The ARF tumor suppressor inhibits tumor cell colonization independent of p53 in a novel mouse model of pancreatic ductal adenocarcinoma metastasis

Pancreatic ductal adenocarcinoma (PDAC) is an incurable, highly metastatic disease that is largely resistant to existing treatments. A better understanding of the genetic basis of PDAC metastasis should facilitate development of improved therapies. To that end, we developed a novel mouse xenograft model of PDAC metastasis to expedite testing of candidate genes associated with the disease. Human PDAC cell lines BxPC-3, MiaPaCa-2, and Panc-1 stably expressing luciferase were generated and introduced by intracardiac injections into immunodeficient mice to model hematogenous dissemination of cancer cells. Tumor development was monitored by bioluminescence imaging. Bioluminescent MiaPaCa-2 cells most effectively recapitulated PDAC tumor development and metastatic distribution in vivo. Tumors formed in nearly 90% of mice and in multiple tissues, including normal sites of PDAC metastasis. Effects of p14ARF, a known suppressor of PDAC, were tested to validate the model. In vitro, p14ARF acted through a CtBP2-dependent, p53-independent pathway to inhibit MiaPaCa-2-invasive phenotypes, which correlated with reduced tumor cell colonization in vivo. These findings establish a new bioluminescent mouse tumor model for rapidly assessing the biological significance of suspected PDAC metastasis genes. This system may also provide a valuable platform for testing innovative therapies.     

Hsa-miRNA-23a-3p promotes atherogenesis in a novel mouse model of atherosclerosis

Of the known regulators of atherosclerosis, miRNAs have been demonstrated to play critical roles in lipoprotein homeostasis and plaque formation. Here, we generated a novel animal model of atherosclerosis by knocking in LDLR^W483X in C57BL/6 mice, as the W483X mutation in LDLR is considered the most common newly identified pathogenic mutation in Chinese familial hypercholesterolemia (FH) individuals. Using the new in vivo mouse model combined with a well-established atherosclerotic in vitro human cell model, we identified a novel atherosclerosis-related miRNA, miR-23a-3p, by microarray analysis of mouse aortic tissue specimens and human aortic endothelial cells (HAECs). miR-23a-3p was consistently downregulated in both models, which was confirmed by qPCR. Bioinformatics analysis and further validation experiments revealed that the TNFα-induced protein 3 (TNFAIP3) gene was the key target of miR-23a-3p. The miR-23a-3p-related functional pathways were then analyzed in HAECs. Collectively, the present results suggest that miR-23a-3p regulates inflammatory and apoptotic pathways in atherogenesis by targeting TNFAIP3 through the NF-κB and p38/MAPK signaling pathways.     

The role of p21-activated kinases in hepatocellular carcinoma metastasis

The p21-activated kinases (PAKs) are downstream effectors of the Rho family small GTPases as well as a wide variety of mitogenic factors and have been implicated in cancer formation, development and metastasis. PAKs phosphorylate a wide spectrum of substrates to mediate extracellular signals and regulate cytoskeletal remodeling, cell motility and survival. In this review, we aim to summarize the findings regarding the oncogenic role and the underlying mechanisms of PAKs signaling in various cancers, and in particular highlight the prime importance of PAKs in hepatocellular carcinoma (HCC) progression and metastasis. Recent studies exploring the potential therapeutic application of PAK inhibitors will also be discussed.     

Overexpression of RhoGDI,a novel predictor of distant metastasis,promotes cell proliferation and migration in hepatocellular carcinoma

RhoGDI (Rho GDP-dissociation inhibitor alpha, or RhoGDIα) was identified as a regulator of Rho GTPases, but its role in cancer remains controversial. In this study, increased expression of RhoGDI was detected in hepatocellular carcinoma (HCC) cell lines and tissues with highly metastatic potential. RhoGDI overexpression correlated with postoperative distant metastasis. Enforced expression of RhoGDI in HCC cells significantly enhanced cell proliferation and migration. Conversely, knockdown of RhoGDI caused an inhibition of the aggressive phenotypes of HCC cells. Furthermore, RhoGDI up-regulated Rho, but not Rac, and enhanced PI3K/AKT and MAPK pathway activity. Our findings suggest that RhoGDI overexpression is a predictor of distant metastasis and plays an important role in the progression of HCC.     

