Cooperation among Numb, MDM2 and p53 in the development and progression of pancreatic cancer

We study the expression of Numb, MDM2 and p53 for clinical significance in pancreatic cancer (PC) and their functional relationship in regulating biological behaviors of PC cells. IHC, IB and qRT-PCR were used to detect Numb, MDM2 and p53 expression in PC. Transfection and drug intervention were used to investigate their functional relationship in PC cells. IHC showed that Numb expression was negatively associated with tumor size, differentiation and UICC stage, while expression of MDM2 and p53 was positively associated with tumor T and UICC stages, respectively (P?<?0.05). Numb was an independent prognostic indicator in PC (P?<?0.05). Patients with Numb-positive expression or combined with MDM2-negative expression had a significantly better overall survival (P?<?0.05). Altered expression of Numb can regulate wild-type but not mutant p53 expression, while MDM2 knockdown increased Numb but not mutant p53 protein level. Meanwhile, Numb knockdown increased chemoresistance but decreased activated p53 and cleaved-caspase-3 protein expression in gemcitabine-treated Capan-2 cells. Moreover, Numb co-immunoprecipitated with p53 to prevent p53 ubiquitin-dependent protein degradation and this ubiquitin-dependent regulation plays an important role in the coordinate function of these three proteins on cell invasion and migration in PC cells. Our study is the first to demonstrate the clinical significance and functional cooperation among Numb, MDM2 and p53 involved in the development and progression of PC.     

Selective COX-2 inhibitor (celecoxib) decreases cellular growth in prostate cancer cell lines independent of p53

Abstract

Celecoxib is a clinically available COX-2 inhibitor that has been reported to have antineoplastic activity. It has been proposed as a preventative agent for several types of early neoplastic lesions. Earlier studies have shown that sensitivity of prostatic carcinoma (PCa) to celecoxib is associated with apoptosis; however, these studies have not demonstrated adequately whether this effect is dependent on p53 status. We studied the relation between sensitivity to celecoxib and the phenotypic p53 status of PCa cells lines, LNCaP (wild type p53), PC3 (null p53) and DU145 (mutated p53). Cellular growth was assessed at 24, 48, 72 and 96 h after celecoxib treatment at concentrations of 0, 10, 30, 50, 70 and 100 μM using an MTT assay. Cellular proliferation (Ki-67 expression) was determined by immunocytochemistry. Phenotypic expression of p53 was analyzed by western blotting. The effects of celecoxib on cellular growth and its association with p53 were assessed after down-regulation of p53 using synthetic interfering RNAs (siRNA) in LNCaP cells. Expression of p53 and COX-2 at mRNA levels was assessed by quantitative real time polymerase reaction (qRT-PCR). We found that celecoxib inhibited cellular growth and proliferation in a dose-dependent manner in all three cell lines; LNCaP cells with a native p53 were the most sensitive to celecoxib. We observed a down- regulation effect on p53 in LNCaP cells exposed to ≥ 30 μM celecoxib for 72 h, but found no significant changes in the p53 levels of DU145 cells, which have a mutated p53. Reduced COX-2 expression was found with decreased p53 in LNCaP and PC-3 cells that were exposed to ≥ 20 μM of celecoxib for 72 h, but COX-2 expression was increased in DU145 cells. All three cell lines demonstrated pan-cytotoxicity when exposed to 100 μM celecoxib. When p53 expression was inhibited using siRNA in LNCaP cells, the inhibitory effects on cellular growth usually exerted by celecoxib were not changed significantly. Celecoxib reduces the growth of prostate cancer cell lines in part by decreasing proliferation, which suggests that the inhibition of growth of LNCaP cells by celecoxib is independent of normal levels of native p53.     

