Lixing Zhang

BMP4 Administration Induces Differentiation of CD133+ Hepatic Cancer Stem Cells, Blocking Their Contributions to Hepatocellular Carcinoma

CD133+ cancer stem cells (CSC) contribute to hepatocellular carcinoma (HCC) progression and resistance to therapy. Bone morphogenetic protein BMP4 plays an important role in hepatogenesis and hepatic stem cell differentiation, but little is known about its function in hepatic CSCs. In this study, we showed that high-dose exogenous BMP4 promotes CD133+ HCC CSC differentiation and inhibits the self-renewal, chemotherapeutic resistance, and tumorigenic capacity of these cells. Interestingly, we found that low-dose exogenous BMP4 upregulated CD133 protein expression in vitro, and endogenous BMP4 was preferentially expressed in CD133+ HCC CSCs, suggesting that low doses of BMP4 may facilitate CSC maintenance. A reduction in endogenous BMP4 levels decreased CD133 protein expression in vitro. In HCC tissues, expression of the BMP4 signaling target gene SMAD6 was positively correlated with CD133 expression. Activation of the Erk1/2 signaling pathway led to BMP4-mediated reduction in CD133 expression, which was reversed by treatment with MEK inhibitors. Taken together, our findings indicated that BMP4 might be a potent therapeutic agent in HCC that targets CSCs.

Isocorydine Targets the Drug-Resistant Cellular Side Population through PDCD4-Related Apoptosis in Hepatocellular Carcinoma

Isocorydine (ICD), an anticancer agent under current evaluation, decreased the percentage of side population (SP) cells significantly in hepatocellular carcinoma (HCC) cell lines. ICD treatment sensitized cancer cells to doxorubicin (DXR), a conventional clinical chemotherapeutic drug for HCC. We found that ICD decreased the percentage of SP cells in HCC cell lines by preferentially killing SP cells. In the early stage of treatment, ICD inhibited SP cell growth by arresting cells in G2/M; later, it induced apoptosis. Our xenograft model confirmed that ICD selectively reduced the size and weight of SP-induced tumor masses in vivo. Furthermore, it was found that programmed cell death 4 (PDCD4), a tumor suppressor gene, was relatively low when expressed in SP cells compared with non-SP cells, and its expression level was remarkably elevated when cells were treated with ICD. Taken together, these data suggest that ICD is a drug that may target the SP cells of HCC.

G Protein-Coupled Receptor 87 (GPR87) Promotes the Growth and Metastasis of CD133+ Cancer Stem-Like Cells in Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is a prevalent disease worldwide, and the majority of HCC-related deaths occur due to local invasion and distant metastasis. Cancer stem cells (CSCs) are a small subpopulation of cancer cells that have been hypothesized to be responsible for metastatic disease. Recently, we and others have identified a CSC population from human HCC cell lines and xenograft tumors characterized by their expression of CD133. However, the precise molecular mechanisms by which CD133+ cancer stem-like cells mediate HCC metastasis have not been sufficiently analyzed. Here, we have sorted HCC cells using CD133 as a cancer stem cell (CSC) marker by magnetic-activated cell sorting (MACS) and demonstrated that the CD133+ HCC cells not only possess greater migratory and invasive capacity in vitro but are also endowed with enhanced metastatic capacity in vivo and in human HCC specimens when compared to CD133− HCC cells. Gene expression analysis of the CD133+ and CD133− cells of the HCC line SMMC-7721 revealed that G protein-coupled receptor 87 (GPR87) is highly expressed in CD133+ HCC cells. In this study, we explored the role of GPR87 in the regulation of CD133 expression. We demonstrated that the overexpression of GPR87 up-regulated CD133 expression, promoted CSC-associated migratory and invasive properties in vitro, and increased tumor initiation in vivo. Conversely, silencing of GPR87 expression reduced the levels of CD133 expression. Conclusion: GPR87 promotes the growth and metastasis of CD133+ cancer stem-like cells, and our findings may reveal new targets for HCC prevention or therapy.

