Chun Dai

Human mesenchymal stem cells inhibit metastasis of a hepatocellular carcinoma model using the MHCC97-H cell line

The effects of mesenchymal stem cells (MSC) on the growth and metastasis of human malignancies including hepatocellular carcinoma (HCC) are controversial, and the underlying mechanisms are not yet understood. The aim of this study was to explore the role of MSC in the progression of HCC. We investigated the effect of MSC on in vitro proliferation and invasion and in vivo tumor growth and pulmonary metastasis of MHCC97‐H HCC cells with a high metastatic potential. The mRNA and protein levels of transforming growth factor‐beta 1 (TGFβ1) and MMP, and their association with the effects of MSC on HCC cells were also evaluated. Co‐culture of MHCC97‐H cells with MSC conditioned medium significantly enhanced in vitro proliferation but inhibited invasiveness. Following MSC treatment of a nude mouse model bearing human HCC, the MSC were predominantly located in the HCC tissues. Compared with controls, MSC‐treated mice exhibited significantly larger tumors (3080.51 ± 1234.78 mm3vs 2223.75 ± 1000.60 mm3, P = 0.045), but decreased cellular numbers of lung metastases (49.75 ± 18.86 vs 227.22 ± 74.67, P = 0.046). Expression of TGFβ1 and MMP‐2 was significantly downregulated in the MSC‐treated HCC cells. TGFβ siRNA concurrently downregulated expression of TGFβ and MMP‐2 in HCC cells and blocked the MSC‐induced proliferation and invasiveness of MHCC97‐H cells. The MSC enhanced tumor growth but significantly inhibited the invasiveness and metastasis of HCC, possibly through downregulation of TGFβ1. These findings suggest that MSC could be useful in controlling metastatic recurrence of HCC. (Cancer Sci 2010; 101: 2546–2553)

Osteopontin promoter polymorphisms at locus -443 significantly affect the metastasis and prognosis of human hepatocellular carcinoma.

Osteopontin (OPN) plays a crucial role in hepatocellular carcinoma (HCC) metastasis. However, little is known about the impact of OPN polymorphisms on cancer progression. In this study, we first identified the single nucleotide polymorphisms (SNPs) in the OPN promoter region by direct sequencing in 30 HCCs, and then evaluated the prognostic values of the selected ones in two large cohorts of 826 HCC patients. The identified SNPs were functionally analyzed using in vitro and in vivo assays and their correlations with OPN levels were also evaluated. Only SNP at locus ‐443 and their related haplotypes (Ht2: ‐1748A/‐616G/‐443T/‐155* [*indicates base deletion]; Ht3: ‐1748A/‐616G/‐443C/‐155*) were significantly associated with overall survival (OS) and time to recurrence (TTR). The patients with the ‐443TT/TC genotype or Ht2 had a shorter OS and TTR compared with those with ‐443CC genotype or Ht3. This was further confirmed in the validation cohort. Moreover, this correlation remained significant in patients with small HCCs (≤5 cm). Multivariate analyses indicated that the prognostic performance of the ‐443 genotypes (OS, P = 0.031; TTR, P = 0.005) and their related haplotypes (OS, P = 0.002; TTR, P = 0.001) was independent of other clinicopathological factors. The Ht2 and ‐443TT genotype could significantly increase the promoter transcriptional activity and expression level of OPN compared with the Ht3 or ‐443CC genotype, and lead to an obvious increase in both in vitro invasion and in vivo tumor growth and lung metastasis of HCC cells (P < 0.05). Conclusion: The genetic variation at locus ‐443 of the OPN promoter plays important roles in the regulation of OPN expression and cancer progression of HCCs, which is a novel determinant and target for HCC metastasis and prognosis. (HEPATOLOGY 2013)

Thrombin is a therapeutic target for metastatic osteopontin‐positive hepatocellular carcinoma

We previously identified osteopontin (OPN) as a promoter and thus a potential therapeutic target for hepatocellular carcinoma (HCC) metastasis. The serine protease thrombin interacts with OPN and can modify its biological activity. To explore the role of thrombin alone or in conjunction with OPN in HCC, we studied the correlation of thrombin levels to HCC prognosis in patients with various OPN levels, and evaluated the effects of OPN fragments generated by thrombin cleavage on proliferation and adhesion of HCC cells. We found that the thrombin level was strongly associated with the metastatic potential of HCC cell lines, and that thrombin was remarkably overexpressed in HCC tissue compared with adjacent nontumor tissue. In addition, HCC tissue from patients with recurrent disease displayed much higher thrombin levels, particularly in those with elevated OPN levels. Only HCCs with elevated OPN levels had a significant correlation between high thrombin levels and overall survival (OS; P < 0.01), or time to recurrence (TTR; P < 0.0001) of HCC. Multivariate analysis revealed that thrombin was an independent prognostic indicator. In vitro assays demonstrated that thrombin promotes the proliferation and adhesion of OPN+ HCC cells. Furthermore, thrombin activated the focal adhesion kinase (FAK) pathway of OPN+ HCC cells, which was blocked by the inhibition of integrin β1. Conclusion: Thrombin plays an important role in OPN‐mediated aggressive phenotype of HCC through activation of integrin β1‐FAK signaling, and is an independent poor prognostic factor for HCC. Thus, thrombin may be a potential therapeutic target to inhibit HCC metastasis in OPN+ patients (HEPATOLOGY 2010.)