A gain of function p53 mutant promotes both genomic instability and cell survival in a novel p53-null mammary epithelial cell model.

Approximately 40% of human breast cancers contain alterations in the tumor suppressor p53. The p53 172R-H gain-of-function mutant (equivalent to the common 175R-H human breast cancer mutant) has been shown to promote aneuploidy and tumorigenesis in the mammary gland in transgenic mice and may affect genomic stability in part by causing centrosome abnormalities. The precise mechanism of action of these gain-of-function mutants is not well understood, and has been studied primarily in fibroblast cell lines. A novel p53-null mouse mammary epithelial cell line developed from p53-null mice has been used in adenovirus-mediated transient transfection experiments to study the properties of this p53 mutant. Marked centrosome amplification and an increased frequency of aberrant mitoses were observed within 72 h of introduction of p53 172R-H. However, few cells with aberrant centrosome numbers were observed in cells stably expressing the p53 172R-H mutant. Furthermore, stable expression of this p53 mutant reduced both basal and DNA damage-induced apoptosis. This result may be mediated in part through abrogation of p73 function. The p53 172R-H mutant, therefore, appears to influence tumorigenesis at the molecular level in two distinct ways: promoting the development of aneuploidy in cells while also altering their apoptotic response after DNA damage.     

PIAS4, upregulated in hepatocellular carcinoma,promotes tumorigenicity and metastasis

Protein inhibitor of activated STAT4 (PIAS4) protein has been implicated in regulating various biological activities including protein posttranslational modification, such as SUMOylation. In this study, we explored the roles of PIAS4 in hepatocellular carcinoma (HCC). We analyzed the PIAS4 expression in cancer tissues and paracancerous tissues from 38 HCC patients and its correlation with patients' prognosis. In vitro, PIAS4 was overexpressed or knockdowned in Huh-7 and HepG-2 cells. Then Cell Counting Kit-8 assay, flow cytometry, and Transwell assay were performed to assess cell viability, apoptosis, migration, and invasion, respectively. Furthermore, SUMOylation of AMPKα and NEMO mediated by PIAS4 was investigated. The results showed that the PIAS4 expression was significantly upregulated in cancer tissues and was correlated with poor prognosis in HCC patients. PIAS4 silencing blocked the SUMOylation of AMPKα and NEMO, leading to enhanced cell proliferation, migration, and invasion. In addition, inhibition of AMPKα or NEMO by siRNAs attenuated the effect of PIAS4 silencing on Huh-7 and HepG-2 cells. In summary, our findings suggest that PIAS4 promotes tumorigenicity and metastasis of HCC cells by promoting the SUMOylation of AMPKα and NEMO.     

p53 regulates ERK activation in carboplatin induced apoptosis in cervical carcinoma: a novel target of p53 in apoptosis

In general, the activation of extracellular recognition kinase (ERK) cascade is implicated in exerting tumorigenic effects. Conversely, recent studies suggest that ERK activation may also have role in DNA-damage induced apoptosis [Wang, X., Martindale, J.L. and Holbrook, N.J. (2000) Requirement for ERK activation in cisplatin-induced apoptosis. J. Biol. Chem. 275, 39435-39443; Schweyer S., Soruri A., Meschter O., Heintze A., Zschunke F., Miosge N., Thelen P., Schlott T., Radzun H.J. and Fayyazi, A. (2004) Cisplatin-induced apoptosis in human malignant testicular germ cell lines depends on MEK/ERK activation. Br. J. Cancer 91, 589-598]. Here we observed an essential requirement of ERK activation in carboplatin (Carb) induced apoptosis in SiHa and CaSki cells. Under similar treatment conditions p53 was also involved in Carb induced apoptosis in these cells. Therefore, we investigated the relation between p53 and ERK in Carb induced apoptosis in these cells. Abrogation of p53 transactivation activity by pifithrin alpha or dominant-negative mutant of p53 resulted in decrease in activation of ERK in Carb treated cells. The present study for the first time proposes that p53 may act as one of the upstream regulators of ERK activation for the induction of apoptosis in Carb treated cervical cancer cells.     

