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RNA interference against MDM2 suppresses tumor growth and metastasis in pancreatic carcinoma SW1990HM cells
Abstract
In our previous study, the mouse double minute 2 (MDM2) was identified as one of the leading genes that promote the metastasis of pancreatic cancer (PC). However, the mechanism by which MDM2 promotes metastasis of PC is not understood. In this study, we show that down-regulation of MDM2 through lentivirus-mediated RNA interference could also suppress in vitro proliferation and in vivo tumor growth, and led to an obvious inhibition of both in vitro invasion and in vivo live metastases of SW1990HM cells which had an over-expression of MDM2 and a higher metastatic potential. Moreover, we also show that the down-regulation of MDM2 induced a significant decrease in MMP9, Ki-67 and increase in P53, E-Cadherin expression, and results in an altered expression of genes involved in metastasis, apoptosis, and cell proliferation. Our results suggest that MDM2 plays an important role in metastasis as well as tumor growth of PC. MDM2 could be a hopeful target for the control of PC.
... Bioinformatics and luciferase assays indicated that miR-579-3p targeted to 3'UTR of MDM2 and inhibited MDM2 expression at both mRNA AGING and protein levels. MDM2 upregulation was observed in numerous human tumors, including pancreatic carcinoma [32]. Additionally, previous studies have revealed that MDM2 was identified as one of the primary genes that accelerate the metastasis of pancreatic carcinoma [32,33]. ...
... MDM2 upregulation was observed in numerous human tumors, including pancreatic carcinoma [32]. Additionally, previous studies have revealed that MDM2 was identified as one of the primary genes that accelerate the metastasis of pancreatic carcinoma [32,33]. Importantly, our PCR results presented that MDM2 expression in pancreatic carcinoma samples was notably increased. ...
... Moreover, MDM2 expression was positively related to HCG11 expression and negatively associated with miR-579-3p expression. Additionally, consistent with the findings from Shi et al. [32], we also discovered that upregulation of MDM2 promoted cell growth, relieved cell cycle arrest, suppressed cell apoptosis and increased cell mobility, which further confirming the carcinogenic effect of MDM2 in pancreatic carcinoma. ...
Background:
Increasing reports have revealed that dysregulated expression of long non-coding RNAs (lncRNAs) is involved in pancreatic carcinoma progression. This study intends to explore the function and molecular mechanism of lncRNA HLA complex group 11 (HCG11) in pancreatic carcinoma.
Methods:
The expression profiles of HCG11 in pancreatic carcinoma samples were detected by qPCR. Bioinformatics analysis was applied to detect the associations among HCG11/miR-579-3p/MDM2. The malignant properties of pancreatic carcinoma cells were measured by numerous biological assays. Xenograft model was exploited to detect the effect of HCG11 on tumor growth.
Results:
A significant increase of HCG11 was occurred in pancreatic carcinoma samples. Knockdown of HCG11 suppressed the progression of pancreatic carcinoma cells. Bioinformatics analysis revealed that HCG11 upregulated MDM2 expression by competitively targeting miR-579-3p. The rescue assays showed that miR-579-3p reversed cell behaviors caused by HCG11, and MDM2 reversed cell properties induced by miR-579-3p. The Notch1 intracellular domain (NICD) and Hes1 protein levels were increased by overexpression of HCG11/MDM2. The tumor growth was suppressed after depletion of HCG11, followed by suppressing Ki67, PCNA and Vimentin expression, increasing TUNEL-positive cells and E-cadherin expression.
Conclusions:
Our observations highlighted that HCG11 contributed to the progression of pancreatic carcinoma by promoting growth and aggressiveness, and inhibiting apoptosis via miR-579-3p/MDM2/Notch/Hes1 axis.
... Conversely, Ecadherin is decreased as cells transition into a mesenchymal phenotype. Moreover, we have shown that the oncogene Mouse double minute 2 (Mdm2) is also positively correlated with EMT (1)(2)(3)(4)(5). Clinically, mdm2 gene amplification occurs in approxi-mately 10% of all human cancers, yet more importantly, several studies have detected the Mdm2 protein in 40% to 80% of highgrade human tumors (6,7). ...
... Only two studies thus far have explored the importance of Mdm2 in metastasis in vivo. One study, conducted in pancreatic carcinoma cells, found that silencing Mdm2 resulted in decreased proliferation (2). This study, along with another that used tail vein injection of transiently transfected KHT-C murine cells (11), employed approaches that did not directly assess the initiation or other phases of metastasis to define the role of Mdm2. ...
Metastasis of cancer cells to distant organ systems is a complex process that is initiated with the programming of cells in the primary tumor. The formation of distant metastatic foci is correlated with poor prognosis and limited effective treatment options. We and others have correlated Mouse double minute 2 (Mdm2) with metastasis; however, the mechanisms involved have not been elucidated. Here, it is reported that shRNA-mediated silencing of Mdm2 inhibits epithelial–mesenchymal transition (EMT) and cell migration. In vivo analysis demonstrates that silencing Mdm2 in both post-EMT and basal/triple-negative breast cancers resulted in decreased primary tumor vasculature, circulating tumor cells, and metastatic lung foci. Combined, these results demonstrate the importance of Mdm2 in orchestrating the initial stages of migration and metastasis.
Implication: Mdm2 is the major factor in the initiation of metastasis. Mol Cancer Res; 15(11); 1598–607. ©2017 AACR.
... The process of EMT enabling dissemination and invasion of the cells include the acquisition of mobility, invasiveness, and potential to disintegrate the complex network of extracellular matrix (ECM) [84,85]. Although EMT possesses similarity in key cellular events, the critical details differ according to tissue nature and site [86]. ...
Murine double minute 2 (MDM2) is a well-recognized molecule for its oncogenic potential. Since its identification, various cancer-promoting roles of MDM2 such as growth stimulation, sustained angiogenesis, metabolic reprogramming, apoptosis evasion, metastasis, and immunosuppression have been established. Alterations in the expression levels of MDM2 occur in multiple types of cancers resulting in uncontrolled proliferation. The cellular processes are modulated by MDM2 through transcription, post-translational modifications, protein degradation, binding to cofactors, and subcellular localization. In this review, we discuss the precise role of deregulated MDM2 levels in modulating cellular functions to promote cancer growth. Moreover, we also briefly discuss the role of MDM2 in inducing resistance against anti-cancerous therapies thus limiting the benefits of cancerous treatment.
... In PAAD, MDM2 is frequently overexpressed [82] and is associated with the T stage of cancer [83], thus playing a key role in drug resistance and the malignant properties of PAAD cells [84]. Knocking down MDM2 can inhibit the growth of PAAD cell lines, as well as tumor invasion and metastasis [85]. Combining MDM2 inhibition with nuclear factor κ-B inhibition can enhance the antitumor effect of radiotherapy in wild-type p53 PAAD [86]. ...
Background:
The murine double minute 2 (MDM2) gene is a crucial factor in the development and progression of various cancer types. Multiple rigorous scientific studies have consistently shown its involvement in tumorigenesis and cancer progression in a wide range of cancer types. However, a comprehensive analysis of the role of MDM2 in human cancer has yet to be conducted.
