AK4 Promotes the Progression of HER2-Positive Breast Cancer by Facilitating Cell Proliferation and Invasion.

Breast cancer (BC) is a type of malignant tumor originating from the epithelial tissue of the mammary gland, and about 20% of breast cancers are human epidermal growth factor receptor 2 positive (HER2+), which is a subtype with more aggression. Recently, HER2-positive breast cancer is often accompanied by poor prognosis of patients, and targeted therapy showed a promising prospect. To combat this disease, novel therapeutic targets are still needed. Adenylate kinase 4 (AK4) is a member of the adenylate kinase family and is expressed in the mitochondrial matrix. AK4 is involved in multiple cellular functions such as energy metabolism homeostasis. Interestingly, AK4 was observed highly expressed in several tumor tissues, and the involvement of AK4 in cancer development was generally revealed. However, the possible role of AK4 on the growth and development of breast cancer is still unclear. Here, we investigated the possible functions of AK4 on the progression of HER2-positive breast cancer. We found the high expression of AK4 in HER2-positive breast cancer tissues from patients who received surgical treatment. Additionally, AK4 expression levels were obviously correlated with clinical-pathological features, including pTNM stage (P = 0.017) and lymph node metastasis (P = 0.046). We mechanically confirmed that AK4 depletion showed the obvious impairment of cell proliferation and invasion in MCF7 and MDA-MB-231 cells. AK4 also facilitates tumor growth and metastasis of HER2-positive breast cancer in vivo. In conclusion, we identified and mechanically confirmed that AK4 is a novel therapeutic target of HER2-positive breast cancer.

Hepatitis C virus core protein increases Snail expression and induces epithelial-mesenchymal transition through the signal transducer and activator of transcription 3 pathway in hepatoma cells.

AIM: Aberrant expression of Snail, a mediator of epithelial-mesenchymal transition (EMT), is crucial for cancer invasiveness and metastasis. Although hepatitis C virus (HCV) core protein has been implicated in hepatocarcinogenesis, the relationship between HCV core and Snail expression has not been clarified. METHODS: HepG2 and Huh7 stable cell lines were established by transfection with pcDNA-HCVc. HepG2-HCVc and Huh7-HCVc cells were co-administered with AG490. Cell migration and invasiveness were tested. STAT3 and Snail expression was analyzed by Real-time PCR and Western blot. RESULTS: We found that HCV core is capable of increasing Snail expression and inducing EMT in hepatoma cells. HCV core-induced Snail expression was accompanied by activation of signal transducer and activator of transcription 3 (STAT3), inhibition of STAT3 abrogated HCV core-induced Snail expression and EMT. Furthermore, chromatin immunoprecipitation showed that phosphorylated STAT3 directly binds to the Snail promoter. CONCLUSION: Collectively, these results suggest that HCV core would play a role in hepatocellular carcinoma invasiveness and metastasis by activating the STAT3 pathway, increasing Snail expression and subsequently triggering EMT. These findings would advance the understanding of HCV-mediated invasiveness and metastasis, and might provide a new potential therapeutic target for HCV-related hepatocellular carcinoma.

Hepatitis C virus core protein regulates OCT4 expression and promotes cell cycle progression in hepatocellular carcinoma.

Hepatitis C virus (HCV) core protein plays an important role in the development of hepatocellular carcinoma. octamer-binding protein 4 (OCT4) is critically essential for the pluripotency and self-renewal of embryonic stem cells. Abnormal expression of OCT4 has been detected in several human solid tumors. However, the relationship between HCV core and OCT4 remains uncertain. In the present study, we found that HCV core is capable of upregulating OCT4 expression. The effect of HCV core-induced OCT4 overexpression was abolished by RNAi-mediated scilencing of HCV core. In addition, HCV core-induced OCT4 overexpression resulted in enhanced cell proliferation and cell cycle progression. Inhibition of OCT4 reduced the CCND1 expression and induced G0/G1 cell cycle arrest. Furthermore, OCT4 protein directly binds to CCND1 promoter and transactivates CCND1. These findings suggest that HCV core protein regulates OCT4 expression and promotes cell cycle progression in hepatocellular carcinoma providing new insight into the mechanism of hepatocarcinogenesis by HCV infection.

