DLC-1与肝癌关系研究

DLC-1基因是一种肿瘤抑制基因,位于人类染色体8p21．3-22。它是RhoA特异性GTP酶的激动蛋白,与调控细胞增殖和粘附的信号传导通路关系密切,在人类多种肿瘤中呈低表达或表达缺失。研究发现DLC-1基因在原发性肝癌(HCC)及肝癌细胞系中表达缺失,提示该基因在原发性肝癌中抑制了肝癌细胞的增殖。DLC-1表达的恢复引起了caspase-3介导的细胞凋亡,抑制肝癌细胞的生长和癌细胞的浸润,从而在肝细胞癌的转移、侵袭及肿瘤细胞的生物特性方面发挥作用。因其与肝癌发生,转移乃至复发关系密切,使其在肝癌早期发现,早期预测肝癌的转移复发及肝癌的预后方面发挥重要角色。     

Mouse model for probing tumor suppressor activity of protein phosphatase 2A in diverse signaling pathways

Evidence that protein phosphatase 2A (PP2A) is a tumor suppressor in humans came from the discovery of mutations in the genes encoding the Aα and Aβ subunits of the PP2A trimeric holoenzymes, Aα-B-C and Aβ-B-C. One point mutation, Aα-E64D, was found in a human lung carcinoma. It renders Aα specifically defective in binding regulatory B’ subunits. Recently, we reported a knock-in mouse expressing Aα-E64D and an Aα knockout mouse. The mutant mice showed a 50–60% increase in the incidence of lung cancer induced by benzopyrene. Importantly, PP2A’s tumor suppressor activity depended on p53. These data provide the first direct evidence that PP2A is a tumor suppressor in mice. In addition, they suggest that PP2A is a tumor suppressor in humans. Here, we report that PP2A functions as a tumor suppressor in mice that develop lung cancer triggered by oncogenic K-ras. We discuss whether PP2A may function as a tumor suppressor in diverse tissues, with emphasis on endometrial and ovarian carcinomas, in which Aα mutations were detected at a high frequency. We propose suitable mouse models for examining whether PP2A functions as tumor suppressor in major growth-stimulatory signaling pathways, and we discuss the prospect of using the PP2A activator FTY720 as a drug against malignancies that are driven by these pathways.     

Loss of hepatic aldolase B activates Akt and promotes hepatocellular carcinogenesis by destabilizing the Aldob/Akt/PP2A protein complex

Loss of hepatic fructose-1, 6-bisphosphate aldolase B (Aldob) leads to a paradoxical up-regulation of glucose metabolism to favor hepatocellular carcinogenesis (HCC), but the upstream signaling events remain poorly defined. Akt is highly activated in HCC, and targeting Akt is being explored as a potential therapy for HCC. Herein, we demonstrate that Aldob suppresses Akt activity and tumor growth through a protein complex containing Aldob, Akt, and protein phosphatase 2A (PP2A), leading to inhibition of cell viability, cell cycle progression, glucose uptake, and metabolism. Interestingly, Aldob directly interacts with phosphorylated Akt (p-Akt) and promotes the recruitment of PP2A to dephosphorylate p-Akt, and this scaffolding effect of Aldob is independent of its enzymatic activity. Loss of Aldob or disruption of Aldob/Akt interaction in Aldob R304A mutant restores Akt activity and tumor-promoting effects. Consistently, Aldob and p-Akt expression are inversely correlated in human HCC tissues, and Aldob down-regulation coupled with p-Akt up-regulation predicts a poor prognosis for HCC. We have further discovered that Akt inhibition or a specific small-molecule activator of PP2A (SMAP) efficiently attenuates HCC tumorigenesis in xenograft mouse models. Our work reveals a novel nonenzymatic role of Aldob in negative regulation of Akt activation, suggesting that directly inhibiting Akt activity or through reactivating PP2A may be a potential therapeutic approach for HCC treatment.

Loss of hepatic fructose bisphosphate aldolase B (Aldob) leads to a paradoxical upregulation of glucose metabolism, favoring hepatocellular carcinogenesis, but the upstream signaling events are unclear. This study shows that Aldob inhibits the activity of the protein kinase Akt and tumor growth in hepatocellular carcinoma via a protein complex containing Aldob, Akt and protein phosphatase 2A.     

