Promotes Hepatocellular Carcinoma Growth Research Articles

MicroRNA-455 regulates migration and invasion of human hepatocellular carcinoma by targeting Runx2.

MicroRNA-455(miR-455) has been considered as a novel cancer-related miRNA and dysregulated expression frequently occurs in various human types of cancer. However, its clinical significance, its biological function and the underlying molecular signaling involved in hepatocellular carcinoma(HCC) remain to be elucidated. In the present study, we found that the expression level of miR-455 was significantly downregulated in both HCC tissues and cell lines. Low expression of miR-455 was significantly associated with poor prognostic features including multiple tumor nodes, high Edmondson‑Steiner grading, advanced tumor-node‑metastasis(TNM) stage and venous infiltration. In addition, our data revealed that miR-455 was a novel prognostic indicator for predicting the 5-year overall and disease-free survival of HCC patients. The gain- and loss-of-function studies revealed that miR-455 significantly suppressed migration and invasion of HCC cells invitro. miR-455 was inversely correlated with runt-related transcription factor2(Runx2) expression in HCC samples. Moreover, we identified that miR-455 inversely regulated Runx2 expression in HCC cells. In this investigation, Runx2 was found to be a direct downstream target of miR-455. Evidently, alteration in Runx2 expression suppressed the effect of miR-455 on HCC cell migration and invasion. In conclusion, our data demonstrated that miR-455 promotes HCC growth by targeting Runx2 and can potentially be regarded as a novel prognostic indicator and valuable therapeutic strategy for HCC.

GOLM1 Modulates EGFR/RTK Cell-Surface Recycling to Drive Hepatocellular Carcinoma Metastasis

The mechanism of cancer metastasis remains poorly understood. Using gene profiling of hepatocellular carcinoma (HCC) tissues, we have identified GOLM1 as a leading gene relating to HCC metastasis. GOLM1 expression is correlated with early recurrence, metastasis, and poor survival of HCC patients. Both gain- and loss-of-function studies determine that GOLM1 acts as a key oncogene by promoting HCC growth and metastasis. It selectively interacts with epidermal growth factor receptor (EGFR) and serves as a specific cargo adaptor to assist EGFR/RTK anchoring on the trans-Golgi network (TGN) and recycling back to the plasma membrane, leading to prolonged activation of the downstream kinases. These findings reveal the functional role of GOLM1, a Golgi-related protein, in EGFR/RTK recycling and metastatic progression of HCC.

Proteinase-activated receptor 2 (PAR2) in hepatic stellate cells – evidence for a role in hepatocellular carcinoma growth in vivo

BackgroundPrevious studies have established that proteinase-activated receptor 2 (PAR2) promotes migration and invasion of hepatocellular carcinoma (HCC) cells, suggesting a role in HCC progression. Here, we assessed the impact of PAR2 in HCC stromal cells on HCC growth using LX-2 hepatic stellate cells (HSCs) and Hep3B cells as model.MethodsPAR2 expression and function in LX-2 cells was analysed by RT-PCR, confocal immunofluorescence, electron microscopy, and [Ca2+]i measurements, respectively. The impact of LX-2-expressed PAR2 on tumour growth in vivo was monitored using HCC xenotransplantation experiments in SCID mice, in which HCC-like tumours were induced by coinjection of LX-2 cells and Hep3B cells. To characterise the effects of PAR2 activation in LX-2 cells, various signalling pathways were analysed by immunoblotting and proteome profiler arrays.ResultsFollowing verification of functional PAR2 expression in LX-2 cells, in vivo studies showed that these cells promoted tumour growth and angiogenesis of HCC xenografts in mice. These effects were significantly reduced when F2RL1 (encoding PAR2) was downregulated by RNA interference (RNAi). In vitro studies confirmed these results demonstrating RNAi mediated inhibition of PAR2 attenuated Smad2/3 activation in response to TGF-β1 stimulation in LX-2 cells and blocked the pro-mitotic effect of LX-2 derived conditioned medium on Hep3B cells. Furthermore, PAR2 stimulation with trypsin or a PAR2-selective activating peptide (PAR2-AP) led to activation of different intracellular signalling pathways, an increased secretion of pro-angiogenic and pro-mitotic factors and proteinases, and an enhanced migration rate across a collagen-coated membrane barrier. Silencing F2RL1 by RNAi or pharmacological inhibition of Src, hepatocyte growth factor receptor (Met), platelet-derived growth factor receptor (PDGFR), p42/p44 mitogen activated protein kinase (MAPK) or matrix-metalloproteinases (MMPs) blocked PAR2-AP-induced migration.ConclusionPAR2 in HSCs plays a crucial role in promoting HCC growth presumably by mediating migration and secretion of pro-angiogenic and pro-mitotic factors. Therefore, PAR2 in stromal HSCs may have relevance as a therapeutic target of HCC.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-016-0538-y) contains supplementary material, which is available to authorized users.