An isocorydine derivative (d-ICD) inhibits drug resistance by downregulating IGF2BP3 expression in hepatocellular carcinoma

In our previous studies, we reported that CD133+ cancer stem cells (CSCs) were chemoresistant in hepatocellular carcinoma (HCC) and that isocorydine treatment decreased the percentage of CD133+ CSCs. Here, we found that a derivative of isocorydine (d-ICD) inhibited HCC cell growth, particularly among the CD133+ subpopulation, and rendered HCC cells more sensitive to sorafenib treatment. d-ICD inhibited IGF2BP3 expression in a time-dependent manner, and IGF2BP3 expression negatively correlated with d-ICD-induced growth suppression. IGF2BP3 overexpression enriched the CD133+ CSC subpopulation in HCC, enhanced tumor sphere formation and suppressed the cytotoxic effects of sorafenib and doxorubicin. The expression of drug resistance-related genes, including ABCB1 and ABCG2, and the CSC marker CD133 expression was increased after IGF2BP3 overexpression. The significance of these observations was underscored by our findings that high IGF2BP3 expression predicted poor survival in a cohort of 236 patients with HCC and positively correlated with ABCG2 and CD133 expression in vivo. These results suggested that the d-ICD may inhibit HCC cells growth by IGF2BP3 decrease and that IGF2BP3 may serve as a therapeutic target for HCC.

NRBP2 overexpression increases the chemosensitivity of hepatocellular carcinoma cells via Akt signaling

Hepatocellular carcinoma is highly resistant to chemotherapy. Research data supported that cancer stem cells (CSC) may be responsible for the chemoresistance and strategies that suppress CSCs stemness could also inhibit the drug resistance. In this study, we found that nuclear receptor binding protein 2 (NRBP2) expression was downregulated in the CD133+ hepatocellular carcinoma CSCs. Most adjacent noncancerous liver tissue analyzed expressed higher level of NRBP2 compared with cancerous tissue in hepatocellular carcinoma patients, and high NRBP2 expression indicated a better prognosis. Real-time PCR results showed that NRBP2 negatively correlated with stemness-related genes, including Oct3/4, Nanog, Notch1, Ep300, and CD133 mRNA expression. High NRBP2 expression in hepatocellular carcinoma cells downregulated CK19 protein expression, inhibited tumorsphere formation, and tumorigenesis ability, indicating that high NRBP2 expression restrains the hepatocellular carcinoma cell stemness. Overexpression of NRBP2 reduced the IC50 of sorafenib in hepatocellular carcinoma cells, and NRBP2 expression was negatively correlated with hepatocellular carcinoma cell resistance to the chemotherapy agents, including cisplatin and the Akt signaling inhibitor perifosine. Coimmunoprecipitation results showed that NRBP2 could bind with Annexin A2 (ANXA2) and inhibit ANXA2 expression. Coexpression of ANXA2 restored the chemoresistant ability in NRBP2-overexpressing hepatocellular carcinoma cells. Further analysis showed that NRBP2 downregulated Akt and its downstream signaling target Bad phosphorylation level. ANXA2 coexpression partially restored the Akt phosphorylation. Analysis of the expression of Bcl2 family proteins showed that NRBP2 may increase hepatocellular carcinoma cell chemosensitivity by regulating expression of survival proteins involved in the Akt and Bcl2 pathway. These results suggest that NRBP2 plays an important role in the tumor progression and chemotherapeutic resistance of hepatocellular carcinoma. Cancer Res; 76(23); 7059-71. ©2016 AACR.

CXCL3 contributes to CD133(+) CSCs maintenance and forms a positive feedback regulation loop with CD133 in HCC via Erk1/2 phosphorylation.

Although the chemotactic cytokine CXCL3 is thought to play an important role in tumor initiation and invasion, little is known about its function in hepatocellular carcinoma (HCC). In our previous study, we found that Ikaros inhibited CD133 expression via the MAPK pathway in HCC. Here, we showed that Ikaros may indirectly down-regulate CXCL3 expression in HCC cells, which leads to better outcomes in patients with CD133+ cancer stem cell (CSC) populations. CD133 overexpression induced CXCL3 expression, and silencing of CD133 down-regulated CXCL3 in HCC cells. Knockdown of CXCL3 inhibited CD133+ HCC CSCs’ self-renewal and tumorigenesis. The serum CXCL3 level was higher in HCC patients’ samples than that in healthy individual. HCC patients with higher CXCL3 expression displayed a poor prognosis, and a high level of CXCL3 was significantly associated with vascular invasion and tumor capsule formation. Exogenous CXCL3 induced Erk1/2 and ETS1 phosphorylation and promoted CD133 expression, indicating a positive feedback loop between CXCL3 and CD133 gene expression in HCC cells via Erk1/2 activation. Together, our findings indicated that CXCL3 might be a potent therapeutic target for HCC.