The expression and regulation of DFNA5 in human hepatocellular carcinoma DFNA5 in hepatocellular carcinoma

Hepatocellular carcinoma is a primary malignancy of hepatocytes which accounts for 80 % of all primary liver cancers. DFNA5 has been identified as a tumor suppressor gene with an important role in several frequent forms of cancers, while little is known about its role in hepatocellular carcinoma. Through comparison of the DFNA5 protein expression in hepatocellular carcinoma cells (HepG2) with human fetal lung fibroblast cells (MRC5), we found that the DFNA5 protein expression in hepatocellular carcinoma cells was significantly lower than that in normal cells. The transfection of DFNA5 gene into HepG2 cells could increase DFNA5 protein expression, which subsequently led to inhibition of cell proliferation. Underlying mechanism study revealed that decreased proliferation was due to increased apoptosis and cell cycle arrest. In view of the important role of DFNA5 gene in carcinogenesis, these findings are expected to provide new understanding on development and treatment of human hepatocellular carcinoma.     

SIRT1 and c-Myc Promote Liver Tumor Cell Survival and Predict Poor Survival of Human Hepatocellular Carcinomas

The increased expression of SIRT1 has recently been identified in numerous human tumors and a possible correlation with c-Myc oncogene has been proposed. However, it remains unclear whether SIRT1 functions as an oncogene or tumor suppressor. We sought to elucidate the role of SIRT1 in liver cancer under the influence of c-Myc and to determine the prognostic significance of SIRT1 and c-Myc expression in human hepatocellular carcinoma. The effect of either over-expression or knock down of SIRT1 on cell proliferation and survival was evaluated in both mouse and human liver cancer cells. Nicotinamide, an inhibitor of SIRT1, was also evaluated for its effects on liver tumorigenesis. The prognostic significance of the immunohistochemical detection of SIRT1 and c-Myc was evaluated in 154 hepatocellular carcinoma patients. SIRT1 and c-Myc regulate each other via a positive feedback loop and act synergistically to promote hepatocellular proliferation in both mice and human liver tumor cells. Tumor growth was significantly inhibited by nicotinamide in vivo and in vitro. In human hepatocellular carcinoma, SIRT1 expression positively correlated with c-Myc, Ki67 and p53 expression, as well as high á-fetoprotein level. Moreover, the expression of SIRT1, c-Myc and p53 were independent prognostic indicators of hepatocellular carcinoma. In conclusion, this study demonstrates that SIRT1 expression supports liver tumorigenesis and is closely correlated with oncogenic c-MYC expression. In addition, both SIRT1 and c-Myc may be useful prognostic indicators of hepatocellular carcinoma and SIRT1 targeted therapy may be beneficial in the treatment of hepatocellular carcinoma.     

PTEN对SMMC—7721人肝癌细胞迁移增殖及粘着斑激酶磷酸化水平的影响

抑癌基因PTEN编码产物具有双专一性磷酸酶活性 ,并与细胞骨架张力蛋白同源。PTEN可参与粘着斑的形成和解聚而影响细胞迁移。现用PTEN表达质粒转染SMMC 772 1肝癌细胞 ,研究SMMC 772 1细胞运动能力的变化及PTEN与粘着斑激酶 (FAK)酪氨酸磷酸化水平之间的关系。PTEN过表达能够显著抑制细胞在Fn基质上的活动 :细胞在Fn基质上的迁移下降了 35 % ;在 30min和 6 0min两个时间点 ,Fn基质上细胞铺展分别降低了 2 9%和 2 6 % ;而在多聚赖氨酸基质上细胞铺展并没有变化。运用免疫沉淀和Western印迹方法 ,分析FAK及其酪氨酸磷酸化水平 ,发现PTEN过表达不影响FAK表达 ,但显著降低Fn诱导的FAK酪氨酸磷酸化水平 ,两者水平呈负相关。流式细胞仪分析细胞周期结果表明 ,PTEN抑制细胞 ,S期细胞下降了 16 %。上述结果提示 ,PTEN抑制肝癌细胞迁移铺展和增殖 ;PTEN对细胞运动的影响可能通过调节FAK酪氨酸磷酸化水平而实现。     