Lack of p53 function promotes radiation-induced mitotic catastrophe in mouse embryonic fibroblast cells

Background  

We have demonstrated that in some human cancer cells both chronic mild heat and ionizing radiation exposures induce a transient block in S and G2 phases of the cell cycle. During this delay, cyclin B1 protein accumulates to supranormal levels, cyclin B1-dependent kinase is activated, and abrogation of the G2/M checkpoint control occurs resulting in mitotic catastrophe (MC).     

Pirh2, a p53-induced ubiquitin-protein ligase,promotes p53 degradation

The p53 tumor suppressor exerts anti-proliferative effects in response to various types of stress including DNA damage and abnormal proliferative signals. Tight regulation of p53 is essential for maintaining normal cell growth and this occurs primarily through posttranslational modifications of p53. Here, we describe Pirh2, a gene regulated by p53 that encodes a RING-H2 domain-containing protein with intrinsic ubiquitin-protein ligase activity. Pirh2 physically interacts with p53 and promotes ubiquitination of p53 independently of Mdm2. Expression of Pirh2 decreases the level of p53 protein and abrogation of endogenous Pirh2 expression increases the level of p53. Furthermore, Pirh2 represses p53 functions including p53-dependent transactivation and growth inhibition. We propose that Pirh2 is involved in the negative regulation of p53 function through physical interaction and ubiquitin-mediated proteolysis. Hence, Pirh2, like Mdm2, participates in an autoregulatory feedback loop that controls p53 function.     

Functional interactions between retinoblastoma and c-MYC in a mouse model of hepatocellular carcinoma

Inactivation of the RB tumor suppressor and activation of the MYC family of oncogenes are frequent events in a large spectrum of human cancers. Loss of RB function and MYC activation are thought to control both overlapping and distinct cellular processes during cell cycle progression. However, how these two major cancer genes functionally interact during tumorigenesis is still unclear. Here, we sought to test whether loss of RB function would affect cancer development in a mouse model of c-MYC-induced hepatocellular carcinoma (HCC), a deadly cancer type in which RB is frequently inactivated and c-MYC often activated. We found that RB inactivation has minimal effects on the cell cycle, cell death, and differentiation features of liver tumors driven by increased levels of c-MYC. However, combined loss of RB and activation of c-MYC led to an increase in polyploidy in mature hepatocytes before the development of tumors. There was a trend for decreased survival in double mutant animals compared to mice developing c-MYC-induced tumors. Thus, loss of RB function does not provide a proliferative advantage to c-MYC-expressing HCC cells but the RB and c-MYC pathways may cooperate to control the polyploidy of mature hepatocytes.     

Microfilament regulatory protein MENA increases activity of RhoA and promotes metastasis of hepatocellular carcinoma

Mammalian enabled (MENA), usually known as a direct regulator of microfilament polymerization and bundling, promotes metastasis in various cancers. Here we focus on the role of MENA in hepatocellular carcinoma (HCC) metastasis and the relevant mechanism from the view of RhoA activity regulation. By HCC tissue microarray analysis, we found that MENA expression was positively associated with satellite lesions (P<0.01) and vascular invasion (P<0.01). Cases with membrane reinforcement of MENA staining in HCC tissues had significantly higher rates of early recurrence in the intermediate MENA expression group. Knockdown of MENA significantly suppressed HCC cell migration and invasion in vitro, as well as their intrahepatic and distant metastasis in vivo. Knockdown of MENA also decreased filopodia and stress fibers in SMMC-7721 cells. Furthermore, a decrease of RhoA activity was detected by a pull-down assay in SMMC-7721-shMENA cells. The ROCK inhibitor, Y-27632, suppressed migration of both MENA knockdown SMMC-7721 cells and control cells, but diminished their difference. Thus, our findings suggest that MENA promotes HCC cell motility by activating RhoA.     