MicroRNA-23a downregulates the expression of interferon regulatory factor-1 in hepatocellular carcinoma cells.

Interferon regulatory factor-1 (IRF-1) is a tumor-suppressor gene induced by interferon-γ (IFNγ) and plays an important role in the cell death of hepatocellular carcinoma (HCC). HCC tumors evade death in part by downregulating IRF-1 expression, yet the molecular mechanisms accounting for IRF-1 suppression in HCC have not yet been characterized. Previous studies have shown that microRNA-23a (miR-23a) can suppress apoptosis by targeting IRF-1. Therefore, we hypothesized that miR-23a promotes HCC growth by down-regulating IRF-1. For the in vivo studies, 7 cases of resected HCC and adjacent liver samples were analyzed. For the in vitro studies, IRF-1 mRNA and protein were examined in HepG2 and Huh-7 HCC cells after IFNγ stimulation by real-time PCR and western blotting, respectively. To determine the role of miR-23a in regulating IRF-1, HepG2 cells were transfected with an miR-23a mimic or inhibitor, and IRF-1 expression was examined. Binding of miR-23a was assessed by cloning the 528-bp human IRF-1 3′-untranslated region (3′UTR) into luciferase reporter plasmid pMIR-IRF-1-3′UTR. The results showed that IRF-1 mRNA expression was down-regulated in the human HCC tumor tissues compared to that in the adjacent background liver tissues. IFNγ-induced IRF-1 protein was less in the HepG2 tumor cells compared to that in the primary human hepatocytes. miR-23a expression was inversely correlated with IRF-1, and addition of the miR-23a inhibitor increased basal IRF-1 mRNA and protein. Likewise, the miR-23a mimic downregulated IFNγ-induced IRF-1 protein expression, while the miR-23a inhibitor increased IRF-1. Furthermore, the miR-23a mimic repressed IRF-1-3′UTR reporter activity, while the miR-23a inhibitor increased the reporter activity. These results demonstrated that IRF-1 expression is downregulated in human HCC tumors compared to that noted in the background liver. miR-23a downregulates the expression of IRF-1 in HCC cells, and the IRF-1 3′UTR has an miR-23a binding site that binds miR-23a and decreases reporter activity. These findings suggest that the targeting of IRF-1 by miR-23a may be the molecular basis for IRF-1 downregulation in HCC and provide new insight into the regulation of HCC by miRNAs.

MiR-592/WSB1/HIF-1α axis inhibits glycolytic metabolism to decrease hepatocellular carcinoma growth.

Hepatocellular carcinoma (HCC) cells rapidly switch their energy source from oxidative phosphorylation to glycolytic metabolism in order to efficiently proliferate. However, the molecular mechanisms responsible for this switch remain unclear. In this study, we found that miR-592 was frequently downregulated in human HCC tissues and cell lines, and its downregulation was closely correlated with aggressive clinicopathological features and poor prognosis of HCC patients. Overexpression of miR-592 inhibited aerobic glycolysis and proliferation in HCC cells in vitro. Conversely, knockdown of miR-592 promoted HCC growth in both subcutaneous injection and orthotopic liver tumor implantation models in vivo. Mechanistically, miR-592 downregulation in human HCCs was correlated with an upregulation of WD repeat and SOCS box containing 1 (WSB1). We further showed that miR-592 directly binds to the 3′-UTR of the WSB1 gene, thus disrupting hypoxia inducible factor-1α (HIF-1α) protein stabilization. In turn, overexpression of WSB1 in HCC cells rescued decreased HIF-1α expression, glucose uptake, and HCC growth induced by miR-592. Collectively, our clinical data and functional studies suggest that miR-592 is a new robust inhibitor of the Warburg effect and a promising therapeutic target for HCC treatment.