Elevated levels of Ser/Thr protein phosphatase 5 (PP5) in human breast cancer

Ser/Thr protein phosphatase 5 (PP5) regulates several signaling-cascades that suppress growth and/or facilitate apoptosis in response to genomic stress. The expression of PP5 is responsive to hypoxia inducible factor-1 (HIF-1) and estrogen, which have both been linked to the progression of human breast cancer. Still, it is not clear if PP5 plays a role in the development of human cancer. Here, immunostaining of breast cancer tissue-microarrays (TMAs) revealed a positive correlation between PP5 over-expression and ductal carcinoma in situ (DCIS; P value 0.0028), invasive ductal carcinoma (IDC; P value 0.012) and IDC with metastases at the time of diagnosis (P value 0.0001). In a mouse xenograft model, the constitutive over-expression of PP5 was associated with an increase in the rate of tumor growth. In a MCF-7 cell culture model over-expression correlated with both an increase in the rate of proliferation and protection from cell death induced by oxidative stress, UVC-irradiation, adriamycin, and vinblastine. PP5 over-expression had no apparent effect on the sensitivity of MCF-7 cells to taxol or rapamycin. Western analysis of extracts from cells over-expressing PP5 revealed a decrease in the phosphorylation of known substrates for PP5. Together, these studies indicate that elevated levels of PP5 protein occur in human breast cancer and suggest that PP5 over-expression may aid tumor progression.     

直接注射法制作ICR小鼠肝癌原位移植瘤模型

目的：培养鼠肝癌H22细胞,直接注射法制作ICR小鼠肝癌原位移植瘤,为后续实验奠定基础。方法：鼠肝癌H22细胞体外培养,将调整好的对数生长期的肝癌细胞直接注射小鼠肝脏,2周后解剖观察,并进行组织HE染色。结果：所有实验小鼠均可见肿瘤生长,HE染色示肝细胞肝癌。结论：直接注射法制作ICR小鼠肝癌原位移植瘤模型简便易行,值得推广应用。     

A Src family inhibitor (PP1) potentiates tumor-suppressive effect of connexin 32 gene in renal cancer cells

Connexin (Cx) genes exert negative growth effects on tumor cells with certain cell specificity. We have recently reported that Cx32 acts as a tumor suppressor gene in renal cancer cells due to the inhibition of Src-dependent signaling. In line with the previous study, here we examined if a Src family inhibitor (PP1) could potentiate tumor-suppressive effect of Cx32 in Caki-2 cell from human renal cell carcinoma. In order to clarify the potentialization of PP1, using Cx32-transfected Caki-2 cells and mock-transfected Caki-2 cells, we estimated difference in cytotoxic effect of PP1 on the two cell clones in vitro as well as in vivo. PP1 showed more cytotoxic effect on Caki-2 cells having Cx32 positive expression than that of Cx32 negative expression at lower doses. This potentialization was also observed in xenograft model of nude mice. The potentialization of the effect mainly depended on the induction of apoptosis but not the control of cell growth. In conjugation with this event, the reduction of anti-apoptotic molecules (Bcl-2 and Bcl-xL) was caused by the combination of Cx32 expression and PP1 treatment in Caki-2 cells. These results suggest that PP1 potentiates tumor-suppressive effect of connexin 32 gene in renal cancer cells through the reduction of anti-apoptotic molecules.     

直接注射法制作ICR 小鼠肝癌原位移植瘤模型

目的:培养鼠肝癌H22细胞,直接注射法制作ICR小鼠肝癌原位移植瘤,为后续实验奠定基础。方法:鼠肝癌H22细胞体外培养,将调整好的对数生长期的肝癌细胞直接注射小鼠肝脏,2周后解剖观察,并进行组织HE染色。结果:所有实验小鼠均可见肿瘤生长,HE染色示肝细胞肝癌。结论:直接注射法制作ICR小鼠肝癌原位移植瘤模型简便易行,值得推广应用。     

Deleted in liver cancer-1 (DLC-1): a tumor suppressor not just for liver

Deleted in liver cancer 1 (DLC-1), as its name implied, was originally isolated as a potential tumor suppressor gene often deleted in hepatocellular carcinoma. Further studies have indicated that down-expression of DLC-1 either by genomic deletion or DNA methylation is associated with a variety of cancer types including lung, breast, prostate, kidney, colon, uterus, ovary, and stomach. Re-expression of DLC-1 in cancer cells regulates the structure of actin cytoskeleton and focal adhesions and significantly inhibits cell growth, supporting its role as a tumor suppressor. This tumor suppressive function relies on DLC-1's RhoGTPase activating protein (RhoGAP) activity and steroidogenic acute regulatory (StAR)-related lipid transfer (START) domain, as well as its focal adhesion localization, which is recruited by the Src Homology 2 (SH2) domains of tensins in a phosphotyrosine-independent fashion. Therefore, the expression and subcellular localization of DLC-1 could be a useful molecular marker for cancer prognosis, whereas DLC-1 and its downstream signaling molecules might be therapeutic targets for the treatment of cancer.     