Methods:
We used various databases, including TIMER2.0, TCGA, GTEx and STRING, to analyze MDM2 expression and its correlation with clinical outcomes, interacting genes and immune cell infiltration. We also investigated the association of MDM2 with immune checkpoints and performed gene enrichment analysis using DAVID tools.
Results:
The pan-cancer MDM2 analysis found that MDM2 expression and mutation status were observably different in 25 types of cancer tissue compared with healthy tissues, and prognosis analysis showed that there was a significant correlation between MDM2 expression and patient prognosis. Furthermore, correlation analysis showed that MDM2 expression was correlated with tumor mutational burden, microsatellite instability and drug sensitivity in certain cancer types. We found that there was an association between MDM2 expression and immune cell infiltration across cancer types, and MDM2 inhibitors might enhance the effect of immunotherapy on breast cancer, bladder cancer and ovarian cancer.
Conclusions:
The first systematic pan-cancer analysis of MDM2 was conducted, and it demonstrated that MDM2 was a reliable prognostic biomarker and was closely related to cancer immunity, providing a potential immunotherapeutic target for breast cancer, bladder cancer and ovarian cancer.
... In this respect, multiple groups, have investigated the correlation between MDM2 and MMPs, which are known to influence VEGF pathways. However, many of the previously reported studies were done utilizing both in vitro and in vivo models, including pancreatic, liver, breast, and prostate cancer samples [14][15][16][17][18]. So far, none of the studies have assessed the role of MDM2 inhibition on MMPs expression nor its activities. ...
The Murine Double Minute 2 (MDM2) amplification or overexpression has been found in many tumors with high metastatic and angiogenic ability. Our experiments were designed to explore the impact of MDM2 overexpression, specifically on the levels of angiogenesis-related genes, which can also play a major role in tumor propagation and increase its metastatic potential. In the present study, we have used the human angiogenesis RT2 profiler PCR array to compare the gene expression profile between LNCaP and LNCaP-MST (MDM2 transfected) prostate cancer cells, along with LNCaP-MST cells treated with Nutlin-3, an MDM2 specific inhibitor. As a result of the overexpression of MDM2 gene in LNCaP-MST (10.3-fold), Thrombospondin 1 (THBS1), Tumor necrosis factor alpha (TNF-α) and Matrix metallopeptidase 9 (MMP9) were also found to be significantly up-regulated while genes such as Epiregulin (EREG), Tissue inhibitor of metalloproteinases 1 (TIMP1) were down-regulated. Also, we determined the total MMP activity and MMP9 expression in LNCaP, LNCaP-MST and SJSA-1 cells. Our results indicated that MDM2 level is positively correlated with MMP activity and MMP9 secretion. Our findings offer strong supporting evidence that MDM2 can impact growth and metastatic potential of cancer cells through tilting the balance towards pro-angiogenic mechanisms.
... Wade et al. described that a 2-fold increase in MDM2 expression level is enough to prohibit p53 activation [80]. As expected, shRNA-mediated knockdown of MDM2 suppressed proliferation rate and tumor growth potential of highly metastatic pancreatic cancer cells [81]. Under their experimental conditions, depletion of MDM2 caused an up-and down-regulation of p53 and MMP9, respectively. ...
Background:
Despite the remarkable advances in the early diagnosis and treatment, overall 5-year survival rate of patients with pancreatic cancer is less than 10%. Gemcitabine (GEM), a cytidine nucleoside analogue and ribonucleotide reductase inhibitor, is a primary option for patients with advanced pancreatic cancer; however, its clinical efficacy is extremely limited. This unfavorable clinical outcome of pancreatic cancer patients is at least in part attributable to their poor response to anti-cancer drugs such as GEM. Thus, it is urgent to understand the precise molecular basis behind the drug-resistant property of pancreatic cancer and also to develop a novel strategy to overcome this deadly disease.
Review:
Accumulating evidence strongly suggests that p53 mutations contribute to the acquisition and/or maintenance of drug-resistant property of pancreatic cancer. Indeed, certain p53 mutants render pancreatic cancer cells much more resistant to GEM, implying that p53 mutation is one of the critical determinants of GEM sensitivity. Intriguingly, runt-related transcription factor 2 (RUNX2) is expressed at higher level in numerous human cancers such as pancreatic cancer and osteosarcoma, indicating that, in addition to its pro-osteogenic role, RUNX2 has a pro-oncogenic potential. Moreover, a growing body of evidence implies that a variety of miRNAs suppress malignant phenotypes of pancreatic cancer cells including drug resistance through the down-regulation of RUNX2. Recently, we have found for the first time that forced depletion of RUNX2 significantly increases GEM sensitivity of p53-null as well as p53-mutated pancreatic cancer cells through the stimulation of p53 family TAp63/TAp73-dependent cell death pathway.
Conclusions:
Together, it is likely that RUNX2 is one of the promising molecular targets for the treatment of the patients with pancreatic cancer regardless of their p53 status. In this review article, we will discuss how to overcome the serious drug-resistant phenotype of pancreatic cancer.
... Notably, the two subgroups with extreme OS difference had opposite status of MDM2 and p53 expression. Therefore, we can assume that the balance between MDM2 and p53 mediates the tumor aggressiveness; as prior research similarly revealed, the downregulation of MDM2 in SW1990HM pancreatic adenocarcinoma cells increases levels of E-cadherin and decreases levels of matrix metallopeptidase 9 and Ki-67 [38]. Downregulation and induction of the autoubiquitination of MDM2 with SP141 inhibit pancreatic adenocarcinoma both in vitro and in vivo. ...
This study evaluated the prognostic roles of murine double minute 2 (MDM2) and p53 in pancreatic cancer patients treated with gemcitabine-based chemotherapy. A total of 137 advanced or recurrent adenocarcinoma patients who were treated with gemcitabine-based palliative chemotherapy were reviewed, selected from 957 patients with pancreatic malignancy between 2008 and 2013 at our hospital. Immunohistochemical staining for MDM2 and p53 with formalin-fixed, paraffin-embedded tumor tissues was independently reviewed. Nuclear or cytoplasmic expression of MDM2 and p53 was found in tumor cells of 30 (21.9%) and 71 (51.8%) patients, respectively. Patients with MDM2 expression had shorter median overall survival (OS) (3.7 vs 5.8 mo; P = .048) and median progression-free survival (PFS) (1.5 vs 2.5 mo; P < .001); by contrast, p53 expression was not correlated with OS or PFS. In the multivariate analysis, MDM2 expression (hazard ratio = 1.731; P = .025) was an independent and unfavorable prognostic factor of OS. Additionally, MDM2 expression was significantly associated with progressive disease (PD) and death (P = .015) following first-line gemcitabine-based therapy. In advanced pancreatic cancer patients, MDM2 expression is associated with shorter OS and PFS after gemcitabine-based chemotherapy.
... [50] Also, in human breast cancer, MDM2 expression is positively correlated with that of MMP-9, and is also negatively correlated with disease-free survival. [51] Moreover, knockdown of MDM2 in pancreatic carcinoma SW1990HM cells results in reduced MMP-9 protein expression, [52] and MDM2 promotes invasion of both MCF7 and MDA-MB-231 cell lines by increasing the MMP-9 promoter activity. [51] Although there is definite clinical and functional correlation between MMP-9 and MDM2, it remains unclear whether MDM2 induces invasion and metastasis in liver cancer through upregulation of MMP-9. ...