Establishment of NOD/SCID mouse models of human hepatocellular carcinoma via subcutaneous transplantation of histologically intact tumor tissue

Hepatocellular carcinoma (HCC) is one of the most deadly human cancers, but it is very difficult to establish an animal model by using surgical specimens. In the present experiment, histologically intact fresh surgical specimens of HCC were subcutaneously transplanted in non-obese diabetic/severe combined immunodeficienccy (NOD/SCID) mice. The biological characteristics of the original and the corresponding transplanted tumors and cell lines were investigated. The results showed that 5 new animal models and 2 primary cell lines were successfully established from surgical specimens. Hematoxylin-eosin staining showed that xenografts retained major histological features of the original surgical specimens. The two new cell lines had been cultivated for 3 years and successively passaged for more than 100 passages in vitro. The morphological characteristics and biologic features of the two cell lines were genetically similar to the original tumor. The subcutaneous transplant animal models with histologically intact tumor tissue and primary cell lines could be useful for in vivo and in vitro testing of anti-cancer drugs and be ideal models to study various biologic features of HCC.

E3 Ubiquitin Ligase UBR5 Drives the Growth and Metastasis of Triple-Negative Breast Cancer

Patients with triple-negative breast cancers (TNBC) are at high risk for recurrence and metastasis at an early time despite standard treatment, underscoring the need for novel therapeutic modalities. Here, we report for the first time a distinctive and profound role of the E3 ubiquitin ligase UBR5 in the growth and metastasis of TNBC. An analysis of primary TNBC specimen by whole-exon sequencing revealed strong gene amplifications of UBR5 associated with the disease. UBR5 overexpression in TNBC tissues was confirmed at mRNA and protein levels. CRISPR/Cas9-mediated deletion of ubr5 in an experimental murine mammary carcinoma model of TNBC dramatically abrogated tumor growth and metastasis in vivo, which could be reversed completely via reconstitution with wild-type UBR5 but not a catalytically inactive mutant. Loss of UBR5 caused an impairment in angiogenesis within the tumor, associated with increased apoptosis, necrosis, and growth arrest. Absence of UBR5 in the tumor triggered aberrant epithelial-to-mesenchymal transition, principally via abrogated expression of E-cadherin, which resulted in severely reduced tumor metastasis to secondary organs. Use of NOD/SCID mice revealed that tumor-derived UBR5 facilitated tumor growth in a manner completely dependent upon immune cells in the microenvironment, whereas it promoted metastasis in a tumor cell-autonomous fashion. Our findings unveil UBR5 as a novel and critical regulator of tumor growth, metastasis, and immune response and highlight the potential for UBR5 as an effective therapeutic target for the treatment of highly aggressive breast and ovarian cancers that fail conventional therapy. Cancer Res; 77(8); 2090-101. ©2017 AACR.

Id4 promotes cell proliferation in hepatocellular carcinoma

BackgroundHepatocellular carcinoma (HCC) is a common malignant tumor in the world, especially in China. As a member of the inhibitor of differentiation (Id) family, Id4 has been reported to function in many cancer types, but relatively little is known about its role in HCC. The purpose of this study was to investigate the potential relationship between Id4 and HCC development and the underlying mechanism involving the function of Id4 in HCC.MethodsWe used quantitative real-time polymerase chain reaction and Western blotting to examine the RNA and protein expression of Id4. In addition, we used Cell Counting Kit-8 assay and colony formation assay to identify the function of Id4 in the regulation of cell proliferation in human HCC.ResultsWe found that the expression of Id4 protein was up-regulated in tumor tissues from HCC patients. Overexpression of Id4 promoted HCC cell proliferation, clonogenicity in vitro, and tumorigenicity in vivo. Id4 knockdown experiments showed that silencing Id4 blocked the proliferation and colony formation ability of HCC cells in vitro. Furthermore, overexpression of CCAAT/enhancer-binding protein β inhibited Id4 expression in HCC cells.ConclusionId4 may be developed as a potent therapeutic agent for the treatment of HCC, but more details about the underlying mechanisms of action are needed.