Tumor suppressor carcinoembryonic antigen-related cell adhesion molecule 1 potentates the anchorage-independent growth of human hepatoma HepG2 cells

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), an adhesion molecule of the immunoglobulin superfamily, has been characterized as a putative tumor suppressor because it is frequently down-regulated in aggressive types of cancer cells. Recently, however, several studies have shown that CEACAM1 actively contributes to malignant progression or migration in some types of tumor cells, suggesting that the role of CEACAM1 might be diverse among different types of cancer cells. To investigate the functional consequences of CEACAM1 expression in hepatocellular carcinoma, we analyzed the status of CEACAM1 in hepatoma cell lines HLF, PLC/PRF/5, HepG2 and KYN-2. We found that CEACAM1 was only expressed in HepG2 cells, which show a unique property for enhanced anchorage-independent growth. When HepG2 cells were treated with small interfering RNA targeted against CEACAM1, the growth rate in monolayer culture was increased. In contrast, when HepG2 cells were cultured in suspension, inhibition of CEACAM1 expression significantly decreased the growth rate, and the speed of cell-cell attachment was repressed. Hyaluronidase treatment attenuated the growth rate of HepG2 cells in suspension culture, indicating that cell-cell attachment is a requisite for anchorage-independent growth. Our data may reveal the dual role of CEACAM1 on hepatocarcinogenesis, by showing that CEACAM1 acts as a tumor suppressor in HepG2 cells in anchorage-dependent growth conditions, while in anchorage-independent growth conditions, it augments cell proliferation by potentiating the cell-cell attachment.     

Inducible expression of catalytically active type 1 serine/threonine protein phosphatase in a human carcinoma cell line

Background  

One of the major cellular serine/threonine protein phosphatases is protein phosphatase type 1 (PP1). Studies employing many eukaryotic systems all point to a crucial role for PP1 activity in controlling cell cycle progression. One physiological substrate for PP1 appears to be the product of the retinoblastoma susceptibility gene (pRB), a demonstrated tumor suppressor. The growth suppressive activity of pRB is regulated by its phosphorylation state. Of critical importance is the question of the in vivo effect of PP1 activity on pRB and growth regulation. As a first step towards addressing this question, we developed an inducible PP1 expression system to investigate the regulation of PP1 activity.     

Loss of the orphan nuclear receptor SHP is more pronounced in fibrolamellar carcinoma than in typical hepatocellular carcinoma

Hepatocellular carcinoma (HCC) remains a major problem in oncology. The molecular mechanisms which underlie its pathogenesis are poorly understood. Recently the Small Heterodimer Partner (SHP), an orphan nuclear receptor, was suggested to be involved as a tumor suppressor in hepatocellular carcinoma development. To date, there are no such studies regarding fibrolamellar carcinoma, a less common variant of HCC, which usually affects young people and displays distinct morphological features. The aim of our project was to evaluate the SHP levels in typical and fibrolamellar hepatocellular carcinoma with respect to the levels of one of the cell cycle regulators, cyclin D1. We assessed the immunoreactivity levels of SHP and cyclin D1 in 48 typical hepatocellular carcinomas, 9 tumors representing the fibrolamellar variant, 29 non malignant liver tissues and 7 macroregenerative nodules. We detected significantly lower SHP immunoreactivity in hepatocellular carcinoma when compared to non malignant liver tissue. Moreover, we found that SHP immunoreactivity is reduced in fibrolamellar carcinoma when compared to typical hepatocellular carcinoma. We also found that SHP is more commonly lost in HCC which arises in the liver with steatosis. The comparison between the cyclin D1 and SHP expression revealed the negative correlation between these proteins in the high grade HCC. Our results indicate that the impact of loss of SHP protein may be even more pronounced in fibrolamellar carcinoma than in a typical form of HCC. Further investigation of mechanisms through which the loss of SHP function may influence HCC formation may provide important information in order to design more effective HCC therapy.     

