamic monitoring of autophagy in cell migration, invasion and detachment showed that autophagy did not significantly alter in those processes.
Autophagy is activated in metastatic colonization but not in invasion, migration and detachment of HCC cells. Autophagy may play a role in HCC metastasis via promoting metastatic colonization of HCC cells.
PMCID: PMC3772859  PMID: 24058558
22.  Quantitative Proteomic Profiling Identifies DPYSL3 as Pancreatic Ductal Adenocarcinoma-Associated Molecule That Regulates Cell Adhesion and Migration by Stabilization of Focal Adhesion Complex 
PLoS ONE  2013;8(12):e79654.
Elucidation of how pancreatic cancer cells give rise to distant metastasis is urgently needed in order to provide not only a better understanding of the underlying molecular mechanisms, but also to identify novel targets for greatly improved molecular diagnosis and therapeutic intervention. We employed combined proteomic technologies including mass spectrometry and isobaric tags for relative and absolute quantification peptide tagging to analyze protein profiles of surgically resected human pancreatic ductal adenocarcinoma tissues. We identified a protein, dihydropyrimidinase-like 3, as highly expressed in human pancreatic ductal adenocarcinoma tissues as well as pancreatic cancer cell lines. Characterization of the roles of dihydropyrimidinase-like 3 in relation to cancer cell adhesion and migration in vitro, and metastasis in vivo was performed using a series of functional analyses, including those employing multiple reaction monitoring proteomic analysis. Furthermore, dihydropyrimidinase-like 3 was found to interact with Ezrin, which has important roles in cell adhesion, motility, and invasion, while that interaction promoted stabilization of an adhesion complex consisting of Ezrin, c-Src, focal adhesion kinase, and Talin1. We also found that exogenous expression of dihydropyrimidinase-like 3 induced activating phosphorylation of Ezrin and c-Src, leading to up-regulation of the signaling pathway. Taken together, the present results indicate successful application of combined proteomic approaches to identify a novel key player, dihydropyrimidinase-like 3, in pancreatic ductal adenocarcinoma tumorigenesis, which may serve as an important biomarker and/or drug target to improve therapeutic strategies.
PMCID: PMC3855176  PMID: 24339867
23.  Yin Yang-1 suppresses invasion and metastasis of pancreatic ductal adenocarcinoma by downregulating MMP10 in a MUC4/ErbB2/p38/MEF2C-dependent mechanism 
Molecular Cancer  2014;13:130.
Increasing evidence indicates an important role of transcription factor Yin Yang-1 (YY1) in human tumorigenesis. However, its function in cancer remains controversial and the relevance of YY1 to pancreatic ductal adenocarcinoma (PDAC) remains to be clarified.
In this study, we detected YY1 expression in clinical PDAC tissue samples and cell lines using quantitative RT-PCR, immunohistochemistry and western blotting. We also detected MUC4 and MMP10 mRNA levels in 108 PDAC samples using qRT-PCR and analyzed the correlations between YY1 and MUC4 or MMP10 expression. The role of YY1 in the proliferation, invasion and metastatic abilities of PDAC cells in vitro was studied by CCK-8 assay, cell migration and invasion assays. In vivo pancreatic tumor growth and metastasis was studied by a xenogenous subcutaneously implant model and a tail vein metastasis model. The potential mechanisms underlying YY1 mediated tumor progression in PDAC were explored by digital gene expression (DGE) sequencing, signal transduction pathways blockage experiments and luciferase assays. Statistical analysis was performed using the SPSS 15.0 software.
We found that the expression of YY1 in PDACs was higher compared with their adjacent non-tumorous tissues and normal pancreas tissues. However, PDAC patients with high level overexpression of YY1 had better outcome than those with low level overexpression. YY1 expression levels were statistically negatively correlated with MMP10 expression levels, but not correlated with MUC4 expression levels. YY1 overexpression suppressed, whereas YY1 knockdown enhanced, the proliferation, invasion and metastatic properties of BXPC-3 cells, both in vitro and in vivo. YY1 suppresses invasion and metastasis of pancreatic cancer cells by downregulating MMP10 in a MUC4/ErbB2/p38/MEF2C-dependent mechanism.
The present study suggested that YY1 plays a negative role, i.e. is a tumor suppressor, in PDAC, and may become a valuable diagnostic and prognostic marker of PDAC.