Knockdown of NANOG enhances chemosensitivity of liver cancer cells to doxorubicin by reducing MDR1 expression.

Multidrug resistance (MDR) is one of the major reasons for the failure of liver cancer chemotherapy, and its suppression may increase the efficacy of chemotherapy. NANOG plays a key role in the regulation of embryonic stem cell self-renewal and pluripotency. Recent studies reported that NANOG was abnormally expressed in several types of tumors, indicating that NANOG is related to tumor development. However, the correlation between NANOG and liver cancer chemoresistance remains uncertain. In this study, RNA interfere technology was employed to knock down NANOG expression in HepG2 human liver cancer cells. We found that the knockdown of NANOG expression in NANOG siRNA-transfected HepG2 cells resulted in decreased colony formation rate and cell migration compared to control HepG2 cells. In addition, HepG2 cells were treated with doxorubicin to evaluate the chemosensitivity to doxorubicin. We found that the doxorubicin sensitivity of HepG2 cells was increased with downregulation of NANOG expression. The expression of MDR1 at both mRNA and protein levels was decreased in HepG2 cells when NANOG was knocked down. These findings suggest that the knockdown of NANOG in HepG2 human cells resulted in decreased MDR1 expression and increased doxorubicin sensitivity, and NANOG could be used as a novel potential therapeutic target to reverse multidrug resistance of liver cancer.

Hepatitis C virus core protein regulates NANOG expression via the stat3 pathway.

HCV Core plays a role in the development of hepatocellular carcinoma. Aberrant expression of NANOG has been observed in many types of human malignancies. However, relationship between Core and NANOG has not been clarified. In this study, we found that Core is capable of up-regulating NANOG expression. Core-induced NANOG expression was accompanied by enforced expression of phosphorylated stat3 protein and was attenuated by inhibition of stat3 phosphorylation. ChIP showed that phosphorylated stat3 directly binds to the NANOG promoter. Core-induced NANOG expression resulted in enhanced cell growth and cell cycle progression. Knockdown of NANOG blocked the cell cycle at the G0/G1 phases and inhibited the cyclin D1 expression. Our findings provide a new insight into the mechanism of hepatocarcinogenesis by HCV infection.

Overexpression of miR-122 promotes the hepatic differentiation and maturation of mouse ESCs through a miR-122/FoxA1/HNF4a-positive feedback loop.

BACKGROUND & AIMS: microRNA-122 is the only identified liver-specific miRNA and plays a crucial role in liver development, maintenance of hepatic homeostasis as well as tumourigenesis. In our previous differentiation of ESCs into hepatocytes, microRNA-122 (miR-122) was expressed at a relatively low level. Here, we aim to elucidate the effect and underlying mechanisms of miR-122 during differentiation of ESCs into hepatocytes. METHODS: Mouse ESCs were initially induced towards HPCs by activin A, FGF-4 and sodium butyrate and were subsequently transfected with a recombinant adenovirus expressing vector pAV.Ex1d-CMV>miR-122/IRES/eGFP 9 days after induction. Cells were analysed by real-time PCR, immunofluorescence, flow cytometry, microscopy and functional assays. Furthermore, microarray analysis was performed. RESULTS: We demonstrated that overexpression of miR-122 could effectively promote hepatic differentiation and maturation, as assessed by morphological and functional tests. The microarray analysis revealed that 323 genes were down-regulated, whereas 59 were up-regulated. Particularly, two liver-specific transcription factors, FoxA1 and HNF4a, were significantly up-regulated. Moreover, the expression of E-cadherin was dramatically increased and the proliferation of HPCs was suppressed, whereas knockdown of FoxA1 reduced E-cadherin expression and increased the proliferation of HPCs. In addition, the expression levels of FoxA1, HNF4a and E-cadherin in time-course transfection experiments with miR-122 were not significantly increased except in cells in which transfection with miR-122 occurred 9 days after induction. CONCLUSION: Overexpression of miR-122 at an appropriate stage could promote hepatic differentiation and maturation by regulating the balance between proliferation and differentiation, as well as the balance between EMT and MET, partially through a miR-122/FoxA1/HNF4a-positive feedback loop.