Silencing PRDX3 Inhibits Growth and Promotes Invasion and Extracellular Matrix Degradation in Hepatocellular Carcinoma Cells.

PRDX3 is a mitochondrial peroxide reductase that regulates cellular redox state. It has been reported that PRDX3 is overexpressed in liver cancer, but how PRDX3 is involved in hepatocellular carcinoma (HCC) tumorigenesis and progression has not been well-characterized. In the present study, we established two stable cell lines by overexpressing or knocking down PRDX3 in HepG2 cells. We found that PRDX3 silencing decreased the growth rate of HepG2 cells and increased mtDNA oxidation. Quantitative proteomics identified 475 differentially expressed proteins between the PRDX3 knockdown and the control cells. These proteins were involved in antioxidant activity, angiogenesis, cell adhesion, cell growth, ATP synthesis, nucleic acid binding, redox, and chaperones. PRDX3 knockdown led to the down-regulation of ATP synthases and the decreased cellular ATP level, contributing to the slow-down of cell growth. Furthermore, silencing PRDX3 enhanced invasive properties of HepG2 cells via TIMP-1 down-regulation and the increased ECM degradation. Taken together, our results indicate that PRDX3 promotes HCC growth and mediates cell migration and invasiveness and is a potential therapeutic target for HCC treatment.

Histidine-rich calcium binding protein promotes growth of hepatocellular carcinoma in vitro and in vivo.

We have recently shown that the histidine-rich calcium binding protein (HRC) promotes the invasion and metastasis of hepatocellular carcinoma (HCC). In the current study, we evaluated whether HRC may also affect the growth of HCC. We found that ectopic expression of HRC obviously enhanced proliferation and colony formation, while suppression of HRC exhibited inhibitory effects. Furthermore, we demonstrated that HRC promoted tumor growth in nude mice. These effects may result from the ability of HRC to upregulate cyclinD1 and cyclin-dependent kinase 2 (CDK2) expressions and promote G1/S transition. Further study showed that MEK/ERK signaling pathway was involved in HRC-induced cell proliferation. Interestingly, overexpression or depletion of HRC revealed its regulation on endoplasmic reticulum stress (ERS) and apoptosis, which was partially dependent on PERK/ATF4/CHOP signaling pathway. In addition, blocking ERS using 4-phenylbutyric acid (4-PBA) not only downregulated the expression of PERK, ATF4 and CHOP, but also significantly decreased apoptosis induced by HRC silence, whereas ERS inducer thapsigargin (TG) exerted the opposite effects. Our study thus demonstrates a role of HRC in promoting HCC growth, besides its role in inducing HCC metastasis, and highlights HRC as a promising intervention target for HCC.

Abstract 2053: Overexpressed Rab1A is associated poor prognosis and promotes oncogenic growth and metastasis through mTORC1 activation in hepatocellular carcinoma