Bim directly antagonizes Bcl-xl in doxorubicin-induced prostate cancer cell apoptosis independently of p53

Doxorubicin and other anthracycline compounds exert their anti-cancer effects by causing DNA damage and initiating cell cycle arrest in cancer cells, followed by apoptosis. DNA damage generally activates a p53-mediated pathway to initiate apoptosis by increasing the level of the BH3-only protein, Puma. However, p53-mediated apoptosis in response to DNA damage has not yet been validated in prostate cancers. In the current study, we used LNCaP and PC3 prostate cancer cells, representing wild type p53 and a p53-null model, to determine if DNA damage activates p53-mediated apoptosis in prostate cancers. Our results revealed that PC3 cells were 4 to 8-fold less sensitive than LNCaP cells to doxorubicin-inuced apoptosis. We proved that the differential response of LNCaP and PC3 to doxorubicin was p53-independent by introducing wild-type or dominant negative p53 into PC3 or LNCaP cells, respectively. By comparing several apoptosis-related proteins in both cell lines, we found that Bcl-xl proteins were much more abundant in PC3 cells than in LNCaP cells. We further demonstrated that Bcl-xl protects LNCaP and PC3 cells from doxorubicin-induced apoptosis by using ABT-263, an inhibitor of Bcl-xl, as a single agent or in combination with doxorubicin to treat LNCaP or PC3 cells. Bcl-xl rather than p53, likely contributes to the differential response of LNCaP and PC3 to doxorubicin in apoptosis. Finally, co-immunoprecipitation and siRNA analysis revealed that a BH3-only protein, Bim, is involved in doxorubicin-induced apoptosis by directly counteracting Bcl-xl.     

Fractionated Radiation Alters Oncomir and Tumor Suppressor miRNAs in Human Prostate Cancer Cells

We have previously demonstrated that prostate carcinoma cells exposed to fractionated radiation differentially expressed more genes compared to single-dose radiation. To understand the role of miRNA in regulation of radiation-induced gene expression, we analyzed miRNA expression in LNCaP, PC3 and DU145 prostate cancer cells treated with single-dose radiation and fractionated radiation by microarray. Selected miRNAs were studied in RWPE-1 normal prostate epithelial cells by RT-PCR. Fractionated radiation significantly altered more miRNAs as compared to single-dose radiation. Downregulation of oncomiR-17-92 cluster was observed only in the p53 positive LNCaP and RWPE-1 cells treated with single-dose radiation and fractionated radiation. Comparison of miRNA and mRNA data by IPA target filter analysis revealed an inverse correlation between miR-17-92 cluster and several targets including TP53INP1 in p53 signaling pathway. The base level expressions of these miRNAs were significantly different among the cell lines and did not predict the radiation outcome. Tumor suppressor miR-34a and let-7 miRNAs were upregulated by fractionated radiation in radiosensitive LNCaP (p53 positive) and PC3 (p53-null) cells indicating that radiation-induced miRNA expression may not be regulated by p53 alone. Our data support the potential for using fractionated radiation to induce molecular targets and radiation-induced miRNAs may have a significant role in predicting radiosensitivity.     

14-3-3sigma is down-regulated in human prostate cancer

The 14-3-3sigma is a negative regulator of the cell cycle, which is induced by p53 in response to DNA damage. It has been characterized as an epithelium-specific marker and down-regulation of the protein has been shown in breast cancers, suggesting its tumor-suppressive activity in epithelial cells. Here we demonstrate that 14-3-3sigma protein is down-regulated in human prostate cancer cell lines, LNCaP, PC3, and DU145 compared with normal prostate epithelial cells. Immunohistochemical analysis of primary prostate cells shows that the expression of 14-3-3sigma protein is epithelial cell-specific. Among prostate pathological specimens, > 95% of benign hyperplasia samples show significant and diffuse immunostaining of 14-3-3sigma in the cytoplasm whereas < 20% of carcinoma samples show positive staining. In terms of mechanisms for the down-regulation of 14-3-3sigma in prostate cancer cells, hypermethylation of the gene promoter plays a causal role in LNCaP cells as 14-3-3sigma mRNA level was elevated by 5-aza-2'-deoxycytidine demethylating treatment. Intriguingly, the proteasome-mediated proteolysis is responsible for 14-3-3sigma reduction in DU145 and PC3 cells, as 14-3-3sigma protein expression was increased by treatment with a proteasome inhibitor MG132. Furthermore, tumor necrosis factor-related apoptosis-inducing ligand enhances 14-3-3sigma gene and protein expression in DU145 and PC3 cells. These data suggest that 14-3-3sigma expression is down-regulated during the neoplastic transition of prostate epithelial cells.     