Hepatocellular carcinoma (HCC) has emerged as one of the most commonly diagnosed forms of human cancer; yet, the mechanisms underlying HCC progression remain unclear. Unlike other cancers, systematic chemotherapy is not effective for HCC patients, while surgical resection and liver transplantation are the most viable treatment options. Thus, identifying factors or pathways that suppress HCC progression would be crucial for advancing treatment strategies for HCC. The murine double minute 2 (MDM2)-p53 pathway is impaired in most of the cancer types, including HCC, and MDM2 is overexpressed in approximately 30% of HCC. Overexpression of MDM2 is reported to be well correlated with metastasis, drug resistance, and poor prognosis of multiple cancer types, including HCC. Importantly, these correlations are observed even when p53 is mutated. Indeed, p53-independent functions of overexpressed MDM2 in cancer progression have been suitably demonstrated. In this review article, we summarize potential effectors of MDM2 that promote or suppress cancer metastasis and discuss the p53-independent roles of MDM2 in liver cancer metastasis from clinical as well as biological perspectives.
... MMPs are believed to be the main contributor to the invasiveness of certain cancers by giving it the ability to degrade the tissues surrounding it (Duffy et al., 2000;Xiaofeng et al., 2013). MMPs are expressed due to the presence of mouse double minute 2 (MDM2) which have been repeatedly found to slow the proliferation of the many cancers when inhibited (Coll-Mulet et al., 2006;Gu et al., 2008;Kojima et al., 2006;Kojima et al., 2005;Shi et al., 2014;Tabe et al., 2009;Tovar et al., 2006;Vassilev et al., 2004;Watanabe et al., 1994;Watanabe et al., 1996;Xiaofeng et al., 2013). One such study that provides a little light is one performed by Hiroshi Maeda in 1998 that described how bacterial infections induce human MMP activation by insults involving proteases and free radicals (Maeda et al., 1998). ...
Utilization of environmental stimuli for growth is the main factor contributing to the evolution of prokaryotes and eukaryotes, independently and mutualistically. Epigenetics describes an organism's ability to vary expression of certain genes based on their environmental stimuli. The diverse degree of dose-dependent responses based on their variances in expressed genetic profiles makes it difficult to ascertain whether hormesis or oncogenesis has or is occurring. In the medical field this is shown where survival curves used in determining radiotherapeutic doses have substantial uncertainties, some as large as 50% (Barendsen, 1990). Many in-vitro radiobiological studies have been limited by not taking into consideration the innate presence of microbes in biological systems, which have either grown symbiotically or pathogenically. Present in-vitro studies neglect to take into consideration the varied responses that commensal and opportunistic pathogens will have when exposed to the same stimuli and how such responses could act as stimuli for their macro/microenvironment. As a result many theories such as radiation carcinogenesis explain microscopic events but fail to describe macroscopic events (Cohen, 1995). As such, this review shows how microorganisms have the ability to perturb risks of cancer and enhance hormesis after irradiation. It will also look at bacterial significance in the microenvironment of the tumor before and during treatment. In addition, bacterial systemic communication after irradiation and the host's immune responses to infection could explain many of the phenomena associated with bystander effects. Therefore, the present literature review considers the paradigms of hormesis and oncogenesis in order to find a rationale that ties them all together. This relationship was thus characterized to be the microbiome.
Copyright © 2015 Elsevier Inc. All rights reserved.
... TNBC shares clinical and pathologic features with hereditary BRCA1-related breast cancers (29). MDM2 is an important negative regulator of TP53 and has been shown to be integral to metastasis and tumor growth (30). Moreover studies have shown that inhibition of MDM2 reduced systemic inflammation and reduced autoantibody production (31). ...
The repertoire of antigens associated with the development of an autoimmune response in breast cancer has relevance to detection and treatment strategies. We have investigated the occurrence of autoantibodies associated with the development of triple negative breast cancer (TNBC) in the prior to diagnosis setting and in samples collected at the time of diagnosis of TNBC. Lysate arrays containing protein fractions from the TNBC MDA-MB-231 cell line were hybridized with TNBC plasmas from the Women's Health Initiative cohort, collected prior to clinical diagnosis and with plasmas from matched controls. An immune response directed against spliceosome and glycolysis proteins was observed with case plasmas as previously reported in ER+ breast cancer. Importantly autoantibodies directed against networks involving BRCA1, TP53 and cytokeratin proteins associated with a mesenchymal/basal phenotype were distinct to TNBC prior to diagnosis samples. Concordant autoantibody findings were observed with mouse plasma samples collected prior to occurrence of palpable tumors from a C3(1)-T triple negative mouse model. Plasma samples collected at the time of diagnosis of stage II TNBC and from matched healthy controls were subjected to proteomic analysis by mass spectrometry to identify Ig bound proteins yielding a predominance of cytokeratins including several associated with a mesenchymal/basal phenotype among cases compared to controls. Our data provide evidence indicative of a dynamic repertoire of antigens associated with a humoral immune response reflecting disease pathogenesis in TNBC.
Copyright © 2015, American Association for Cancer Research.
... A mouse xenograft tumor model that overexpressed beta-arrestin, a known regulator of MDM2 [33], showed increased MMP9 activity with more aggressive tumors [34]. Furthermore, in vitro studies using pancreatic cell lines showed a direct link between MDM2 downregulation and the suppression of MMP9 expression [35]. ...
The molecular mechanisms that underpin invasive ductal breast cancer (IDC) invasion and metastasis are incompletely understood. The oncogene, mouse double minute 2 (MDM2), has been implicated in the pathogenesis of numerous cancers, where it stimulates the expression of matrix metalloproteinase 9 (MMP9), an important enzyme in the breakdown of the extracellular matrix. However, its role in breast cancer remains poorly understood. This study assessed the clinical significance of MDM2 expression in IDC and used in vitro expression assays to determine the molecular roles of MDM2. Immunohistochemical staining for MMP9 and MDM2 was performed using archived tumor blocks from 321 women who underwent surgical resection for IDC at the First Affiliated Hospital of Nanjing Medical University, China between January 2002 and December 2003. MCF-7 and MDA-MD-231 cell lines were transfected with siRNA targeted against MDM2, or MDM2 was overexpressed using transiently expressed vectors. The invasion, cell migration and proteolytic capabilities of cells that over- or underexpressed MDM2 was then assessed and compared against control cells, in addition to the consequent effects on MMP9 expression using RT-PCR. In vivo, 54.9% and 49.6% of samples were positive for MMP9 and MDM2 expression, respectively, and their expression was significantly correlated (r(2) = 0.171, P = 0.012). Moreover, MDM2 expression was markedly correlated with disease-free survival (HR 2.56, 95% CI 1.02-6.40, P = 0.038). In vitro, MDM2 overexpression significantly enhanced cell invasion, migration and proteolysis compared with control cells, and the converse effects were observed after MDM2-siRNA treatment. MDM2 overexpression induced MMP9 expression in a dose-dependent manner. Taken together, these results suggest that high levels of MDM2 are associated with a poorer prognosis in IDC. This might result from increased tumor invasiveness due to enhanced MMP9 expression causing increased extracellular matrix breakdown.