Abstract Background: The mammalian target of rapamycin complex 1 (mTORC1) is a pivotal regulator of cell growth and proliferation in response to nutrition and various factor. In recent years, studies have focus on over-activation of mTORC1 in normal cells leading to tumorigenesis and malignant tumor development. However, the precise factors of mTORC1 hyperactivation to promote the growth of hepatocellular carcinoma (HCC) or other cancers are still not well characterized. Thus, in this study, we aimed to clarify a novel gene -Rab1A, and it's expression and functional role in mTORC1 signal in of HCC. Methods: We analyzed the Rab1A protein in HCC and para-cancer samples from 143 patients and the correlation with its survival. To identify Rab1A's role in tumor progression, a serious of functional study were applied including overexpression and knockdown Rab1A in different cell lines both in vivo and in vitro. For mechanism research, western bolt was demonstrated to identify the signal dysregulation between Rab1A abnormity and mTORC1 pathway under involvement of irritants. Results: Our results showed that, compared with normal livers, Rab1A was frequently overexpressed in HCC and significantly associated with HCC prognosis. Functional study demonstrated that overexpression of Rab1A in HCC cells increased cell growth, cell migration, cell cycle progression and tumor formation in vivo and/or in vitro, all of which were effectively inhibited when Rab1A was silenced by shRNA. We further provided evidences that Rab1A promotes HCC growth and migration through phosphorylation of S6K, the consequence of activating mTORC1 signaling pathway. Mechanism research of the signal pathway between Rab1A and mTORC1 suggest that Rab1A is likely to stimulate mTORC1 directly, with the cooperation of Amino acid stimulation but not depend on the pathway of PI3K/AKT and MAPK/ERK.. Citation Format: Bi-Hong Xu, Xiao-Xing Li, Hui-yun Wang, X.F. Steven Zheng. Overexpressed Rab1A is associated poor prognosis and promotes oncogenic growth and metastasis through mTORC1 activation in hepatocellular carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2053. doi:10.1158/1538-7445.AM2015-2053

PTPN11/Shp2 overexpression enhances liver cancer progression and predicts poor prognosis of patients

We have previously reported that Shp2, a tyrosine phosphatase previously known as a pro-leukemogenic molecule, suppresses the initiation of hepatocellular carcinoma (HCC). However, the role of Shp2 in HCC progression remains obscure. Shp2 expression was determined in human HCC using real-time PCR, immunoblotting and immunohistochemistry. Clinical significance of Shp2 expression was analyzed in 301 HCC tissues with clinico-pathological characteristics and follow-up information. Short hairpin RNA was utilized to investigate the function of Shp2 in hepatoma cell behavior. Role of Shp2 in HCC progression was monitored through nude mice xenograft assay. Kinase activity assay and co-immunoprecipitation were used for mechanism analysis. Elevated expression of Shp2 was detected in 65.9% (394/598) of human HCCs, and its levels were even higher in metastasized foci. Overexpression of Shp2 correlated well with the malignant clinico-pathological characteristics of HCC and predicted the poor prognosis of patients. Interference of Shp2 expression suppressed the proliferation of hepatoma cells in vitro and inhibited the growth of HCC xenografts in vivo. Down-regulation of Shp2 attenuated the adhesion and migration of hepatoma cells and diminished metastasized HCC formation in mice. Our data demonstrated that Shp2 promotes HCC growth and metastasis by coordinately activating Ras/Raf/Erk pathway and PI3-K/Akt/mTOR cascade. Moreover, down-regulation of Shp2 enhanced the sensitivity of hepatoma cells upon sorafenib treatment, and patients with low Shp2 expression exhibited superior prognosis to sorafenib. Shp2 promotes the progression of HCC and may serve as a prognostic biomarker for patients.

Hepatic stellate cells promote tumor progression by enhancement of immunosuppressive cells in an orthotopic liver tumor mouse model

The immunosuppressive properties of hepatic stellate cells (HSCs) contribute to the occurrence and development of hepatocellular carcinoma (HCC). The accumulation of cells with immune suppressive activities, such as myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs) is a key mechanism for tumor immune evasion. However, the impact of HSCs on immune cell populations in tumor-bearing hosts is unclear. In this study, we established an orthotopic liver tumor mouse model for studying the complex tumor–host interactions in HCC. The activated HSCs promoted HCC growth not only induced tumor angiogenesis and lymphangiogenesis, but also significantly increased the suppressive immune cell population of Tregs and MDSCs in the spleen, bone marrow, and tumor tissues of the tumor-bearing mice. Murine HCC cell line H22-activated HSCs also expanded the expression of Tregs and MDSCs in vitro. In conclusion, our study suggests a novel role for HSCs in the HCC microenvironment. HSCs can promote HCC progression by enhancement of the immunosuppressive cell population. Targeting HSCs, which is a new concept in adjuvant immunotherapy, may be introduced in the near future to improve the outcome of patients with HCC.