p53 regulates the expression of the tumor suppressor gene maspin

Maspin has been shown to inhibit tumor cell invasion and metastasis in breast tumor cells. Maspin expression was detected in normal breast and prostate epithelial cells, whereas tumor cells exhibited reduced or no expression. However, the regulatory mechanism of maspin expression remains unknown. We report here a rapid and robust induction of maspin expression in prostate cancer cells (LNCaP, DU145, and PC3) and breast tumor cells (MCF7) following wild type p53 expression from an adenovirus p53 expression vector (AdWTp53). p53 activates the maspin promoter by binding directly to the p53 consensus-binding site present in the maspin promoter. DNA-damaging agents and cytotoxic drugs induced endogenous maspin expression in cells containing the wild type p53. Maspin expression was refractory to the DNA-damaging agents in cells containing mutant p53. These results, combined with recent studies of the tumor metastasis suppressor gene KAI1 and plasminogen activator inhibitor 1 (PAI1), define a new category of molecular targets of p53 that have the potential to negatively regulate tumor invasion and/or metastasis.     

Biological aggressiveness evaluation in prostate carcinomas:immunohistochemical analysis of PCNA and p53 in a series of Gleason 6 (3+3) adenocarcinomas

We selected 63 prostate tumors with Gleason's grade 6 (3+3), commonly showing both tubular and cribrous patterns. We compared in both patterns the expression of two of the most used biologic markers: PCNA and p53, with the aim to verify the validity of the Gleason's grading system to compare the morphologic grade with biologic aggressiveness and prognostic value. We did not find any statistical difference in the protein immunopositivity, indicating that both patterns could have identical biologic behaviour; then we confirmed the validity of Gleason's system for considering both tubular and cribrous patterns as an intermediate grade of tumoral differentiation. Moreover, we found a linear relationship between the increase of PCNA and the accumulation of mutated p53; this datum could confirm the hypothesis that p53 mutation is a late event in prostate carcinogenesis.     

肺癌组织中PCNA、p63和p53蛋白的表达及临床意义

目的:检测蛋白增殖细胞核抗原(PCNA)、p63和p53在肺癌组织中的表达情况,以探讨三者在肺癌的发生、发展中的生物学作用和临床意义。方法:选取195例肺癌组织(其中57例有癌旁组织),应用组织芯片技术和免疫组织化学方法观察三种蛋白的表达情况,并研究三者之间及其与临床病理参数的关系。结果:PCNA、p63和p53蛋白在肺癌组织中的阳性表达率分别为96.41%、38.46%及58.46%,但三者在癌旁组织中均无表达,差异有统计学意义(均P0.05);在肺癌组织中,PCNA、p63和p53蛋白的表达情况均与组织分型有关(P0.05),且PCNA、p53蛋白表达与分化程度有关(P0.05),分化越差,表达越高;p53表达与PCNA表达呈正相关(r=0.352,P=0.043),p63与p53、PCNA的表达不相关(P0.05)。结论:肺癌组织中PCNA、p63和p53蛋白的表达升高,三者均在肺癌的发生、发展中发挥着重要作用,并且临床可通过检测三者的蛋白水平,作为鉴别肺鳞状细胞癌与其他类型癌的重要参考指标,为病理诊断提供依据。     

Effect of p53 on centrosome amplification in prostate cancer cells.

Chromosomal instability (CIN) is one of the common features in prostate cancer, especially in advanced stages. Recently, the involvement of p53 in CIN through the regulation of centrosome amplification has been proposed in certain tumor types. In this study, we investigated the relationship between p53 and centrosome amplification in prostate cancer cells. Increased centrosome number and size were observed in DU145 and PC3 containing nonfunctional p53 compared to LNCap which expressed wild-type p53. Transfection of p53 into PC3 cells resulted in a decreased cell growth rate, G2/M arrest and decreased centrosome abnormalities. We provide the first evidence on a correlation between loss of p53 function and centrosome amplification in prostate cancer cells. Our results indicate that p53 may play a role in the regulation of centrosome amplification and loss of p53 may be one of the mechanisms involving CIN in prostate cancer cells.     