Fine particulate matter less than 2.5 micrometers in diameter (PM2.5) is regarded as one of carcinogenic factors, but the mechanism is left unexplored nowadays. Our goal was to reveal the carcinogenic mechanism at gene and protein level under the inhalational air-liquid interface (ALI) condition. Herein, we developed an ALI organ-level lung microfluidic platform (ALI-OLMP) carrying lung epithelial cell line BEAS-2B and human pulmonary microvascular endothelial cells (HPMEC), the cell viability was above 98% within 14 days on this system, which was used to mimic the practical alveolar microenvironment for the multi-omics analysis, to identify the global genes and proteins expression after exposure to PM2.5 in Shanghai, China from 2014 to 2015. The combined RNA-Seq and iTRAQ analysis indicated that the same set unique was 2532 genes at 10 ug/cm2 of PM2.5, and there were also at least 25 same identical activated signal transduction cascades including bladder cancer, transcriptional dysregulation in cancer, the TP53 (p53) signaling pathway, Jak-STAT signaling pathway, and PI3K-Akt signaling pathway, which could lead to the block of differentiation, cell proliferation and survival, and sustained angiogenesis. The images obtained by the transmission electron microscopy (TEM) showed that the particles could enter the mitochondria, and even get into the nucleus. Pearson’s correlation coefficient test elucidated that inorganics (EC), organics (OC, PAHs and Alkane), and metals (Cr, Mn and Sb) were significantly correlated to the dysregulated oncoproteins (VEGF, IL6, MDM2, AKT1, STAT, and P53). The findings may to some extent explore the molecular mechanism of carcinogenicity caused by fine-particle exposure.
Pancreatic cancer shows a remarkable preference for the liver to establish secondary tumors. Selective metastasis to the liver is attributed to the development of potential microenvironment for the survival of pancreatic cancer cells. This review aims to provide a full understanding of the hepatic metastatic process from circulating pancreatic cancer cells to their settlement in the liver, serving as a basic theory for efficient prediction and treatment of metastatic diseases.
A systematic search of relevant original articles and reviews was performed on PubMed, EMBASE and Cochrane Library for the purpose of this review.
Three interrelated phases are delineated as the contributions of the interaction between pancreatic cancer cells and the liver to hepatic metastasis process. Chemotaxis of disseminated pancreatic cancer cells and simultaneous defensive formation of platelets or neutrophils facilitate specific metastasis toward the liver. Remodeling of extracellular matrix and stromal cells in hepatic lobules and angiogenesis induced by proangiogenic factors support the survival and growth of clinical micrometastasis colonizing the liver. The bimodal role of the immune system or prevalence of cancer cells over the immune system makes metastatic progression successfully proceed from micrometastasis to macrometastasis.
Pancreatic cancer is an appropriate research object of cancer metastasis representing more than a straight cascade. If any of the successive or simultaneous phases, especially tumor-induced immunosuppression, is totally disrupted, hepatic metastasis will be temporarily under control or even cancelled forever. To shrink cancers on multiple fronts and prolong survival for patients, novel oral or intravenous anti-cancer agents covering one or different phases of metastatic pancreatic cancer are expected to be integrated into innovative strategies on the premise of safety and efficacious biostability.
To study the expression of Gli1, MDM2 and p53 for clinical significance in pancreatic cancer (PC), and their functional relationship in regulating the biological behaviour of PC cells.
Immunohistochemistry showed that the expression of Gli1, MDM2 and p53 was much higher in 57 cases of PC than in paired normal pancreatic tissues, and was positively associated with tumour UICC stage and T stage (P < 0.05). Patients with expression of Gli1 only or coexpression of Gli1 and MDM2 had significantly worse overall survival than patients with negative expression (P < 0.05). RNA interference showed that p53 knockdown increased the protein level of Gli1 but decreased the level of MDM2, and enhanced cell invasion and migration in wild-type p53 Capan-2 cells; whereas Gli1 or MDM2 knockdown did not change p53 expression, but decreased the protein level of MDM2 or Gli1, respectively, and inhibited cell invasion and migration in mutant p53 PANC-1 cells.
Overexpression of Gli1, MDM2 and mutant p53 contributes to the development and progression of PC, and plays an important role in predicting PC patients' prognosis. Moreover, we report a positive association between Gli1 and MDM2 in PC cells, but their relationship with p53 is dependent on wild-type or mutant p53 status.
The present study is the first to show in pancreatic cancer (PC) the growth inhibition and apoptosis by novel MDM2 inhibitors (MI-319 & 219) through reactivation of p53 pathway. Our results highlight two new secondary targets of MDM2 inhibitor 'SIRT1' and Ku70. SIRT1 which has a role in ageing and cancer and is known to regulate p53 signaling through acetylation. Ku70 is a key component of non-homologous end joining machinery in the DNA damage pathway and is known to regulate apoptosis by blocking Bax entry into mitochondria. Growth inhibition and apoptosis by MI-219, MI-319 was accompanied by increase in levels of p53 along with p21(WAF1) and the proapoptotic Puma. SiRNA against p21(WAF1) abrogated the growth inhibition of PC cells confirming p21(WAF1) as a key player downstream of activated p53. Immunoprecipitation-western blot analysis revealed reduced association of MDM2-p53 interaction in drug exposed PC cells. In combination studies, the inhibitors synergistically augmented anti-tumor effects of therapeutic drug gemcitabine both in terms of cell growth inhibition as well as apoptosis. Surface plasmon resonance studies confirmed strong binding between MI-319 and Ku70 (K(D) 170 nM). Western blot revealed suppression of SIRT1 and Ku70 with simultaneous upregulation of acetyl-p53 (Lys379) and Bax. Co-Immunoprecipitation studies confirmed that MI-319 could disrupt Ku70-Bax and SIRT1-Bax interaction. Further, using wt-p53 xenograft of Capan-2, we found that oral administration of MI-319 at 300 mg/kg for 14 days resulted in significant tumor growth inhibition without any observed toxicity to the animals. No tumor inhibition was found in mut-p53 BxPC-3 xenografts. In light of our results, the inhibitors of MDM2 warrant clinical investigation as new agents for PC treatment.
Tumor Protein p53 (p53), cyclin-dependent kinase inhibitor 1A (p21/WAF1), and murine double minute 2 (MDM2) participate in the regulation of cell growth. Altered expression of these gene products has been found in malignant tumors and has been associated with poor prognosis. Our aim was to investigate the expression of the 3 proteins in hepatocellular carcinoma (HCC) and their prognostic significance.
We examined p53, p21/WAF1, and MDM2 expression in 181 pairs of HCC tissues and the adjacent hepatic tissues by performing immunohistochemistry and examined the expression of the 3 proteins in 7 pairs of HCC tissues and the adjacent hepatic tissues by using western blot analysis.