肝细胞癌组织中OATP1B3的表达及其作用的初步研究

摘要 目的：探讨有机阴离子转运多肽1B3（OATP1B3）在肝细胞癌（肝癌）组织中的表达及作用。方法：通过免疫组化实验和免疫印迹试验（Western blot）检测肝癌组织及其癌旁组织中OATP1B3情况,并分析其与患者临床病理特征的相关性。采用实时荧光定量聚合酶链反应（qRT-PCR）检测OATP1B3在多株肝癌细胞中的表达情况,选择表达量相对较低的人肝癌细胞（HepG2和Huh7）细胞进行过表达实验,细胞毒实验（MTT）和流式细胞术分别检测OATP1B3对细胞增殖及凋亡的影响。结果：肝癌组织中OATP1B3表达水平明显低于癌旁组织（P<0.05）,且与患者恶性肿瘤国际临床病期分类（TNM分期）、肿瘤分化程度、有无肿瘤复发显著相关（P<0.05）。过表达OATP1B3可抑制HepG2和Huh7细胞增殖,促进细胞凋亡。结论：OATP1B3在肝癌组织中低表达,上调其表达可抑制肝癌细胞增殖和促进细胞凋亡。OATP1B3可能是肝癌的抑癌基因,对肝癌的发生、发展具有重要作用。     

Spinophilin: a new tumor suppressor at 17q21

The scaffold protein spinophilin (SPN) is a regulatory subunit of phosphatase 1a (PP1a) located at 17q21.33. This region is frequently associated with microsatellite instability and LOH and contains a relatively high density of known tumor suppressor genes, and several unidentified candidate tumor suppressor genes located distal to BRCA1. Spn is located in this locus and proposed to be a new tumor suppressor. Loss of Spn induces a proliferative response by increasing pRb phosphorylation, which in turn activates p53, thereby, neutralizing the proliferative response. The absence of p53 bypasses this barrier and enhances the malignant phenotype. Furthermore, the ectopic expression of SPN in human tumor cells from different types of malignancies greatly reduced cell growth. Spn knock-out mice had decreased lifespan with increased cellular proliferation in tissues such as the mammary ducts and early appearance of tumors. Furthermore, the combined loss of Spn and mutant p53 activity led to increased mammary carcinomas, confirming the functional relationship between p53 and Spn. In human tumors, Spn is absent in 20% and reduced in another 37% of human lung tumors. Spn reduction correlates with malignant grade and p53 mutations. Furthermore, Spn mRNA is lost in a percentage of renal carcinomas and lung adenocarcinomas. Finally, lower levels of Spn mRNA correlate with higher grade of ovarian carcinoma and chronic myelogenous leukemia. Therefore, Spn may be the tumor suppressor gene that is located at 17q21.33 and that its tumor suppressive function is dependent on the absence of p53.     

Human cancer-associated mutations in the Aα subunit of protein phosphatase 2A increase lung cancer incidence in Aα knock-in and knockout mice

Strong evidence has indicated that protein phosphatase 2A (PP2A) is a tumor suppressor, but a mouse model for testing the tumor suppressor activity was missing. The most abundant forms of trimeric PP2A holoenzyme consist of the scaffolding Aα subunit, one of several regulatory B subunits, and the catalytic Cα subunit. Aα mutations were discovered in a variety of human carcinomas. All carcinoma-associated mutant Aα subunits are defective in binding the B or B and C subunits. Here we describe two knock-in mice expressing cancer-associated Aα point mutants defective in binding B' subunits, one knockout mouse expressing truncated Aα defective in B and C subunit binding, and a floxed mouse for generating conditional Aα knockouts. We found that the cancer-associated Aα mutations increased the incidence of cancer by 50 to 60% in lungs of FVB mice treated with benzopyrene, demonstrating that PP2A acts as a tumor suppressor. We show that the effect of Aα mutation on cancer incidence is dependent on the tumor suppressor p53. The finding that the Aα mutation E64D, which was detected in a human lung carcinoma, increases the lung cancer incidence in mice suggests that this mutation also played a role in the development of the carcinoma in which it was discovered.