TRAIP modulates the IGFBP3/AKT pathway to enhance the invasion and proliferation of osteosarcoma by promoting KANK1 degradation

Osteosarcoma is one of the most common primary malignancies in bones and is characterized by high metastatic rates. Circulating tumor cells (CTCs) derived from solid tumors can give rise to metastatic lesions, increasing the risk of death in patients with cancer. Here, we used bioinformatics tools to compare the gene expression between CTCs and metastatic lesions in osteosarcoma to identify novel molecular mechanisms underlying osteosarcoma metastasis. We identified TRAIP as a key differentially expressed gene with prognostic significance in osteosarcoma. We demonstrated that TRAIP regulated the proliferation and invasion of osteosarcoma cells. In addition, we found that TRAIP promoted KANK1 polyubiquitination and subsequent degradation, downregulating IGFBP3 and activating the AKT pathway in osteosarcoma cells. These results support the critical role of the TRAIP/KANK1/IGFBP3/AKT signaling axis in osteosarcoma progression and suggest that TRAIP may represent a promising therapeutic target for osteosarcoma.Subject terms: Bone cancer, Oncogenes     

Design,synthesis and biological evaluation of ezrin inhibitors targeting metastatic osteosarcoma

Respiratory failure due to pulmonary metastasis is the major cause of death for patients with osteosarcoma. However, the molecular basis for metastasis of osteosarcoma is poorly understood. Recently, ezrin, a member of the ERM family of proteins, has been associated with osteosarcoma metastasis to the lungs. The small molecule NSC 668394 was identified to bind to ezrin, inhibit in vitro and in vivo cell migration, invasion, and metastatic colony survival. Reported herein are the design and synthesis of analogues of NSC 668394, and subsequent functional ezrin inhibition studies. The binding affinity was characterized by surface plasmon resonance technique. Cell migration and invasion activity was determined by electrical cell impedance methodology. Optimization of a series of heterocyclic-dione analogues led to the discovery of compounds 21k and 21m as potential novel antimetastatic agents.     

Metastatic function of BMP-2 in gastric cancer cells: the role of PI3K/AKT, MAPK, the NF-κB pathway, and MMP-9 expression

Bone morphogenetic proteins (BMPs) have been implicated in tumorigenesis and metastatic progression in various types of cancer cells, but the role and cellular mechanism in the invasive phenotype of gastric cancer cells is not known. Herein, we determined the roles of phosphoinositide 3-kinase (PI3K)/AKT, extracellular signal-regulated protein kinase (ERK), nuclear factor (NF)-κB, and matrix metalloproteinase (MMP) expression in BMP-2-mediated metastatic function in gastric cancer. We found that stimulation of BMP-2 in gastric cancer cells enhanced the phosphorylation of AKT and ERK. Accompanying activation of AKT and ERK kinase, BMP-2 also enhanced phosphorylation/degradation of IκBα and the nuclear translocation/activation of NF-κB. Interestingly, blockade of PI3K/AKT and ERK signaling using LY294002 and PD98059, respectively, significantly inhibited BMP-2-induced motility and invasiveness in association with the activation of NF-κB. Furthermore, BMP-2-induced MMP-9 expression and enzymatic activity was also significantly blocked by treatment with PI3K/AKT, ERK, or NF-κB inhibitors. Immunohistochemistry staining of 178 gastric tumor biopsies indicated that expression of BMP-2 and MMP-9 had a significant positive correlation with lymph node metastasis and a poor prognosis. These results indicate that the BMP-2 signaling pathway enhances tumor metastasis in gastric cancer by sequential activation of the PI3K/AKT or MAPK pathway followed by the induction of NF-κB and MMP-9 activity, indicating that BMP-2 has the potential to be a therapeutic molecular target to decrease metastasis.     