The expression of p53, p21/WAF1, and MDM2 in the HCC tissues was significantly higher than those in the adjacent hepatic tissues (P < 0.05). A statistical correlation was observed between p53 and p21/WAF1 expression in HCC tissues (R = 0.195, P = 0.008). A statistical correlation was observed between expression of p53 and p21/WAF1 (R = 0.380, P = 0.000), p53 and MDM2 (R = 0.299, P = 0.000), p21/WAF1 and MDM2 (R = 0.285, P = 0.000) in 181 liver tissues adjacent to the tumor. Patients with a low pathologic grade HCC (I+II) had a higher tendency to express p53 on tumor cells than the patients with high pathologic grade HCC (III+IV) (P = 0.007). Survival analysis showed that positive p21/WAF1 expression or/and negative MDM2 expression in HCC was a predictor of better survival of patients after tumor resection (P < 0.05).
The proteins p53, p21/WAF1, and MDM2 were overexpressed in all the HCC cases in this study, and p53 and p21/WAF1 overexpression were positively correlated. The expression of p21/WAF1 and MDM2 can be considered as 2 useful indicators for predicting the prognosis of HCC.
The E3 ubiquitin ligase human murine double minute (HDM2) is overexpressed in 40%-80% of late-stage metastatic cancers in the absence of gene amplification. Hdm2 regulates p53 stability via ubiquitination and has also been implicated in altering the sensitivity of cells to TGF-beta1. Whether TGF-beta1 signaling induces Hdm2 expression leading to HDM2-mediated destabilization of p53 has not been investigated. In this study, we report that TGF-beta1-activated SMA- and MAD3 (Smad3/4) transcription factors specifically bound to the second promoter region of HDM2, leading to increased HDM2 protein expression and destabilization of p53 in human cancer cell lines. Additionally, TGF-beta1 expression led to Smad3 activation and murine double minute 2 (Mdm2) expression in murine mammary epithelial cells during epithelial-to-mesenchymal transition (EMT). Furthermore, histological analyses of human breast cancer samples demonstrated that approximately 65% of late-stage carcinomas were positive for activated Smad3 and HDM2, indicating a strong correlation between TGF-beta1-mediated induction of HDM2 and late-stage tumor progression. Identification of Hdm2 as a downstream target of TGF-beta1 represents a critical prosurvival mechanism in cancer progression and provides another point for therapeutic intervention in late-stage cancer.
The MDM2 oncogene is overexpressed in various human cancers. Its expression correlates with the phenotypes of high-grade, late-stage, and more resistant tumors. The auto-regulatory loop between MDM2 and the tumor suppressor p53 has long been considered the epitome of a rational target for cancer therapy. As such, many novel agents have been generated to interfere with the interaction of the two proteins, which results in the activation of p53. Among these agents are several small molecule inhibitors synthesized based upon the crystal structures of the MDM2-p53 complex. With use of high-throughput screening, several specific and effective agents for inhibition of the protein-protein interaction were discovered. Recent investigations, however, have demonstrated that many proteins regulate the MDM2-p53 interaction, and that MDM2 may have p53-independent oncogenic functions. In order for novel MDM2 inhibitors to be translated to the clinic, it is necessary to obtain a better understanding of the regulation of MDM2 and of the MDM2-p53 interaction. In particular, the implications of various interactions between certain regulator(s) and MDM2/p53 under different circumstances need to be elucidated to determine which pathway(s) represent the best targets for therapy. Targeting both MDM2 itself and regulators of MDM2 and the MDM2-p53 interaction, or use of MDM2 inhibitors in combination with conventional treatments, may improve prospects for tumor eradication.
Purpose
MDM2 regulates p53, which controls cell cycle arrest and apoptosis. Both proteins, along with Ki-67, which is an established strong determinant of metastasis, have shown promise in predicting the outcome of men treated with radiation therapy (RT) with or without short-term androgen deprivation (STAD). This report compares the utility of abnormal expression of these biomarkers in estimating progression in a cohort of men treated on RTOG 92-02.
Patients and Methods
Adequate tissue for immunohistochemistry was available for p53, Ki-67, and MDM2 analyses in 478 patient cases. The percentage of tumor nuclei staining positive (PSP) was quantified manually or by image analysis, and the per-sample mean intensity score (MIS) was quantified by image analysis. Cox regression models were used to estimate overall mortality (OM), and Fine and Gray's regressions were applied to the end points of distant metastasis (DM) and cause-specific mortality (CSM).
Results
In multivariate analyses that adjusted for all markers and treatment covariates, MDM2 overexpression was significantly related to DM (P = .02) and OM (P = .003), and Ki-67 overexpression was significantly related to DM (P < .0001), CSM (P = .0007), and OM (P = .01). P53 overexpression was significantly related to OM (P = .02). When considered in combination, the overexpression of both Ki-67 and MDM2 at high levels was associated with significantly increased failure rates for all end points (P < .001 for DM, CSM, and OM).
Conclusion
Combined MDM2 and Ki-67 expression levels were independently related to distant metastasis and mortality and, if validated, could be considered for risk stratification of patients with prostate cancer in clinical trials.
Tumor hypoxia has been reported to be a negative prognostic factor in a number of tumor sites. Both clinical and experimental studies have suggested a positive correlation between tumor hypoxia and increased metastatic efficiency; however, the mechanisms are not understood. In this study, the mechanisms of hypoxia-enhanced metastasis have been investigated in murine KHT fibrosarcoma and SCC VII cells. We have observed that hypoxia-pretreated KHT-C cells have a higher survival rate than control KHT-C cells after being arrested in mouse lungs. cDNA microarray analysis revealed many hypoxia-regulated genes, most of which have been reported to be involved in cell survival and growth. Among these genes, we have confirmed the up-regulation of Mdm2 by hypoxia and have demonstrated that this up-regulation is p53 independent. The up-regulation of Mdm2 by hypoxia is associated with decreased p53 protein and inhibition of the transactivation of p53 downstream proapoptotic genes. Overexpression of Mdm2 or suppression of p53 by transient transfection increased metastatic efficiency in KHT-C cells. These data suggest that hypoxia can increase tumor cell metastatic efficiency by rendering the tumor cells less sensitive to stress-induced cell death, e.g., through modifying the levels of Mdm2 and p53.
Gene amplification and protein overexpression of MDM2, which is often found in certain types of cancers, indicate that MDM2 plays an important role in tumorigenesis. Interestingly, several clinical reports have demonstrated that amplification of the MDM2 gene correlates with the metastatic stage. Using an antibody array assay, we identified E-cadherin as an MDM2-binding protein and confirmed that E-cadherin is a substrate for the MDM2 E3 ubiquitin ligase. We demonstrate that MDM2 interacts in vivo with E-cadherin, resulting in its ubiquitination and degradation. This regulation appears to be clinically relevant, as we found a significant correlation between high MDM2 and low E-cadherin protein levels in resected tumor specimens recovered from breast cancer patients with lymph node metastases. Ectopic expression of MDM2 in breast cancer cells was found to disrupt cell-cell contacts and enhance cell motility and invasive potential. We found that E-cadherin and MDM2 colocalized on the plasma membrane and in the early endosome, where ubiquitin moieties were attached to E-cadherin. Blocking endocytosis with dominant-negative mutants of dynamin abolished the association of MDM2 with E-cadherin, prevented E-cadherin degradation, and attenuated cell motility as observed by fluorescence microscopy. Thus, we provide evidence to support a novel role for MDM2 in regulating cell adhesions by a mechanism that involves degrading and down-regulating the expression of E-cadherin via an endosome pathway. This novel MDM2-regulated pathway is likely to play a biologically relevant role in cancer metastasis.