Immunohistochemical expression of retinoblastoma gene product (Rb), p53 protein, MDM2, c-erbB-2, HLA-DR and proliferation indices in human urinary bladder carcinoma

Archival biopsy specimens from transitional cell bladder cancers (n=88) were analysed immunohistochemically for the expression of the retinoblastoma (Rb) gene protein, p53, mdm2, c-erbB-2, HLA-DR antigen and proliferation indices. An altered nuclear expression of Rb, p53 and mdm2 was observed in 55.2%, 33.3% and 18.2% of tumors respectively. Cytoplasmic membrane immunoreactivity (>25% tumor cells) for c-erbB-2 was detected in 14.1% of tumors and aberrant HLA-DR antigen cytoplasmic staining (>5% of tumor cells) in 22.2% of the cases. P53 overexpression was associated with higher tumor grade and stage. Aberrant HLA-DR antigen expression and PCNA were also correlated with the grade of differentiation and tumor stage. MIB1 was statistically correlated with stage. pRb scores and HLA-DR antigen expression were correlated with proliferation activity as determined by PCNA and MIB1 immunostaining. p53 protein was also strongly correlated with the proliferation index PCNA. A strong correlation between PCNA and MIB1 (p<0.0001) was also found. In addition a statistically positive correlation between p53 and HLA-DR antigen expression was observed. Our data show that, although pRb and p53 protein expressions are not associated between them, they may contribute to the growth fraction of the bladder cancer. In addition, p53 and HLA-DR antigen expression could be indicators of aggressive behavior of bladder cancer.     

Changes in Biomarker Expression in the Development of Prostatic Adenocarcinoma

In spite of the prevalence of prostatic adenocarcinoma, the development and natural history of this malignancy is poorly understood. This paper reviews the current knowledge of biomarker expression during the development and progression of prostatic adenocarcinoma. Emphasis is placed on the comparison of biomarker expression in benign prostatic epithelium, intraepithelial neoplasia (PIN), a putative preinvasive lesion, and prostatic adenocarcinoma. Within the benign epithelium, the proliferative potential is restricted to the basal cells as demonstrated by the expression of proliferating cellular nuclear antigen (PCNA). The strong expression of the bcl-2 protein, an inhibitor of apoptosis, supports the concept that the basal cells or a subpopulation of the basal cells represent the stem cell of the epithelium. In addition, the strong expression of growth factor receptors such as the epidermal growth factor receptor (EGFr), p185^erbB-2, p180^erbB-3, and c-met suggests that the growth of the basal cells is regulated by autocrine or paracrine factors. The luminal cells express secretory products such as prostate specific antigen and prostatic acid phosphatase, but demonstrate little expression of PCNA as well as growth factor receptors and proto-oncogene products. These observations are consistent with the theory that the luminal cell population is derived from the differentiation of the basal cells. In contrast to the normal epithelium, PCNA expression is frequently detected in the dysplastic luminal cells of the PIN lesion. Likewise, strong expression of p185^erbB-2, p180^erbB-3 and the c-met proto-oncogene product is also detected in the luminal cells of PIN lesions. Other factors which are strongly expressed by the dysplastic luminal cells include the nm23-Hl gene product, tumor associated glycoprotein-72 (TAG-72), fatty acid synthetase (FASE) and proteolytic enzymes. These findings suggest that PIN lesions are derived from an impairment of the differentiation of basal cells. The majority of biomarkers such as PCNA, p185^erbB-2, p180^erbB-3, TAG-72, nm23-Hl and FASE which are strongly expressed in PIN lesions are also expressed in prostatic adenocarcinoma supporting the concept that PIN is a preinvasive lesion. Mutations of the p53 tumor suppressor gene, as well as strong expression of transforming growth factor a and bcl-2 typically occur in advanced stage prostatic adenocarcinomas and therefore likely represent late events in the development of prostatic adenocarcinoma.     