Osteopontin promotes epithelial-mesenchymal transition of hepatocellular carcinoma through regulating vimentin.

Our previous studies have found that osteopontin (OPN) is a promoter for hepatocellular carcinoma (HCC) progression. However, the molecular mechanism by which OPN enhances HCC metastasis remains elusive. Epithelial-mesenchymal transition (EMT) of cancer cells plays a pivotal role in promoting metastatic process. In this study, we demonstrated that OPN promotes HCC metastasis by inducing an EMT-like, more aggressive cellular phenotype in vitro and in vivo. Furthermore, OPN was identified to interact with vimentin by reciprocal OPN and vimentin immunoprecipitation as well as co-immunofluorescence examination. By using deletion mutants, we found that the residues between 246 and 406 in vimentin are required for binding to OPN. Importantly, OPN significantly increased vimentin stability through inhibition of its protein degradation. Knockdown of vimentin neutralized the EMT induced by OPN both in vitro and in vivo. Moreover, a significant correlation between OPN and vimentin levels was found in clinical HCC specimens and their combination had a worse prognosis with shorter overall survival (OS) and time to recurrence (TTR). In multivariate analysis, OPN and their combination were demonstrated to be independent prognostic indicators for OS and TTR of HCC patients. Collectively, this study indicates that OPN can induce EMT of HCC cells through increasing vimentin stability, which provides more in-depth understanding about the molecular mechanisms of OPN in promoting HCC metastasis and opens tantalizing therapeutic possibilities in HCC.

Association of Specific Genotypes in Metastatic Suppressor HTPAP with Tumor Metastasis and Clinical Prognosis in Hepatocellular Carcinoma

The phosphatidic acid phosphatase HTPAP has been defined as a metastatic suppressor of hepatocellular carcinoma (HCC), but little is known about its function or potential applications as a prognostic marker. In this study, we analyzed patterns of HTPAP genetic variation and gene expression in 864 patients who underwent HCC resection, assessing these patterns for correlations to tumor metastasis potential. Focusing on two tagSNPs that were selected (+357G/C and +1838A/G), we found that only the +357G/C genotype was significantly associated with HTPAP mRNA and protein expression levels and the probability of metastasis. In an independent cohort of 665 HCC patients, we determined that the +357G/C genotype was associated with shorter time to recurrence and overall survival. Together, these results indicated that the HTPAP tagSNP +357 GG+GC genotypes may influence HCC metastatic potential and clinical prognosis by down-regulating HTPAP expression. Extending these results, a global expression profiling analysis identified 41 genes including the pro-inflammatory genes IL-8 and TLR2 that were significantly overexpressed in the +357 GG+GC group, as possible coregulated markers with HTPAP. Together, our findings identify an HTPAP genotype and associated gene expression pattern that favors metastasis progression and that could be used to predict tumor metastasis and prognosis in HCC patients.

Anti-cancer effects of p21WAF1/CIP1 transcriptional activation induced by dsRNAs in human hepatocellular carcinoma cell lines

Aim:To investigate the anti-cancer effects of p21WAF1/CIP1 transcriptional activation induced by dsRNAs in hepatocellular carcinoma (HCC) cell lines.Methods:HCC cell lines BEL7402, SMMC-7721, MHCC97L, MHCC97H, and MHCCLM3 were used. HCC cells were treated with dsP21-322 (50 nmol/L), dsControl (50 nmol/L), siP21 (50 nmol/L), or mock transfection. The expression of p21 was detected using quantitative PCR and Western blot. The effects of RNA activation on HCC cells were determined using cell viability assays, apoptosis analyses and clonogenic survival assays. Western blot was also conducted to detect the expression of Bcl-xL, survivin, cleaved caspase-3, cleaved caspase-9 and cleaved PARP.Results:At 72 to 120 h following the transfection, dsP21-322 markedly inhibited the viability of HCC cells and clone formation. At the same times, dsP21-322 caused a significant increase in HCC cell apoptosis, as demonstrated with cytometric analysis. The phenomena were correlated with decreased expression levels of the anti-apoptotic proteins Bcl-xL, surviving, and increased expression of cleaved caspase-3, cleaved caspase-9 and cleaved PARP.Conclusion:RNA-induced activation of p21 gene expression may have significant therapeutic potential for the treatment of hepatocellular carcinoma and other cancers.