Atypical lipomatous tumours (ALTs) represent a distinctive subset of mesenchymal neoplasms featuring mature adipocytic differentiation. Most ALTs are characterized cytogenetically by the presence of supernumerary ring and/or long marker chromosomes derived from the chromosomal region 12q13–15. The 12q13–15 chromosome region contains several genes which may play an important role in human tumorigenesis. A series of ALTs was analysed by investigating the MDM2, CDK4, and HMGI‐C genes and their proteins. The study was extended to a series of ordinary lipomas, to determine whether the immunohistochemical investigation of these gene products might play any diagnostic role. Cytogenetic analysis revealed the presence of various cytogenetic aberrations involving the 12q13–15 region in 11/18 (61%) lipomas and of ring chromosomes in all ALTs. Overexpression of mdm2 protein was observed in 6/12 (50%) atypical lipomatous tumours. All lipomas were mdm2‐negative. cdk4 overexpression was present in 100% of ALTs. Weak cdk4 immunopositivity was detected in 2/18 (11%) ordinary lipomas in a minority of cells. HMGI‐C immunopositivity was observed in 10/12 (83%) ALTs. Positive immunoreactivity was also observed in 8/18 (44%) lipomas. Southern blot analysis revealed amplification of the CDK4 and MDM2 genes in 3/5 ALTs analysed. HMGI‐C was amplified in 3/5 cases and was deleted in one case. Mutation analysis of the CDK4 gene did not demonstrate any mutation. These data support the hypothesis that ordinary lipomas may form a molecular genetic and morphological continuum with ALT. At one end of the spectrum are lipomas characterized by 12q13–15 rearrangements and HMGI‐C activation and at the other end are ALTs with ring chromosomes, 12q13–15 amplification with overrepresentation of the HMGI‐C, CDK4 or MDM2 genes, and aberrant cdk4, mdm2, and HMGI‐C protein expression. These findings not only provide insights into the molecular pathogenesis of lipomatous tumours, but also indicate that the immunohistochemical analysis of mdm2 and cdk4 may help to increase diagnostic accuracy. Copyright © 2000 John Wiley & Sons, Ltd.
Atypical lipomatous tumours (ALTs) represent a distinctive subset of mesenchymal neoplasms featuring mature adipocytic differentiation. Most ALTs are characterized cytogenetically by the presence of supernumerary ring and/or long marker chromosomes derived from the chromosomal region 12q13–15. The 12q13–15 chromosome region contains several genes which may play an important role in human tumorigenesis. A series of ALTs was analysed by investigating the MDM2, CDK4, and HMGI-C genes and their proteins. The study was extended to a series of ordinary lipomas, to determine whether the immunohistochemical investigation of these gene products might play any diagnostic role. Cytogenetic analysis revealed the presence of various cytogenetic aberrations involving the 12q13–15 region in 11/18 (61%) lipomas and of ring chromosomes in all ALTs. Overexpression of mdm2 protein was observed in 6/12 (50%) atypical lipomatous tumours. All lipomas were mdm2-negative. cdk4 overexpression was present in 100% of ALTs. Weak cdk4 immunopositivity was detected in 2/18 (11%) ordinary lipomas in a minority of cells. HMGI-C immunopositivity was observed in 10/12 (83%) ALTs. Positive immunoreactivity was also observed in 8/18 (44%) lipomas. Southern blot analysis revealed amplification of the CDK4 and MDM2 genes in 3/5 ALTs analysed. HMGI-C was amplified in 3/5 cases and was deleted in one case. Mutation analysis of the CDK4 gene did not demonstrate any mutation. These data support the hypothesis that ordinary lipomas may form a molecular genetic and morphological continuum with ALT. At one end of the spectrum are lipomas characterized by 12q13–15 rearrangements and HMGI-C activation and at the other end are ALTs with ring chromosomes, 12q13–15 amplification with overrepresentation of the HMGI-C, CDK4 or MDM2 genes, and aberrant cdk4, mdm2, and HMGI-C protein expression. These findings not only provide insights into the molecular pathogenesis of lipomatous tumours, but also indicate that the immunohistochemical analysis of mdm2 and cdk4 may help to increase diagnostic accuracy. Copyright © 2000 John Wiley & Sons, Ltd.
Nutlin-3, a selective small-molecule inhibitor of the p53-MDM2 interaction, has been shown to have antitumor activities in various tumors with wild-type p53. However, its effect on hepatocellular carcinoma (HCC) with different types of p53 remains unclear. This study is designed to determine nutlin-3's antitumor efficacy and underlying mechanisms of action in human HCC cells.
Cell viability assay, cell cycle analysis, apoptosis assay, western blot, co- immunoprecipitation and siRNA experiments were analyzed in three human HCC cells. Anti-tumoral effects of nutlin-3 targeting the p53 and p73 pathways were evaluated in HCC cell lines.
Nutlin-3 exerted the greatest anti-tumoral effect to three human HCC cells with wild-type p53, mutant p53 and p53-null. Nutlin-3 not only upregulated p53 in HepG2 cells, but also p73 in Huh7 and Hep3B cells, and disrupted p53-MDM2 and p73-MDM2 complexes in HCC cells. The compound inhibited cell proliferation, induced G0/G1 phase arrest, decreased the levels of CyclinD1, CyclinE, CDK2, CDK4, PCNA and E2F-1, and increased the levels of p21 and p27. It also induced apoptosis, increased the Bax/Bcl-2 ratio, then activated caspase-9 and caspase-3.
Nutlin-3 has significant anticancer effects against human HCC cells, regardless of p53 status, indicating that it is a promising therapy for human hepatocellular carcinoma.
Antibody microarrays have been successfully used to determine relative abundance of key proteins in various cancers and other diseases. We have previously showed liver metastatic-related genes between the metastatic pancreatic cancer line (SW1990HM) and its parental line (SW1990). In this study, we searched for potential markers for metastatic progression using antibody microarrays. The SpringBio Antibody Microarrays were used to analysis the different proteomes between SW1990HM and SW1990 cells. A standard ≥2.0-fold cutoff value was used to determine differentially expressed proteins and Western blotting analysis further confirmed the results. Antibody microarrays revealed that 40 proteins were reproducibly altered more than 2-fold between the selected variant and its parental counterpart; 14 of the proteins were up-regulated, and 26 were down-regulated. Most of the up-regulated proteins (7/14) play a role in tumor signal transduction, while a number of down-regulated proteins (10/26) function in cell differentiation; this might be crucial for pancreatic cancer metastasis. Four dysregulated proteins were validated by western blotting in the cell lines. Interestingly, the up-regulation of Glucagon and down-regulation of Prolactin were further confirmed in the culture supernatants by western blotting. These proteomic data are valuable for understanding pancreatic cancer metastasis and searching for potential markers of metastatic progression.