1044 LYSOPHOSPHATIDIC ACID RECEPTOR 6 (LPA6) PROMOTES HEPATOCELLULAR CARCINOMA GROWTH AND PROGRESSION THROUGH ACTIVATION OF PIM-3 PROTO-ONCOGENE KINASE

1044 LYSOPHOSPHATIDIC ACID RECEPTOR 6 (LPA6) PROMOTES HEPATOCELLULAR CARCINOMA GROWTH AND PROGRESSION THROUGH ACTIVATION OF PIM-3 PROTO-ONCOGENE KINASE A. Mazzocca, F. Dituri, F. De Santis, L. Lupo, A. Azzariti, C. Sabba, G. Giannelli. Internal Medicine Unit C. ‘Frugoni’ University of Bari A. Moro, Section of General Surgery and Organ Transplantation, University of Bari Medical School, Department of Experimental Oncology, National Cancer Institute, Bari, Italy E-mail: gianluigi.giannelli@uniba.it

Receptor for activated C kinase 1 promotes hepatocellular carcinoma growth by enhancing mitogen-activated protein kinase kinase 7 activity

c-Jun N-terminal protein kinase (JNK) is a member of the mitogen-activated protein kinase (MAPK) superfamily. The activation of JNK is mediated by sequential protein phosphorylation through a MAPK module, namely, MAPK kinase kinase (MAP3K or MEKK) → MAPK kinase (MAP2K or MKK) → MAPK. Elevated levels of JNK activity have been frequently observed in hepatocellular carcinoma (HCC) and have been demonstrated to contribute to HCC growth by promoting HCC cell proliferation and resistance to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)- or Fas-mediated apoptosis. Chronic inflammation contributes to the up-regulation of JNK activity in HCC. However, it remains unknown whether aberrant JNK activity also results from some cell intrinsic defect(s). Here, we show that receptor for activated C kinase 1 (RACK1), an adaptor protein implicated in the regulation of multiple signaling pathways, could engage in a direct interaction with MKK7, the JNK-specific MAP2K, in human HCC cells. Levels of RACK1 protein show correlation with the activity of the JNK pathway in human HCC tissues and cell lines. RACK1 loss-of-function or gain-of-function analyses indicate that RACK1 enhances MKK7/JNK activity in human HCC cells. Further exploration reveals that the interaction of RACK1 with MKK7 is required for the enhancement of MKK7/JNK activity by RACK1. RACK1/MKK7 interaction facilitates the association of MKK7 with MAP3Ks, thereby enhancing MKK7 activity and promoting in vitro HCC cell proliferation and resistance to TRAIL- or Fas-mediated apoptosis as well as in vivo tumor growth. Overexpressed RACK1 augments JNK activity and thereby promotes HCC growth through directly binding to MKK7 and enhancing MKK7 activity.

GSI-I has a better effect in inhibiting hepatocellular carcinoma cell growth than GSI-IX, GSI-X, or GSI-XXI

Current studies are ongoing to find new drugs for the treatment of hepatocellular carcinoma (HCC). The discovery of drugs depends on the identification of molecules that can play essential roles in the development of liver cancer, for example, Notch pathway molecules. γ-Secretase inhibitors (GSIs) can inhibit the cleavage of intramembranous substrates of all Notch receptors and subsequently suppress Notch signaling. However, whether the inhibition of the Notch pathway can suppress or promote HCC growth is still under debate. In this study, we examined the expression of Notch pathway molecules in 20 pairs of HCC tissue with their normal counterparts and a panel of eight HCC cell lines. We also determined the effects of different types of GSI treatments on the cell growth of those HCC cell lines. Our results showed that the molecules of the Notch pathway were expressed in six of the eight HCC cell lines. Those six HCC cell lines were more sensitive to GSI-I treatment than the nonexpression ones. Among the four inhibitors, GSI-X and GSI-XXI exerted no effect on HCC cells growth at all. GSI-IX inhibited the growth of four HCC cell lines at 40 μmol/l. In contrast, most of these HCC cell lines were susceptible to a low concentration of GSI-I (1.2 μmol/l) treatment. The suppressive effect of GSI-I on cell growth was because of the inhibition of C-Myc, a Notch target gene. In addition, 80% (16/20) of the specimens showed either an increased expression of at least one Notch receptor or an augmented expression of Jagged1 compared with their normal counterparts. Our study reports for the first time that different kinds of GSIs can block the growth of several HCC cell lines. Our finding suggests that GSI-I is a potential chemical reagent and warrants additional testing in liver cancer therapeutics.