Tissue-specific apoptotic effects of the p53 codon 72 polymorphism in a mouse model

Currently there are several dozen human polymorphisms that have been loosely associated with cancer risk. Correlating such variants with cancer risk has been challenging, primarily due to factors such as genetic heterogeneity, contributions of diet and environmental factors, and the difficulty in obtaining large sample sizes for analysis. Such difficulties can be circumvented with the establishment of mouse models for human variants. Recently, several groups have modeled human cancer susceptibility polymorphisms in the mouse. Remarkably, in each case these mouse models have accurately reflected human phenotypes, and clarified the contribution of these variants to cancer risk. We recently reported on a mouse model for the codon 72 polymorphism in p53, and found that this polymorphism regulates the ability to cooperate with NFκB and induce apoptosis. Here-in we present evidence that this polymorphism impacts the apoptotic function of p53 in a tissue-specific manner; such tissue-specific effects of polymorphic variants represent an added challenge to human cancer risk association studies. The data presented here support the premise that modeling human polymorphisms in the mouse represents a powerful tool to assess the impact of these variants on cancer risk, progression and therapy.Key words: p53, polymorphism, apoptosis, codon 72, NFκB     

Down-regulation of MYH10 driven by chromosome 17p13.1 deletion promotes hepatocellular carcinoma metastasis through activation of the EGFR pathway

Somatic copy number alterations (CNAs) are a genomic hallmark of cancers. Among them, the chromosome 17p13.1 deletions are recurrent in hepatocellular carcinoma (HCC). Here, utilizing an integrative omics analysis, we screened out a novel tumour suppressor gene within 17p13.1, myosin heavy chain 10 (MYH10). We observed frequent deletions (~38%) and significant down-regulation of MYH10 in primary HCC tissues. Deletion or decreased expression of MYH10 was a potential indicator of poor outcomes in HCC patients. Knockdown of MYH10 significantly promotes HCC cell migration and invasion in vitro, and overexpression of MYH10 exhibits opposite effects. Further, inhibition of MYH10 markedly potentiates HCC metastasis in vivo. We preliminarily elucidated the mechanism by which loss of MYH10 promotes HCC metastasis by facilitating EGFR pathway activation. In conclusion, our study suggests that MYH10, a candidate target gene for 17p13 deletion, acts as a tumour suppressor and may serve as a potential prognostic indicator for HCC patients.     

Tissue-specific apoptotic effects of the p53 codon 72 polymorphism in a mouse model

Currently there are several dozen human polymorphisms that have been loosely associated with cancer risk. Correlating such variants with cancer risk has been challenging, primarily due to factors such as genetic heterogeneity, contributions of diet and environmental factors, and the difficulty in obtaining large sample sizes for analysis. Such difficulties can be circumvented with the establishment of mouse models for human variants. Recently, several groups have modeled human cancer susceptibility polymorphisms in the mouse. Remarkably, in each case these mouse models have accurately reflected human phenotypes, and clarified the contribution of these variants to cancer risk. We recently reported on a mouse model for the codon 72 polymorphism in p53, and found that this polymorphism regulates the ability to cooperate with NF-kB and induce apoptosis. Here-in we present evidence that this polymorphism impacts the apoptotic function of p53 in a tissue-specific manner; such tissue-specific effects of polymorphic variants represent an added challenge to human cancer risk association studies. The data presented here support the premise that modeling human polymorphisms in the mouse represents a powerful tool to assess the impact of these variants on cancer risk, progression and therapy.