Inhibition of Proliferation and Induction of Autophagy by Atorvastatin in PC3 Prostate Cancer Cells Correlate with Downregulation of Bcl2 and Upregulation of miR-182 and p21

The epidemiologic association between statin use and decreased risk of advanced prostate cancer suggests that statins may inhibit prostate cancer development and/or progression. Studies were performed to determine the effects of a model statin, atorvastatin (ATO), on the proliferation and differentiation of prostate cancer cells, and to identify possible mechanisms of ATO action. ATO inhibited the in vitro proliferation of both LNCaP and PC3 human prostate cancer cells in a dose- and time-dependent fashion. The greater inhibitory activity of ATO in PC3 cells was associated with induction of autophagy in that cell line, as demonstrated by increased expression of LC3-II. miR-182 was consistently upregulated by ATO in PC3 cells, but not in LNCaP cells. ATO upregulation of miR-182 in PC3 cells was p53-independent and was reversed by geranylgeraniol. Transfection of miR-182 inhibitors decreased expression of miR-182 by >98% and attenuated the antiproliferative activity of ATO. miR-182 expression in PC3 cells was also increased in response to stress induced by serum withdrawal, suggesting that miR-182 upregulation can occur due to nutritional stress. Bcl2 and p21 were identified to be potential target genes of miR-182 in PC3 cells. Bcl2 was downregulated and p21 was upregulated in PC3 cells exposed to ATO. These data suggest that miR-182 may be a stress-responsive miRNA that mediates ATO action in prostate cancer cells.     

Interferon-gamma and its functional receptors overexpression in benign prostatic hyperplasia and prostatic carcinoma: parallelism with c-myc and p53 expression

The therapeutic potential of IFN-gamma in prostatic cancer has been documented in several reports, although no immunohistochemical studies of this factor and its receptors in the prostate have been reported. The aim of the present study was to investigate the expression of IFN-gamma and its receptor components (IFN-gamma-Ralpha and IFN-gamma-Rbeta) in normal prostate, benign prostatic hyperplasia (BPH) and prostatic cancer (PC), as well as the possible relationship between this factor and the products of the p53 gene (the wild and mutant forms) and the oncogene c-myc, by means of immunochemical techniques (Western blot, ELISA, and quantification of immunostaining in histological sections). In normal prostate, IFN-gamma and its two receptors were expressed in the basal cells of the epithelium and some stromal cells. In BPH specimens, immunostaining of basal epithelial cells was significantly increased for IFN-gamma and its a receptor, whereas stromal cell immunostaining was significantly increased for IFN-gamma and its b receptor. In addition, columnar epithelial cells immunostained for IFNbeta-Rbeta. PC specimens differed from BPH specimens in the significantly increased immunostaining of epithelial cells for IFN-gamma and its two receptors, and the immunostaining of columnar epithelial cells for IFN-gamma-Ralpha. Immunodetection of wild-p53 was weak and limited to some stromal cells in the three types of specimens. Immunostainings for both mutant-p53 and c-myc were negative in normal prostate, and positive in the epithelium and stromal cells of both BPH and PC specimens. Immunostaining intensity in PC was significantly higher than in BPH. These observations suggest that the expression of both mutant-p53 and c-myc, together with other factors, might be involved in the development of prostatic hyperplasia and neoplasia, while the increased expression of IFN-gamma and its receptors could be regarded as an attempt, although insufficient, to inhibit the uncontrolled cell proliferation.     