CCL2/CCR2 Chemokine Signaling Coordinates Survival and Motility of Breast Cancer Cells through Smad3 Protein- and p42/44 Mitogen-activated Protein Kinase (MAPK)-dependent Mechanisms

Increased cell motility and survival are important hallmarks of metastatic tumor cells. However, the mechanisms that regulate the interplay between these cellular processes remain poorly understood. In these studies, we demonstrate that CCL2, a chemokine well known for regulating immune cell migration, plays an important role in signaling to breast cancer cells. We report that in a panel of mouse and human breast cancer cell lines CCL2 enhanced cell migration and survival associated with increased phosphorylation of Smad3 and p42/44MAPK proteins. The G protein-coupled receptor CCR2 was found to be elevated in breast cancers, correlating with CCL2 expression. RNA interference of CCR2 expression in breast cancer cells significantly inhibited CCL2-induced migration, survival, and phosphorylation of Smad3 and p42/44MAPK proteins. Disruption of Smad3 expression in mammary carcinoma cells blocked CCL2-induced cell survival and migration and partially reduced p42/44MAPK phosphorylation. Ablation of MAPK phosphorylation in Smad3-deficient cells with the MEK inhibitor U0126 further reduced cell survival but not migration. These data indicate that Smad3 signaling through MEK-p42/44MAPK regulates CCL2-induced cell motility and survival, whereas CCL2 induction of MEK-p42/44MAPK signaling independent of Smad3 functions as an alternative mechanism for cell survival. Furthermore, we show that CCL2-induced Smad3 signaling through MEK-p42/44MAPK regulates expression and activity of Rho GTPase to mediate CCL2-induced breast cancer cell motility and survival. With these studies, we characterize an important role for CCL2/CCR2 chemokine signaling in regulating the intrinsic relationships between breast cancer cell motility and survival with implications on the metastatic process.     

Ezrin regulates focal adhesion and invadopodia dynamics by altering calpain activity to promote breast cancer cell invasion

Up-regulation of the cytoskeleton linker protein ezrin frequently occurs in aggressive cancer types and is closely linked with metastatic progression. However, the underlying molecular mechanisms detailing how ezrin is involved in the invasive and metastatic phenotype remain unclear. Here we report a novel function of ezrin in regulating focal adhesion (FA) and invadopodia dynamics, two key processes required for efficient invasion to occur. We show that depletion of ezrin expression in invasive breast cancer cells impairs both FA and invadopodia turnover. We also demonstrate that ezrin-depleted cells display reduced calpain-mediated cleavage of the FA and invadopodia-associated proteins talin, focal adhesion kinase (FAK), and cortactin and reduced calpain-1–specific membrane localization, suggesting a requirement for ezrin in maintaining proper localization and activity of calpain-1. Furthermore, we show that ezrin is required for cell directionality, early lung seeding, and distant organ colonization but not primary tumor growth. Collectively our results unveil a novel mechanism by which ezrin regulates breast cancer cell invasion and metastasis.     

Ezrin expression and cell survival regulation in colorectal cancer

Colorectal cancer (CRC) is the second largest cause of cancer deaths in the United States. A key barrier that prevents better outcomes for this type of cancer as well as other solid tumors is the lack of effective therapies against the metastatic disease. Thus there is an urgent need to fill this gap in cancer therapy. We utilized a 2D-DIGE proteomics approach to identify and characterize proteins that are differentially regulated between primary colon tumor and liver metastatic deposits of the IGF1R-dependent GEO human CRC xenograft, orthotopically implanted in athymic nude mice that may serve as potential therapeutic targets against CRC metastasis. We observed increased expression of ezrin in liver metastasis in comparison to the primary colonic tumor. Increased ezrin expression was further confirmed by western blot and microarray analyses. Ezrin, a cytoskeletal protein belonging to Ezrin–Radixin–Moesin (ERM) family plays important roles in cell motility, invasion and metastasis. However, its exact function in colorectal cancer is not well characterized. Establishment of advanced GEO cell lines with enhanced liver-metastasizing ability showed a significant increase in ezrin expression in liver metastasis. Increased phosphorylation of ezrin at the T567 site (termed here as p-ezrin T567) was observed in liver metastasis. IHC studies of human CRC patient specimens showed an increased expression of p-ezrin T567 in liver metastasis compared to the primary tumors of the same patient. Ezrin modulation by siRNA, inhibitors and T567A/D point mutations significantly downregulated inhibitors of apoptosis (IAP) proteins XIAP and survivin that have been linked to increased aberrant cell survival and metastasis and increased cell death. Inhibition of the IGF1R signaling pathway by humanized recombinant IGF1R monoclonal antibody MK-0646 in athymic mouse subcutaneous xenografts resulted in inhibition of p-ezrin T567 indicating ezrin signaling is downstream of the IGF1R signaling pathway. We identified increased expression of p-ezrin T567 in CRC liver metastasis in both orthotopically implanted GEO tumors as well as human patient specimens. We report for the first time that p-ezrin T567 is downstream of the IGF1R signaling and demonstrate that ezrin regulates cell survival through survivin/XIAP modulation.     