Serum and glucocorticoid kinase 3 at 8q13.1 promotes cell proliferation and survival in hepatocellular carcinoma

Amplification of broad regions of 8q is one of the most frequent genetic alterations in hepatocellular carcinoma (HCC), suggesting the existence of oncogenes in addition to MYC at 8q24.21. In this report we examine the potential role of the candidate amplified oncogene serum and glucocorticoid kinase 3 (SGK3) at 8q13.1 in HCC pathogenesis. We found amplification and overexpression of SGK3 was frequently detected in clinical HCC specimens and that SGK3 genomic activation was significantly associated with poor outcome of patients (P = 0.028). Functionally, we found that overexpression of SGK3 in HCC cells increased cell cycle progression through G(1), cell survival, clonogenicity, anchorage-independent growth, and tumor formation in nude mice. In contrast, RNA interference (RNAi) silencing of SGK3 inhibited its oncogenic effects. We provide evidence that SGK3 promotes HCC growth and survival through inactivating glycogen synthase kinase 3 beta and Bcl-2-associated death promoter, respectively. We also found that expression of SGK3, which like AKT is activated by PI3K/PDK1 signaling, has more significance than overexpression of AKT in predicting poor outcome in HCC patients. Taken together, our findings in the present study suggests that the SGK3 pathway may function in parallel with the AKT pathway and undergoes an AKT-independent signaling pathway in the pathogenesis of HCC. Further characterization of SGK3 may provide a prognostic biomarker for HCC outcome prediction and a novel therapeutic target in HCC treatment.

Activated hepatic stellate cells promote hepatocellular carcinoma development in immunocompetent mice

Activated hepatic stellate cells (HSCs) play a central role in the hepatic fibrosis and cirrhosis. Recently, HSCs were reported to have strong immune modulatory activities. However, the role of HSCs in hepatocellular carcinoma (HCC) remains unclear. In this study, we showed that HSCs could promote HCC growth both in vitro and in vivo. We examined the HSC-mediated inhibition of T-cell proliferation and the ability of conditioned medium from activated HSCs to promote the growth of murine HCC cell lines in vitro. We also assessed the immune suppression by HSCs during the development of HCC in immunocompetent mice. Cotransplantation of HSCs promoted HCC growth and progression by enhancing tumor angiogenesis and tumor cell proliferation and by creating an immunosuppressed microenvironment. Cotransplanted HSCs inhibited the lymphocyte infiltration in tumors and the spleens of mice bearing tumors, induced apoptosis of infiltrating mononuclear cells, and enhanced the expression of B7H1 and CD4(+) CD25(+) Treg cells. The immune modulation by HSCs seemed to be systemic. In conclusion, our data provide new information to support an integral role for HSCs in promoting HCC progression in part via their immune regulatory activities, and suggest that HSCs may serve as a therapeutic target in HCC.

Activated hepatic stellate cells promote tumorigenicity of hepatocellular carcinoma

Liver cirrhosis is the main risk factor for the development of hepatocellular carcinoma (HCC). Activated hepatic stellate cells (HSC) are the effector cells of hepatic fibrosis and also infiltrate the HCC stroma where they might play a critical role in HCC progression. Here we aimed to analyze the effects of activated HSC on the proliferation and growth of HCC cell lines in vitro and in vivo. Conditioned media (CM) collected from HSC significantly induced proliferation and migration of HCC cells cultured in monolayers. In a 3-dimensional spheroid coculture system, HSC promoted HCC growth and diminished the extent of central necrosis. In accordance, in vivo simultaneous implantation of HSC and HCC cells into nude mice promoted tumor growth and invasiveness, and inhibited necrosis formation. As potential mechanism of the tumorigenic effects of HSC we identified activation of NFkappaB and extracellular-regulated kinase (ERK) in HCC cells, two signaling cascades that play a crucial role in HCC progression. In summary, our data indicate that stromal HSC promotes HCC progression and suggest the HSC-HCC interaction as an interesting tumor differentiation-independent target for therapy of this highly aggressive cancer.