[The clinic research of the modified laparoscopic splenectomy for massive splenomegaly in the treatment of children with hematologic diseases].

OBJECTIVE: To summarize the experience and characteristics of the modified laparoscopic splenectomy for massive splenomegaly in the treatment of children with hematologic disease. METHODS: The clinical data of 30 cases of laparoscopic splenectomy for massive splenomegaly of children with hematologic disease from March 2007 to December 2011 was analyzed retrospectively. There were 18 male and 12 female patients, aging from 2 to 14 years. Primary disease included mediterranean anemia (17 cases), hereditary spherocytosis (4 cases) and idiopathic thrombocytopenic purpura (ITP, 9 cases). Dissection started with cutting off the gastrosplenic ligaments and lesser sac to fully reveal the splenic hilum, the splenic artery was clamped twice with 10 mm tiatanum clamp. When most of blood stored in the spleen back to heart through the veins and the splenic volume had already decreased, the splenic vein was ligated with 10 mm titanium clip and cut with ligsure and splenic pedicle separated. The Surgery and complication were recorded. For 1 week after surgery, the hemoglobin and platelet counts were reviewed. RESULTS: Twenty-six cases were performed successfully, and 4 cases were converted to open procedure. Of the 4 cases, 2 cases was obesity because of idiopathic thrombocytopenic purpura, 1 case was ß thalassaemia combined severe liver enlargement, and 1 case was after partial splenic embolization. In cases of laparoscopic splenectomy, operation time was 110 to 130 minutes, with an average of 120 minutes, and blood loss during operation was 35 to 180 ml, with an average of 45 ml. Compared with pre-operation, the hemoglobin of mediterranean anemia and hereditary spherocytosis patients were (92 ± 8) g/L, and blood platelet count of ITP patients was (127 ± 20)×10(9)/L, and they increased obviously at 1 week after operation (t = 4.175 and 8.253, both P = 0.000). CONCLUSION: The modified surgical method make the laparoscopic splenectomy for massive splenomegaly in many children with hematologic diseases possible, which was thought to be impossible in the past.

A modified laparoscopic splenectomy for massive splenomegaly in children with hematological disorder: a single institute retrospective clinical research.

PURPOSE: With the optimal acceptance of its clinical advantages, laparoscopic splenectomy (LS) emerged as a gold standard procedure as compared with open splenectomy (OS). However, it is still controversial and even counted as contraindication for massive splenomegaly. Here, we aim to summarize the experiences, characteristics and trends of modified LS for massive splenomegaly in children with hematological disorders. METHODS: Retrospective series of 57 pediatric patients with massive splenomegaly who underwent splenectomy from March 2007 to December 2011 were designated for this clinical analysis. The main outcome measures were dealt by statistics. For 30 cases of LS, we strictly adhered to the principle of using only three trocars to operate and initial ligation of the splenic artery, followed by retrieving the piecemeal of spleen through an accessory incision of 2-3 cm at 12 mm trocar port site. RESULTS: Of the 57 pediatric patients, 27 underwent OS and 30 underwent LS, respectively. Despite the operative time being shorter for OS than for LS (P < 0.001), the blood loss was lower in LS than in OS (P < 0.001); the time required for oral intake as well as duration of hospital stay was lower in LS than in OS (P < 0.001). Post-operatively, 7 (25.9 %) complications occurred in OS and 3 (10 %) in LS. The conversion rate of LS to OS was 13.33 % in four cases till 2009. CONCLUSIONS: Despite the conflicting reports regarding the safety of LS for massive splenomegaly, we demonstrated that our modified laparoscopic splenectomy in the treatment of children with massive splenomegaly in hematological diseases seemed to achieve the fundamental goal of less invasion; it was safe and feasible.

Selection of hepatocyte-like cells from mouse differentiated embryonic stem cells and application in therapeutic liver repopulation.