Characteristics of the PI3K/AKT and MAPK/ERK pathways involved in the maintenance of self-renewal in lung cancer stem-like cells

Lung cancer is the leading cause of cancer-related mortality worldwide due to its early asymptomatic and late metastasis. While cancer stem cells (CSCs) may play a vital role in oncogenesis and development of lung cancer, mechanisms underlying CSCs self‐renewal remain less clear. In the present study, we constructed a clinically relevant CSCs enrichment recognition model and evaluated the potential functions of phosphatidylinositol 3-kinase (PI3K)/AKT pathway (PI3K/AKT) and mitogen-activated protein kinases/extracellular signal-regulated kinase (MAPK/ERK) pathways in lung cancer via bioinformatic analysis, providing the basis for in depth mechanistic inquisition. Experimentally, we confirmed that PI3K/AKT pathway predominantly promotes proliferation through anti-apoptosis in lung adenocarcinoma cells, while MAPK/ERK pathway has an overwhelming superiority in regulating the proliferation in lung CSCs. Further, utilizing stemness score model, LLC-Symmetric Division (LLC-SD) cells and mouse orthotopic lung transplantation model, we elucidated an intricate cross-talk between the oncogenic pathway and the stem cell reprograming pathway that impact stem cell characteristics as well as cancer biology features of lung CSCs both in vitro and in vivo. In summary, our findings uncovered a new insight that PI3K/AKT and MAPK/ERK pathways as oncogenic signaling pathway and/or stem cell signaling pathway act distinctively and synergistically to regulate lung CSCs self-renewal.     

Discovery and Validation of a Prostate Cancer Genomic Classifier that Predicts Early Metastasis Following Radical Prostatectomy

Purpose

Clinicopathologic features and biochemical recurrence are sensitive, but not specific, predictors of metastatic disease and lethal prostate cancer. We hypothesize that a genomic expression signature detected in the primary tumor represents true biological potential of aggressive disease and provides improved prediction of early prostate cancer metastasis.

Methods

A nested case-control design was used to select 639 patients from the Mayo Clinic tumor registry who underwent radical prostatectomy between 1987 and 2001. A genomic classifier (GC) was developed by modeling differential RNA expression using 1.4 million feature high-density expression arrays of men enriched for rising PSA after prostatectomy, including 213 who experienced early clinical metastasis after biochemical recurrence. A training set was used to develop a random forest classifier of 22 markers to predict for cases - men with early clinical metastasis after rising PSA. Performance of GC was compared to prognostic factors such as Gleason score and previous gene expression signatures in a withheld validation set.

Results

Expression profiles were generated from 545 unique patient samples, with median follow-up of 16.9 years. GC achieved an area under the receiver operating characteristic curve of 0.75 (0.67–0.83) in validation, outperforming clinical variables and gene signatures. GC was the only significant prognostic factor in multivariable analyses. Within Gleason score groups, cases with high GC scores experienced earlier death from prostate cancer and reduced overall survival. The markers in the classifier were found to be associated with a number of key biological processes in prostate cancer metastatic disease progression.

Conclusion

A genomic classifier was developed and validated in a large patient cohort enriched with prostate cancer metastasis patients and a rising PSA that went on to experience metastatic disease. This early metastasis prediction model based on genomic expression in the primary tumor may be useful for identification of aggressive prostate cancer.     

PRRX1 promotes malignant properties in human osteosarcoma

Paired related homeobox 1 (PRRX1) is a marker of limb bud mesenchymal cells, and deficiency of p53 or Rb in Prrx1-positive cells induces osteosarcoma in several mouse models. However, the regulatory roles of PRRX1 in human osteosarcoma have not been defined. In this study, we performed PRRX1 immunostaining on 35 human osteosarcoma specimens to assess the correlation between PRRX1 level and overall survival. In patients with osteosarcoma, the expression level of PRRX1 positively correlated with poor prognosis or the ratio of lung metastasis. Additionally, we found PRRX1 expression on in 143B cells, a human osteosarcoma line with a high metastatic capacity. Downregulation of PRRX1 not only suppressed proliferation and invasion but also increased the sensitivity to cisplatin and doxorubicin. When 143B cells were subcutaneously transplanted into nude mice, PRRX1 knockdown decreased tumor sizes and rates of lung metastasis. Interestingly, forskolin, a chemical compound identified by Connectivity Map analysis using RNA expression signatures during PRRX1 knockdown, decreased tumor proliferation and cell migration to the same degree as PRRX1 knockdown. These results demonstrate that PRRX1 promotes tumor malignancy in human osteosarcoma.     