Renal cell carcinoma (RCC) is the most common type of kidney cancer and follows an unpredictable disease course. To improve prognostication, a better understanding of critical genes associated with disease progression is required. The objective of this review was to focus attention on 2 such genes, p53 and murine double minute 2 (MDM2), and to provide a comprehensive summary and critical analysis of the literature regarding these genes in RCC. Information was compiled by searching the PubMed database for articles that were published or e-published up to April 1, 2009. Search terms included renal cancer, renal cell carcinoma, p53, and MDM2. Full articles and any supplementary data were examined; and, when appropriate, references were checked for additional material. All studies that described assessment of p53 and/or MDM2 in renal cancer were included. The authors concluded that increased p53 expression, but not p53 mutation, is associated with reduced overall survival/more rapid disease progression in RCC. There also was evidence that MDM2 up-regulation is associated with decreased disease-specific survival. Two features of RCC stood out as unusual and will require further investigation. First, increased p53 expression is tightly linked with increased MDM2 expression; and, second, patients who have tumors that display increased p53 and MDM2 expression may have the poorest overall survival. Because there was no evidence to support the conclusion that p53 mutation is associated with poorer survival, it seemed clear that increased p53 expression in RCC occurs independent of mutation. Further investigation of the mechanisms leading to increased p53/MDM2 expression in RCC may lead to improved prognostication and to the identification of novel therapeutic interventions.
Each year, the American Cancer Society estimates the number of new cancer cases and deaths expected in the United States in the current year and compiles the most recent data on cancer incidence, mortality, and survival based on incidence data from the National Cancer Institute, Centers for Disease Control and Prevention, and the North American Association of Central Cancer Registries and mortality data from the National Center for Health Statistics. Incidence and death rates are standardized by age to the 2000 United States standard million population. A total of 1,479,350 new cancer cases and 562,340 deaths from cancer are projected to occur in the United States in 2009. Overall cancer incidence rates decreased in the most recent time period in both men (1.8% per year from 2001 to 2005) and women (0.6% per year from 1998 to 2005), largely because of decreases in the three major cancer sites in men (lung, prostate, and colon and rectum [colorectum]) and in two major cancer sites in women (breast and colorectum). Overall cancer death rates decreased in men by 19.2% between 1990 and 2005, with decreases in lung (37%), prostate (24%), and colorectal (17%) cancer rates accounting for nearly 80% of the total decrease. Among women, overall cancer death rates between 1991 and 2005 decreased by 11.4%, with decreases in breast (37%) and colorectal (24%) cancer rates accounting for 60% of the total decrease. The reduction in the overall cancer death rates has resulted in the avoidance of about 650,000 deaths from cancer over the 15-year period. This report also examines cancer incidence, mortality, and survival by site, sex, race/ethnicity, education, geographic area, and calendar year. Although progress has been made in reducing incidence and mortality rates and improving survival, cancer still accounts for more deaths than heart disease in persons younger than 85 years of age. Further progress can be accelerated by applying existing cancer control knowledge across all segments of the population and by supporting new discoveries in cancer prevention, early detection, and treatment.
Pancreatic cancer with liver metastases has a poor prognosis and the molecular mechanisms remain unclear. In this study, SW1990HM, a highly metastatic human pancreatic carcinoma line was subcloned from SW1990 by intrasplenic injection. In vivo and in vitro tumorigenicity, metastatic potential, in vitro invasion, cell growth curves, plate efficiency and S-phase cell numbers were higher in SW1990HM cells. Gene expression profiles of SW1990HM and SW1990 cells showed 40 metastasis-related genes expressed with a 3-fold difference. Thirteen of these 32.5% (13/40) were adhesion and extracellular-matrix related and twelve 30% (12/40) were cell growth and proliferation related, such as MMP10, MMP9, MMP7, CDH1, MGAT5, CTNNA1, IGF1, IL8RB, ITGA7, MDM2, MET, SSTR2 and VEGF, which were related to the onset and progression of tumor metastasis. Thus, SW1990HM is an attractive model to study metastasis and identify potential therapeutic targets.
We have reconstituted a matrix of basement membrane onto a filter in a Boyden chamber and assessed the ability of various malignant and nonmalignant cells to penetrate through the coated filter. Cells from all the malignant cell lines tested were able to cross the matrix in 5-6 h, whereas human fibroblasts as well as mouse 3T3 and 10T1/2 cell lines, which are not tumorigenic, were not invasive. In addition, normal primary prostate epithelial cells and benign prostatic hyperplasia cells were not invasive when tested in this assay, whereas malignant prostate carcinoma cells were highly invasive. Parallel experiments with these prostatic cells using the intrasplenic assay for metastasis detection in the nude mouse confirmed the benign behavior of the former cells and the metastatic phenotype of the latter ones. These results suggest that this in vitro test allows the rapid and quantitative assessment of invasiveness and a means to screen for drugs which alter the invasive phenotype of tumor cells.
Inactivation of tumour-suppressor genes leads to deregulated cell proliferation and is a key factor in human tumorigenesis. Both p53 and retinoblastoma genes are frequently mutated in human cancers, and the simultaneous inactivation of RB and p53 is frequently observed in a variety of naturally occurring human tumours. Furthermore, three distinct DNA tumour virus groups--papovaviruses, adenoviruses and human papillomaviruses--transform cells by targeting and inactivating certain functions of both the p53 and retinoblastoma proteins. The cellular oncoprotein, Mdm2, binds to and downmodulates p53 function; its human homologue, MDM2, is amplified in certain human tumours, including sarcomas and gliomas. Overproduction of Mdm2 is both tumorigenic and capable of immortalizing primary rat embryo fibroblasts. Here we show that MDM2 interacts physically and functionally with pRB and, as with p53, inhibits pRB growth regulatory function. Therefore, both pRB and p53 can be subjected to negative regulation by the product of a single cellular protooncogene.
The MDM2 proto-oncogene is found amplified in a variety of tumours. The oncogenic capacity of the MDM2 protein is attributed to its ability to bind the p53 tumour-suppressor protein and mask its transcriptional activation potential. Here we show that MDM2 makes a functional contact with two cooperating transcription factors, E2F1 and DP1 (refs 4,5), which are involved in S-phase progression. MDM2 contacts the activation domain of E2F1 using residues conserved in the activation domain of p53. However, in contrast to its repression of p53 activity, MDM2 stimulates the activation capacity of E2F1/DP1. These results indicate that MDM2 not only releases a proliferative block by silencing the tumour suppressor p53, it also positively augments proliferation by stimulating the S-phase inducing transcription factors E2F1/DP1.
The human homologue of the murine double minute 2 gene (MDM2), a p53-binding protein which may act as a regulator of p53 protein function, has recently been cloned. Initial studies of this gene in a variety of human tumors have shown frequent gene amplification in most types of sarcomas, including osteosarcomas. Amplification of the MDM2 gene may produce a functional inactivation of the p53 protein. To examine possible clinical or pathological correlates of MDM2 gene amplification in osteosarcoma, we studied 28 specimens on 26 patients with high grade osteosarcoma (16 primary, 11 metastatic, and 1 local recurrence) for MDM2 gene amplification by Southern blot analysis, using two MDM2 complementary DNA probes isolated by polymerase chain reaction. Four specimens (14%) showed amplification, including 3 metastases and 1 local recurrence. None of the primary osteosarcoma specimens had detectable MDM2 gene amplification. None of the specimens tested showed MDM2 gene rearrangement. In the present series, MDM2 gene amplification was detected significantly more frequently in metastatic or recurrent osteosarcomas than it was in primary osteosarcomas (P = 0.02). Our data suggest that MDM2 gene amplification may be associated with tumor progression and metastasis in osteosarcoma. Further investigation is warranted on the potential clinicopathological correlates of MDM2 gene amplification in osteosarcoma.