UNLABELLED: BACKGROUD/AIM: Because of the oncogenic risk, it is important to gain the homogeneous and purified cells from differentiated ESCs before transplantation. Here, we aim to select hepatocyte-like cells from differentiated ESCs, and investigate their growth, differentiation and neoplastic formation after intrahepatic transplantation. METHODS: Mouse ESCs were primarily induced by Dexamethesone, FGF-4 and HGF sequentially, then placed to a conditioning selection media consisting of 5% cholestatic sera and cultivated for 2 wks. After labeled by CFDA-SE, the selected cells were transplanted into mouse liver in therapeutic liver repopulation models. RESULTS: In the early stage of screening cultivation, most cells were suffered from apoptosis or even death. 1w later, some hepatocyte-like colony-forming units were observed, then the selected cells could grow and tend to be more mature, as assessed by morphological and functional tests. After intrahepatic transplantation, the labeled cells could proliferate and expressed albumin. Moreover, teratoma didn't form over 3 months. CONCLUSION: Our conditioning selection media could not only effectively select hepatocyte-like cells from differentiated ESCs, but further promote their growth and differentiation as well. After intrahepatic transplantation in therapeutic liver repopulation models, the selected cells could grow, differentiate and keep partial hepatic function. In particular, the transplantation was safe.

Selective enrichment of hepatocytes from mouse embryonic stem cells with a culture system containing cholestatic serum.

AIM: There is increasing evidence indicating that embryonic stem (ES) cells are capable of differentiating into hepatocyte-like cells in vitro. However, it is necessary to improve the differentiation efficiency so as to promote the clinical application. Here, we report an efficient culture system to support hepatocyte differentiation from ES cells by utilizing cholestatic serum. METHODS: One week after the induction of E14 mouse ES cells into hepatocytes with sodium butyrate, cholestatic serum was added into the culture system at various concentrations and hepatocyte-like cells were induced to proliferate. The morphological and phenotypic markers of hepatocytes were characterized using light microscopy, immunocytochemistry, and RT-PCR, respectively. The function of glycogen storage of the differentiated cells was detected by Periodic acid-Schiff (PAS) reaction, and the ratio of hepatic differentiation was determined by counting the albumin and PAS-positive cells. RESULTS: In the presence of conditional selective medium containing cholestatic serum, numerous epithelial cells resembling hepatocytes were observed. The RT-PCR analysis showed that undifferentiated ES cells did not express any hepatic-specific markers; however, in the presence of sodium butyrate and conditional selective medium containing cholestatic serum, hepatic differentiation markers were detected. Immunofluorescence staining showed that those ES-derived hepatocytes were alpha-fetoprotein, albumin, and cytokeratin 18 positive, with the ability of storing glycogen. Further determination of the hepatic differentiation ratio showed that the application of cholestatic serum efficiently enriched ES-derived hepatocyte-like cells by inducing lineage differentiation and enhancing lineage proliferation. CONCLUSION: The conditional selective medium containing cholestatic serum is optimal to selectively enrich hepatocyte-like cells from mixed differentiated ES cells, which may provide a novel method to improve the hepatic differentiation ratio of ES cells.

[Study of embryonic stem cells induced to express hepatic cell functions in vitro in a pathologic niche containing cholestatic serum].

OBJECTIVE: To study the role of a pathologic niche inducing mouse embryonic stem cells (ESC) to express hepatic cell functions in vitro. METHODS: Embryoid bodies were developed from 5 to 7 day hanging-drop culture of mouse ESC, and their dissociated cells were planted in three differential systems: nothing added; with 20 ng/ml hepatocyte growth factor (HGF); and 5% rat cholestatic serum plus 20 ng/ml HGF added. Their differentiation was observed with inverted microscopes daily, and their hepatic functions were analyzed against their synthesis of glycogen, triglycerides, albumin, and urea nitrogen, and by their staining of indocyanine green (ICG) and fluorescein diacetate (FDA). RESULTS: ESC spontaneous differentiation was hardly being controlled to form three germ layers. HGF prompted the ESC to develop further into visceral endoderm and mesoderm (myocardium), but both of them only expressed a low level of hepatocyte-specific metabolic functions. With cholestatic serum added into the HGF-induced system, differentiated cells grew into similar angular cells, and had a higher level synthesis of glycogen, triglycerides, albumin and urea nitrogen with positive ICG and FDA staining. CONCLUSIONS: Spontaneous or HGF-induced ESC differentiation has only limited hepatic functions expressed. A pathologic niche in vitro induces ESC to develop into hepatic lineages, with a higher level of hepatic metabolic functions.