FGF9/FGFR1 promotes cell proliferation,epithelial-mesenchymal transition,M2 macrophage infiltration and liver metastasis of lung cancer

Fibroblast growth factors 9 (FGF9) modulates cell proliferation, differentiation and motility for development and repair in normal cells. Abnormal activation of FGF9 signaling is associated with tumor progression in many cancers. Also, FGF9 may be an unfavorable prognostic indicator for non-small cell lung cancer patients. However, the effects and mechanisms of FGF9 in lung cancer remain elusive. In this study, we investigated the FGF9-induced effects and signal activation profiles in mouse Lewis lung carcinoma (LLC) in vitro and in vivo. Our results demonstrated that FGF9 significantly induced cell proliferation and epithelial-to-mesenchymal transition (EMT) phenomena (migration and invasion) in LLC cells. Mechanism-wise, FGF9 interacted with FGFR1 and activated FAK, AKT, and ERK/MAPK signal pathways, induced the expression of EMT key proteins (N-cadherin, vimentin, snail, MMP2, MMP3 and MMP13), and reduced the expression of E-cadherin. Moreover, in the allograft mouse model, intratumor injection of FGF9 to LLC-tumor bearing C57BL/6 mice enhanced LLC tumor growth which were the results of increased Ki67 expression and decreased cleaved caspase-3 expression compared to control groups. Furthermore, we have a novel finding that FGF9 promoted liver metastasis of subcutaneous inoculated LLC tumor with angiogenesis, EMT and M2-macrophage infiltration in the tumor microenvironment. In conclusion, FGF9 activated FAK, AKT, and ERK signaling through FGFR1 with induction of EMT to stimulate LLC tumorigenesis and hepatic metastasis. This novel FGF9/LLC allograft animal model may therefore be useful to study the mechanism of liver metastasis which is the worst prognostic factor for lung cancer patients with distant organ metastasis.     

GFRA1 promotes cisplatin-induced chemoresistance in osteosarcoma by inducing autophagy

Recent progress in chemotherapy has significantly increased its efficacy, yet the development of chemoresistance remains a major drawback. In this study, we show that GFRA1/GFRα1 (GDNF family receptor α 1), contributes to cisplatin-induced chemoresistance by regulating autophagy in osteosarcoma. We demonstrate that cisplatin treatment induced GFRA1 expression in human osteosarcoma cells. Induction of GFRA1 expression reduced cisplatin-induced apoptotic cell death and it significantly increased osteosarcoma cell survival via autophagy. GFRA1 regulates AMPK-dependent autophagy by promoting SRC phosphorylation independent of proto-oncogene RET kinase. Cisplatin-resistant osteosarcoma cells showed NFKB1/NFκB-mediated GFRA1 expression. GFRA1 expression promoted tumor formation and growth in mouse xenograft models and inhibition of autophagy in a GFRA1-expressing xenograft mouse model during cisplatin treatment effectively reduced tumor growth and increased survival. In cisplatin-treated patients, treatment period and metastatic status were associated with GFRA1-mediated autophagy. These findings suggest that GFRA1-mediated autophagy is a promising novel target for overcoming cisplatin resistance in osteosarcoma.     

Matriptase-2 inhibits HECV motility and tubule formation in vitro and tumour angiogenesis in vivo

Ezrin, primarily acts as a linker between the plasma membrane and the cytoskeleton, is involved in many cellular functions, including regulation of actin cytoskeleton, control of cell shape, adhesion, motility, and modulation of signaling pathways. Although ezrin is now recognized as a key component in tumor metastasis, its roles and the underlying mechanisms remain unclear. In the present study, we chose highly metastatic human lung carcinoma 95D cells, which highly express the ezrin proteins, as a model to examine the functional roles of ezrin in tumor suppression. An ezrin-silenced 95D cell line was established using lentivirus-mediated short hairpin RNA method. CCK-8 assay and soft agar assay analysis showed that downregulation of ezrin significantly suppressed the tumorigenicity and proliferation of 95D cells in vitro. cell migration and invasion studies showed that ezrin-specific deficiency in the cells caused the substantial reduction of the cell migration and invasion. In parallel, it also induced rearrangements of the actin cytoskeleton. Flow cytometry assay showed that changes in the ezrin protein level significantly affected the cell cycle distribution and eventual apoptosis. Furthermore, further studies showed that ezrin regulated the expression level of E-cadherin and CD44, which are key molecules involved in cell growth, migration, and invasion. Meanwhile, the suppression of ezrin expression also sensitized cells to antitumor drugs. Altogether, our results demonstrated that ezrin played an important role in the tumorigenicity and metastasis of lung cancer cells, which will benefit the development of therapeutic strategy for lung cancer.     