Downregulation of HTPAP transcript variant 1 correlates with tumor metastasis and poor survival in patients with hepatocellular carcinoma.

Our previous study has identified HTPAP as a novel metastasis suppressor from chromosome 8p which is often deleted in metastatic HCC. We sought to further evaluate the expression levels of transcript variants of HTPAP (HTPAP‐1, HTPAP‐2 and HTPAP‐3) in 67 HCC tumor tissues and 11 normal liver tissues by RT‐PCR with specific TaqMan probes and primer sets, and explore their association with HCC metastasis and survival. We found that the expression levels of three HTPAP transcript variants were quite different in HCCs. Only HTPAP‐1 was found to be significantly associated with HCC metastasis (P = 0.00053), overall survival (P = 0.0023) and time to recurrence (P = 0.010) of HCC. Patients with a lower expression of HTPAP‐1 were inclined to accompany intrahepatic metastases and tumor thrombi (P < 0.05) and had a poor prognosis. In vitro, three fusion pEGFP‐N1 vectors encoding HTPAP‐1, HTPAP‐2 and HTPAP‐3 were introduced into HCC cells respectively to track HTPAPs’ expressions and identify their function. We found overexpression of HTPAP‐1 conferred HCC cells reduced ability of invasion without significant impact on cell proliferation, and also displayed a distinct cell location on cell membrane and in cytoplasm, which were different from two other variants. Consequently, HTPAP‐1 may be the transcript of HTPAP to exhibit a suppressive role on HCC metastasis, and can be a prognostic marker for HCC. (Cancer Sci 2011; 102: 583–590)

Abstract 2695: Osteopontin regulated epithelial-mesenchymal transition via PI3K/AKT signaling pathway in hepatocellular carcinoma.

Our previous studies demonstrated that osteopontin (OPN) plays a crucial role in hepatocellular carcinoma (HCC) metastasis. However, its function in epithelial-mesenchymal transition (EMT) remains obscure. In this study, we reported the critical role of OPN in EMT mediated HCC metastasis. Increased expression of OPN in human HCC predicts poor survival and disease recurrence after surgery. HCC tumors overexpressing OPN were featured by active EMT and motility/invasion of tumor cells both in vitro and in vivo, whereas silencing OPN expression attenuated EMT. OPN overexpression enhanced twist through activation of Akt, a critical EMT regulation of transcription factor. Suppression of the PI3K-AKT-Twist pathway interfered with OPN-mediated EMT and invasion. Consistently, we have observed a significant correlation between OPN expression and twist levels in a cohort of HCC patients, and found that combination of these two parameters is a more powerful predictor of poor prognosis. Conclusions: OPN plays a pivotal role in EMT mediated HCC metastasis and may serve as a novel prognostic biomarker and therapeutic target. Citation Format: Qiongzhu Dong, Xu-Chao Zhu, Chun Dai, Xiao-Fei Zhang, Hu-Liang Jia, Qing-Hai Ye, Lun-Xiu Qin. Osteopontin regulated epithelial-mesenchymal transition via PI3K/AKT signaling pathway in hepatocellular carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2695. doi:10.1158/1538-7445.AM2013-2695

Abstract 1995: The Functional analyses of Oct4 in metastasis of human hepatocellular carcinoma

There is an increasing evidence that in a malignant bulk tumor, a small proportion of ‘cancer stem cells’ exist, leading to tumorigenesis,metastasis or recurrence by their plasticity. However, the functional status and significance of stem cell factors in hepatocellular carcinogenesis is unknown. In this study, we analyzed the expression of 3 stem cell factors: Oct4, Sox2 and Nanog in HCC patients, and found that Oct4 and Sox2, 3 stem cell factors, were expressed in HCCs. Expression of Oct4 correlated with HCC recurrence and metastasis. Through gain- and loss-of-function studies, Oct4 was demonstrated to significantly promote in vitro cell proliferation, invasion, transformation. In addition, Oct4 reduced chemosensitivity of HCC cells. Importantly, Oct4 enhanced in vivo tumor growth and metastasis in nude mice models bearing human HCC. Moreover, Oct4 dramatically stimulated expression of HCC stem cell markers, including Epcam,ELF, KLF4,CD44 and CD133 as well as stem cell signaling pathway (IL-6/surviving, TGF-beta and Wnt / beta-catenin passway). Conclusion: Oct4 plays a critical role in HCC development and progression and its deregulated signaling could be a promising new molecular target for designing novel preventive and therapeutic strategies to control this malignancy Note: This abstract was not presented at the meeting. Citation Format: Qiongzhu Dong, Chun Dai, Huliang Jia, Lunxiu Qin. The Functional analyses of Oct4 in metastasis of human hepatocellular carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1995. doi:10.1158/1538-7445.AM2014-1995