PMCID: PMC4047260  PMID: 24884523
Yin Yang-1; Pancreatic ductal adenocarcinoma; Metastasis; MMP10; MUC4; MEF2C
24.  CD133 facilitates epithelial-mesenchymal transition through interaction with the ERK pathway in pancreatic cancer metastasis 
Molecular Cancer  2014;13:15.
Pancreatic cancer is a lethal disease due to the high incidence of metastasis at the time of detection. CD133 expression in clinical pancreatic cancer correlates with poor prognosis and metastasis. However, the molecular mechanism of CD133-regulated metastasis remains unclear. In recent years, epithelial-mesenchymal transition (EMT) has been linked to cancer invasion and metastasis. In the present study we investigated the role of CD133 in pancreatic cancer metastasis and its potential regulatory network.
A highly migratory pancreatic cancer cell line, Capan1M9, was established previously. After shRNA was stable transducted to knock down CD133 in Capan1M9 cells, gene expression was profiled by DNA microarray. Orthotopic, splenic and intravenous transplantation mouse models were set up to examine the tumorigenesis and metastatic capabilities of these cells. In further experiments, real-time RT-PCR, Western blot and co-immunoprecipitate were conducted to evaluate the interactions of CD133, Slug, N-cadherin, ERK1/2 and SRC.
We found that CD133+ human pancreatic cancer cells were prone to generating metastatic nodules in in vivo models using immunodeficient mice. In contrast, CD133 knockdown suppressed cancer invasion and metastasis in vivo. Gene profiling analysis suggested that CD133 modulated mesenchymal characteristics including the expression of EMT-related genes, such as Slug and N-cadherin. These genes were down-regulated following CD133 knockdown. Moreover, CD133 expression could be modulated by the extracellular signal-regulated kinase (ERK)1/2 and SRC signaling pathways. The binding of CD133 to ERK1/2 and SRC acts as an indispensable mediator of N-cadherin expression.
These results demonstrate that CD133 plays a critical role in facilitating the EMT regulatory loop, specifically by upregulating N-cadherin expression, leading to the invasion and metastasis of pancreatic cancer cells. Our study provides a novel insight into the function of CD133 in the EMT program and a better understanding of the mechanism underlying the involvement of CD133 in pancreatic cancer metastasis.
PMCID: PMC3931313  PMID: 24468059
Pancreatic cancer; CD133; Epithelial-mesenchymal transition (EMT); ERK1/2; N-cadherin; Cancer metastasis
25.  iTRAQ-Based Proteomic Analysis of Polyploid Giant Cancer Cells and Budding Progeny Cells Reveals Several Distinct Pathways for Ovarian Cancer Development 
PLoS ONE  2013;8(11):e80120.
Polyploid giant cancer cells (PGCCs) are a morphologically distinct subgroup of human tumor cells with increased nuclear size or multiple nuclei, but they are generally considered unimportant because they are presumed to be nondividing and thus nonviable. We have recently shown that these large cancer cells are not only viable but also can divide asymmetrically and yield progeny cancer cells with cancer stem-like properties via budding division. To further understand the molecular events involved in the regulation of PGCCs and the generation of their progeny cancer cells, we comparatively analyzed the proteomic profiles of PGCCs, PGCCs with budding daughter cells, and regular control cancer cells from the HEY and SKOv3 human ovarian cancer cell lines with and without CoCl2. We used a high-throughput iTRAQ-based proteomic methodology coupled with liquid chromatography-electrospray ionization tandem mass spectroscopy to determine the differentiated regulated proteins. We performed Western blotting and immunohistochemical analyses to validate the differences in the expression patterns of a variety of proteins between PGCCs or budding PGCCs and regular cancer cells identified by iTRAQ approach and also a selected group of proteins from the literature. The differentially regulated proteins included proteins involved in response to hypoxia, stem cell generation, chromatin remodeling, cell-cycle regulation, and invasion and metastasis. In particular, we found that HIF-1alpha and its known target STC1 are upregulated in PGCCs. In addition, we found that a panel of stem cell-regulating factors and epithelial-to-mesenchymal transition regulatory transcription factors were upregulated in budding PGCCs, whereas expression of the histone 1 family of nucleosomal linker proteins was consistently lower in PGCCs than in control cells. Thus, proteomic expression patterns provide valuable insight into the underlying mechanisms of PGCC formation and the relationship between PGCCs and cancer stem cells in patients with ovarian cancers.
PMCID: PMC3858113  PMID: 24348907

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