cAMP-response-element-binding protein positively regulates breast cancer metastasis and subsequent bone destruction

cAMP-response-element-binding protein (CREB) signaling has been reported to be associated with cancer development and poor clinical outcome in various types of cancer. However, it remains to be elucidated whether CREB is involved in breast cancer development and osteotropism. Here, we found that metastatic MDA-MB-231 breast cancer cells exhibited higher CREB expression than did non-metastatic MCF-7 cells and that CREB expression was further increased by several soluble factors linked to cancer progression, such as IL-1, IGF-1, and TGF-β. Using wild-type CREB and a dominant-negative form (K-CREB), we found that CREB signaling positively regulated the proliferation, migration, and invasion of MDA-MB-231 cells. In addition, K-CREB prevented MDA-MB-231 cell-induced osteolytic lesions in a mouse model of cancer metastasis. Furthermore, CREB signaling in cancer cells regulated the gene expression of PTHrP, MMPs, and OPG, which are closely involved in cancer metastasis and bone destruction. These results indicate that breast cancer cells acquire CREB overexpression during their development and that this CREB upregulation plays an important role in multiple steps of breast cancer bone metastasis.     

Targeting Insulin-Like Growth Factor 1 Receptor Inhibits Pancreatic Cancer Growth and Metastasis

Pancreatic cancer is one of the most lethal cancers. Increasing incidence and mortality indicates that there is still much lacking in detection and management of the disease. This is partly due to a lack of specific symptoms during early stages of the disease. Several growth factor receptors have been associated with pancreatic cancer. Here, we have investigated if an RNA interference approach targeted to IGF-IR could be effective and efficient against pancreatic cancer growth and metastasis. For that, we evaluated the effects of IGF-1R inhibition using small interfering RNA (siRNAs) on tumor growth and metastasis in HPAC and PANC-1 pancreatic cancer cell lines. We found that silencing IGF-1R inhibits pancreatic cancer growth and metastasis by blocking key signaling pathways such AKT/PI3K, MAPK, JAK/STAT and EMT. Silencing IGF-1R resulted in an anti-proliferative effect in PANC-1 and HPAC pancreatic cancer cell lines. Matrigel invasion, transwell migration and wound healing assays also revealed a role for IGF-1R in metastatic properties of pancreatic cancer. These results were further confirmed using Western blotting analysis of key intermediates involved in proliferation, epithelial mesenchymal transition, migration, and invasion. In addition, soft agar assays showed that silencing IGF-1R also blocks the colony forming capabilities of pancreatic cancer cells in vitro. Western blots, as well as, flow cytometric analysis revealed the induction of apoptosis in IGF-1R silenced cells. Interestingly, silencing IGF-1R also suppressed the expression of insulin receptor β. All these effects together significantly control pancreatic cancer cell growth and metastasis. To conclude, our results demonstrate the significance of IGF-1R in pancreatic cancer.     

Metastatic breast tumors downregulate miR-145 regulating the hypoxia-induced vasculogenic mimicry