The human oncoprotein MDM2 (hMDM2) overexpresses in various human tumors. If amplified, the mdm2 gene can enhance the tumorigenic potential of murine cells. Here, we present evidence to show that the full-length human or mouse MDM2 expressed from their respective cDNA can inhibit the G0/G1-S phase transition of NIH 3T3 and normal human diploid cells. The protein harbors more than one cell-cycle-inhibitory domain that does not overlap with the p53-interaction domain. Deletion mutants of hMDM2 that lack the cell-cycle-inhibitory domains can be stably expressed in NIH 3T3 cells, enhancing their tumorigenic potential. The tumorigenic domain of hMDM2 overlaps with the p53-interaction domain. Some tumor-derived cells, such as Saos-2, H1299 or U-2OS, are relatively insensitive to the growth-inhibitory effects of hMDM2. These observations suggest that hMDM2 overexpression in response to oncogenic stimuli would induce growth arrest in normal cells. Elimination or inactivation of the hMDM2-induced G0/G1 arrest may contribute to one of the steps of tumorigenesis.
The endothelium is one of the largest cellular compartments of the human body and has a high proliferative potential. However, angiosarcomas are among the rarest malignancies. Despite this interesting contradiction, data on growth and angiogenesis control mechanisms of angiosarcomas are scarce. In this study of 19 angiosarcomas and 10 benign vascular control lesions we investigated the sequence and expression of the p53 tumor suppressor gene and the expression of the mdm-2 proto-oncogene, which is a negative regulator of p53 activity and of the vascular endothelial growth factor (VEGF), whose expression, among other factors, is regulated by the p53/MDM-2 pathway. Ten sarcomas (53%) exhibited clear nuclear p53 protein accumulation. Two of these cases revealed mutations in the sequence-specific DNA binding domain of the p53 gene. Thirteen angiosarcomas (68%) showed an increased amount of MDM-2 protein. Elevated expression of p53 and MDM-2 protein correlated with increased VEGF expression, which was found in nearly 80% of the angiosarcoma cases. Negative or clearly lower immunostaining was obtained in cases from the benign control collective. Only one case of a juvenile hemangioma reached the cutoff value of p53 positivity coincidentally with high VEGF expression. Our data suggest that the p53/ MDM-2 pathway is impaired in about two-thirds (14/ 19) of the angiosarcomas. This may be a key event in the pathogenesis of human angiosarcomas. The increased VEGF expression observed supports this hypothesis.
Pancreatic ductal adenocarcinoma is an aggressive and devastating disease, which is characterized by invasiveness, rapid progression and profound resistance to treatment. Advances in pathological classification and cancer genetics have improved our descriptive understanding of this disease; however, important aspects of pancreatic cancer biology remain poorly understood. What is the pathogenic role of specific gene mutations? What is the cell of origin? And how does the stroma contribute to tumorigenesis? A better understanding of pancreatic cancer biology should lead the way to more effective treatments.
Mouse double minute 2 (MDM2) is a key negative regulator of the p53 activity. Recently, a polymorphism in the MDM2 intronic promoter, SNP309, was shown to influence MDM2 expression and p53 activity. We examined whether the SNP309 was related to the risk of developing nasopharyngeal carcinoma (NPC) among Chinese populations.
We genotyped the SNP309 in two independent case-control populations in southern China, one is from Guangxi province (including 593 NPC patients and 480 controls) and the other is from Guangdong province (including 239 patients and 286 controls), by PCR direct sequencing. Multivariate logistic regression analysis was used to calculate adjusted odds ratio (OR) and 95% confidence interval (95% CI).
We observed that compared with the TT genotype, the genotypes containing G allele (GT + GG genotype) were associated with significant increased susceptibility to NPC in both Guangxi (OR, 1.43; 95% CI, 1.04-1.91) and Guangdong population (OR, 1.53; 95% CI, 1.00-2.36). When these two sample sets were combined, the OR of the GT + GG genotype developing NPC was 1.45 (95% CI, 1.12-1.85) compared with the TT genotype. Furthermore, compared with the TT genotype, the GT + GG genotype was also significantly associated with the advanced lymph node metastasis (OR, 1.84; 95% CI, 1.09-3.05).
Our findings suggest that the MDM2 SNP309 may be a risk factor for the occurrence and advanced neck lymph node metastasis of NPC in Chinese population.
Several studies have reported that p53/mdm2 distortions play a pivotal role in the development and progression of various human malignancies. However, the number of reports having evaluated simultaneously the components of the P53-pathway alterations in advanced-stage human endometrial carcinomas (EC) is low. In this study, we examined the expression of P53/MDM2 proteins in primary and metastatic ECs, and analyzed the clinicopathological characteristics as well as the survival outcome of patients in relation to P53/MDM2 overexpression. The study group comprised 36 patients with advanced EC, whose primary and metastatic tumor slides were sufficient for analysis. Immunohistochemical assessment was made by applying anti-human P53 and MDM2 antibodies and a highly sensitive EnVision(+)/HPR visualization system. Nuclear P53 overexpression was seen in 11 (31%) primary ECs and 12 (33%) metastatic tumors. There was a significant correlation between P53 overexpression (in primary cancers and metastatic tumors) and MDM2 overexpression in metastatic tumors. Nuclear MDM2 overexpression was noted in 42% (15/36) of primary carcinomas and in 47% (17/36) of metastatic tumors. A significant association existed between MDM2 overexpression and histological grading (G1 + G2 versus G3, P = 0.043). P53/MDM2 overexpression occurred simultaneously in 7 out of 36 (19%) primary ECs and in 9 out of 36 (25%) metastatic lesions. Concomitant overexpression of these proteins was reported in 7 out of 36 (19%) cases and tended to be higher in tumors showing VSI compared to neoplasms lacking vascular space invasion (P = 0.051). P53 overexpression, either in primary ECs (P < 0.0001) or metastatic lesions (P < 0.0001), was significantly associated with poor survival in univariate analysis. Moreover, the log-rank test demonstrated that simultaneous P53/MDM2 overexpression was also correlated with decreased length of survival. There was no correlation between MDM2 overexpression and patient survival. Multivariate Cox regression analysis revealed that only P53 overexpression is an independent predictor of survival. In conclusion, our data support the view that patients with P53 overexpression are significantly associated with an unfavorable outcome, whereas MDM2 overexpression is not related to decreased survival length in women operated on for advanced-stage EC.
P53 and MDM2 in renal cell car-cinoma
- Noon Ap
- N Vlatkovic
- R Polan
- M Maguire
- H Shawki
- K Parsons
- Boyd
- Mt
Noon AP, Vlatkovic 0 N, Polan 0 ski R, Maguire M, Shawki H, Parsons K, Boyd MT (2010) P53 and MDM2 in renal cell car-cinoma. Cancer 116:780–790