Gonadotropin-Releasing Hormone Agonists Sensitize,and Resensitize,Prostate Cancer Cells to Docetaxel in a p53-Dependent Manner

Gonadotropin-releasing hormone (GnRH) receptors are expressed in prostate cancer, specifically in the most aggressive stage of the tumor (castration-resistant prostate cancer, CRPC) for which the standard treatment, docetaxel-based chemotherapy, can only improve the median survival time by few months. We previously showed that GnRH agonists exert an antitumor activity in CRPC cells; however, a link between GnRH receptors and the apoptotic machinery remains to be defined. Aim of this study was to evaluate whether, in CRPC cells, GnRH agonists might affect the expression/activity of apoptosis-related proteins and might sensitize, or resensitize, cancer cells to chemotherapeutics. We demonstrated that, in p53-positive DU145 cells, GnRH agonists: a) increase the expression of the proapoptotic protein Bax; this effect is mediated by the phosphorylation (activation) of p53, triggered by the p38 MAPK; b) potentiate the antiproliferative/proapoptotic activity of docetaxel; c) resensitize docetaxel-resistant cells to the antitumor activity of the cytotoxic drug. These data indicate that GnRH agonists sensitize and, more importantly, resensitize DU145 CRPC cells to chemotherapy in a p53-dependent manner. To confirm the crucial role of p53 in the activity of GnRH agonists, experiments were performed in p53-null PC3 cells. We found that GnRH agonists fail to increase Bax expression and do not potentiate the cytotoxic activity of docetaxel. These results may provide a rationale for novel combination treatment strategies, especially for docetaxel-resistant CRPC patients expressing a functional p53 protein.     

Study of p53 protein expression in prostate cancer

The aim of this study was the immunohistochemical evaluation of p53 protein expression in localized prostate cancer (Pca) following radical prostatectomy and analysis of its relationship to chosen anatomo-clinical and morphological parameters of the tumours. The present investigation included material from 28 randomly selected patients undergoing radical prostatectomy. Tissue sections were fixed in 10% buffered formaldehyde solution, embedded in paraffin and stained immunohistochemically with the anti-human p53 protein monoclonal antibody. The immunolocalization of p53 protein was performed using the Labelled Streptavidyn Biotin (LSAB) method. The p53 protein expression was semiquantitatively assessed in neoplastic cells and the reaction present in more then 25% of tumour cells was accepted as the threshold of positivity. No correlation was found between p53 protein expression and Gleason score, pT stage, lymph node metastases, seminal vesicles invasion, positive or negative surgical resection margins, age of patients. However, p53 protein expression and capsular penetration was found statistically significantly correlated.     

Proliferating cell nuclear antigen in normal urothelium and urothelial lesions of the urinary bladder: a quantitative assessment using a true color image analysis system

To evaluate proliferating cell nuclear antigen (PCNA) staining for assessing proliferative activity in routine pathology specimens of urinary bladder, the bladder carcinoma cell line J82 and a total of 122 specimens of normal bladder and urothelial lesions were stained with the antibody clone PC10 against proliferating cell nuclear antigen. In in vitro plateau cultures the proportion of PCNA-positive cells exceeded that of Ki-67-positive cells, and only very few cells were negative. In formalin-fixed tissues, the PCNA staining pattern, which should be confined to replicon units in the nucleus, was optimized by 1 h postfixation in an organic solvent (methacarn). Sections showed positive nuclear staining confined to basal and some suprabasal cells in normal urothelium and grade 1 dysplasias, but more generalized nuclear staining in all other neoplastic lesions. In addition, stromal cells adjacent to invasive tumors showed nuclear positivity in some instances. Using quantitative true color image analysis of sections counterstained with hemalum, the degree of brown staining of the PCNA reaction product is contrasted with the blue staining of the nuclear area. With this method low contrast specific staining not appreciated optically can be reliably detected. Image analysis data confirmed observations made on noncounterstained sections and showed significant differences between grade 1 and 2 dysplasias as well as between grade 1 dysplasia and all grades of papillary tumor. Furthermore, a significant difference in PCNA staining indices was found between grade 1 and 3 bladder carcinomas. The results indicate that PCNA staining using the PC10 antibody is not confined to the proliferative fraction of neoplastic urothelium. In contrast with data from normal tissue and malignant hematological neoplasms, the amount of PCNA is regulated differently in urothelial neoplasms, emphasizing the biological differences between the following two sets: mild dysplasia and moderate dysplasia; mild dysplasia and papillary carcinomas. The use of image analysis to standardize the detection process after controlled staining conditions is advisable in order to provide reliable data. Supported by the DFG project: Knuechel/Urothelcarcinom 263