Tumor cells grow in three-dimensional (3D) channels-like structures denoted as vasculogenic mimicry (VM), which provides a route for nutrients and oxygen acquisition. VM is activated by hypoxia and associated with metastasis and poor prognosis. MetastamiRs are microRNAs regulating metastasis, however, if they control VM in breast cancer remains poorly understood. The aim of this study was to evaluate the expression of VM-associated microRNAs in tumors of metastatic breast cancer patients. Firstly, we constructed microRNAs/mRNAs coregulation networks using expression data from TCGA databases. Dozens of microRNAs regulating genes involved in VM and metastasis were found. Of these, we selected 10 microRNAs for further characterization. The presence of VM in histological samples from patients with or without metastasis was evaluated using CD31-/PAS+ immunophenotyping. Remarkably, data showed that VM was significantly increased in tumors from patients with metastasis in comparison with no-metastatic group. Gene expression analysis indicated that miR-145, miR-142-3p, miR-31, miR-148a, miR-200b-3p and miR-526b were downregulated in primary tumors from patients with metastatic disease and positive for VM. Moreover, modulated microRNAs showed a predictive clinical value in overall survival in a cohort (n=1262) of breast cancer patients. Of these, we evaluated the role of miR-145 in formation of hypoxia-induced 3D channels-like using an in vitro model that recapitulates the early stages of VM. Data showed that miR-145 mimics was able to abolish the VM development in both metastatic Hs578t and MDA-MB-231 breast cancer cells. In conclusion, manipulation of miR-145 levels may represent a therapeutic approach in metastatic breast cancer patients that developed VM.     

Downregulation of AKT3 Increases Migration and Metastasis in Triple Negative Breast Cancer Cells by Upregulating S100A4

Background

Treatment of breast cancer patients with distant metastases represents one of the biggest challenges in today’s gynecological oncology. Therefore, a better understanding of mechanisms promoting the development of metastases is of paramount importance. The serine/threonine kinase AKT was shown to drive cancer progression and metastasis. However, there is emerging data that single AKT isoforms (i.e. AKT1, AKT2 and AKT3) have different or even opposing functions in the regulation of cancer cell migration in vitro, giving rise to the hypothesis that inhibition of distinct AKT isoforms might have undesirable effects on cancer dissemination in vivo.

Methods

The triple negative breast cancer cell line MDA-MB-231 was used to investigate the functional roles of AKT in migration and metastasis. AKT single and double knockdown cells were generated using isoform specific shRNAs. Migration was analyzed using live cell imaging, chemotaxis and transwell assays. The metastatic potential of AKT isoform knockdown cells was evaluated in a subcutaneous xenograft mouse model in vivo.

Results

Depletion of AKT3, but not AKT1 or AKT2, resulted in increased migration in vitro. This effect was even more prominent in AKT2,3 double knockdown cells. Furthermore, combined downregulation of AKT2 and AKT3, as well as AKT1 and AKT3 significantly increased metastasis formation in vivo. Screening for promigratory proteins revealed that downregulation of AKT3 increases the expression of S100A4 protein. In accordance, depletion of S100A4 by siRNA approach reverses the increased migration induced by knockdown of AKT3.

Conclusions

We demonstrated that knockdown of AKT3 can increase the metastatic potential of triple negative breast cancer cells. Therefore, our results provide a rationale for the development of AKT isoform specific inhibitors.     

Genomic Instability of Osteosarcoma Cell Lines in Culture: Impact on the Prediction of Metastasis Relevant Genes

BackgroundOsteosarcoma is a rare but highly malignant cancer of the bone. As a consequence, the number of established cell lines used for experimental in vitro and in vivo osteosarcoma research is limited and the value of these cell lines relies on their stability during culture. Here we investigated the stability in gene expression by microarray analysis and array genomic hybridization of three low metastatic cell lines and derivatives thereof with increased metastatic potential using cells of different passages.ConclusionsConsiderable instability during culture in terms of gene expression and chromosomal aberrations was observed in osteosarcoma cell lines. The use of cells from different passages and a search for genes consistently regulated in early and late passages allows the analysis of metastasis-relevant genes despite the observed instability in gene expression in osteosarcoma cell lines during culture.     

Proteomic study explores AGR2 as pro-metastatic protein in HCC

Hepatocellular carcinoma (HCC) is one of the most common and aggressive malignant tumors worldwide. The prognosis of patients with HCC still remains very dismal, mainly due to metastasis. We found that high-expression levels of AGR2 existed in metastatic HCC cell lines and patient samples. Overexpression of AGR2 was found to be correlated to the metastatic status of HCC cells, and inhibition of AGR2 by siRNA resulted in a dramatic decline in invasion abilities in metastatic cells in vitro. Overexpression of AGR2 increased the invasion of HCC cells in vitro and also in vivo with a nude mouse model. The tandem affinity purification (TAP) identified 18 AGR2-binding proteins and IPA analysis revealed that these proteins focus on MAPK and Caspase pathway. Therefore, we speculate that the overexpression of AGR2 can promote HCC metastasis, possibly by affecting MAPK and Caspase pathway through AGR2-interacting proteins.