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.     

Circular RNA circ-VANGL1 as a competing endogenous RNA contributes to bladder cancer progression by regulating miR-605-3p/VANGL1 pathway

Increasing reports indicate that circular RNAs (circRNAs) are very important regulators in human diseases, including cancers. In bladder cancer (BC), several circRNAs have been reported to be involved in tumor progressions, such as circ-ITCH and circTCF25. However, the functions of most circRNAs in BC still remains largely unknown. In this study, we identified a novel circRNA termed as circ-VANGL1 by bioinformatics analysis. We found that circ-VANGL1 was highly expressed in BC tissues compared with adjacent normal tissues. Furthermore, we showed that circ-VANGL1 could serve as a prognostic marker for patients with BC. Through functional experiments, we found that circ-VANGL1 knockdown significantly suppressed BC cell proliferation, cell cycle, migration, and invasion in vitro. Besides, circ-VANGL1 silence inhibited BC cell propagation in vivo. Mechanistically, we identified circ-VANGL1 as a sponge of miR-605-3p which targeted VANGL1 in BC cells. Through repressing miR-605-3p availability, circ-VANGL1 contributes to VANGL1 expression, consequently leading to BC cell proliferation, migration, and invasion. Taken together, our study demonstrated circ-VANGL1/miR-605-3p/VANGL1 as a novel essential signaling pathway involved in BC progression.     

CCL17-CCR4 axis promotes metastasis via ERK/MMP13 pathway in bladder cancer

As an important chemokine receptor, the role of CCR4 in the progression of bladder cancer (BC) remains unknown. In this study, we have shown that CCR4 expression was upregulated in bladder carcinoma tissues compared with adjacent nontumor tissues. Kaplan-Meier survival analysis revealed that CCR4 expression was an independent prognostic risk factor in BC patients, and the addition of CCL17 induced CCR4 production and promoted migration and invasion of BC cells. In addition, CCR4 knockdown significantly attenuated the migratory and invasive capabilities of BC cells. Mechanistically, CCL17-CCR4 axis is involved in ERK1/2 signaling and could mediate the migration and invasion of BC cells by regulating MMP13 activation. This study suggests that CCR4 might represent a promising prognostic biomarker and a potential therapeutic option for BC.     

β-catenin is up-expressed and predicts poor overall survival of breast cancer

Breast cancer (BC) is one of the most common malignant tumors in women.The majority of BC cells contain at least one or more up-expressed oncogene. β-catenin is found overexpressed in various epithelial cell cancers and has the function of inducing cancer cell proliferation, invasion and metastasis. However, the expression of β-catenin and its prognostic value in BC is not yet clear. In this study, mRNA and β-catenin proteins expressed in BC tissues have been explored. Quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC) on tissue microarrays (TMA) were performed to examine the level of β-catenin mRNA and protein in BC tissues. The association between β-catenin and clinical characteristics and prognostic value were also explored. β-catenin mRNA and protein were found over-expressed in BC tissues when compared with matched tumor neighbor tissues. A high degree of β-catenin staining in BC tissues was significantly associated with tumor size, Ki67 expression, lymph node status and TNM stage. β-catenin up-expression was also able to predict poor overall survival (OS) rates. These results indicated that β-catenin may be a useful prognostic molecular biomarker for BC patients.     

High Level of COP1 Expression is Associated with Poor Prognosis in Primary Gastric Cancer

COP1 (constitutive photomorphogenic 1, also known as RFWD2) is a p53-targeting E3 ubiquitin ligase containing RING-finger, coiled-coil, and WD40-repeat domains. Recent studies have identified that COP1 is overexpressed in several cancer types and that increased COP1 expression promotes cell proliferation, cell transformation, and tumor progression. In the present study, we investigated the expression and prognostic value of COP1 in primary gastric cancer. To investigate the role of the COP1 gene in primary gastric cancer pathogenesis, real-time quantitative PCR and western blotting were performed to examine COP1 expression in paired cancerous and matched adjacent noncancerous gastric tissues. The results revealed high COP1 mRNA (P=0.030) and protein (P=0.008) expression in most tumor-bearing tissues compared with the matched adjacent non-tumor tissues. The correlated protein expression analysis revealed a negative correlation between COP1 and p53 in gastric cancer samples (P=0.005, r=-0.572). Immunohistochemical staining of gastric cancer tissues from the same patient showed a high COP1 expression and a low p53 expression. To further investigate the clinicopathological and prognostic roles of COP1 expression, we performed immunohistochemical analysis of 401 paraffin-embedded gastric cancer tissue blocks. The data revealed that high COP1 expression was significantly correlated with T stage (P=0.030), M stage (P=0.048) and TNM stage (P=0.022). Consistent with these results, we found that high expression of COP1 was significantly correlated with poor survival in gastric cancer patients (P<0.001). Cox regression analyses showed that COP1 expression was an independent predictor of overall survival (P<0.001). Our data suggest that COP1 could play an important role in gastric cancer and might serve as a valuable prognostic marker and potential target for gene therapy in the treatment of gastric cancer.     

Investigation of oncogene amplification or deletion,and oncoprotein expression in papillary thyroid cancer

AIM: Assessment of occurrence and possible prognostic significance of c-myc and Ha-ras amplification, p53 deletion and overexpression of cyclin D1, p53 and p21 in papillary thyroid cancer. MATERIALS AND METHODS: Formalin-fixed, paraffin-embedded tumor tissue from 24 patients were investigated. Dot-blot DNA hybridization was used to detect oncogene amplification or deletion. The expression of oncoproteins was determined by immunohistochemical method. RESULTS: In our samples neither Ha-ras amplification nor p53 deletion were found. Low c-myc amplification (mean: 2.55) occured in 4 cases (17%). p53 protein was detected in 16 samples (66.6%), with p21 expression (chi(2)=7.02, p<0.01) in 6 cases (25%). The p53 expression did not influence the tumor fenotype. Cyclin D1 overexpression was found in 12 cases (50%), it was often associated with p21 expression (chi2=10.1, p<0.001) and in inverse relation to the tumor lymphocytic infiltration (chi(2)=5.35, p<0.05). Increased expression of estrogen receptor was shown in 4 cyclin D1 positive samples (17%). CONCLUSIONS: The p53 detected in our study is likely not to be mutant protein in all cases because its presence was associated with p21 expression that the mutant protein cannot induce and also it did not mean more aggressive tumor phenotype. The connection of cyclin D1 overexpression with the lymphocytic infiltration of the tumor suggests that the increased expression of cyclin D1 means poor prognosis. The coexpression of cyclin D1 and p21 raises the modulative character of the p21 protein, thought to be a tumor suppressor originally, but we find a CDK-independent, estrogen receptor mediated effect of cyclin D1 more likely, which has been described in breast cancer and is also proved by the coexpression of cyclin D1 and estrogen receptor detected here.     

Evaluation of thymidylate synthase protein expression by Western blotting and immunohistochemistry on human colon carcinoma xenografts in nude mice.

In this study we investigated the relationship between thymidylate synthase (TS) protein expression, evaluated by Western blotting analysis and by immunohistochemistry (IHC), and growth rate in human colon xenograft tumors in nude mice. Human colon cancer cell lines were used to induce xenograft tumors and the tumor mass growth rate was calculated by measuring tumor size variations over time. TS 106 monoclonal antibody was used for both Western blotting and IHC TS detection. Tumor cell growth fraction was measured by Ki67/MIB1 immunolabeling and tumor cell growth rate by evaluating the mean nucleolar size in silver-stained sections. TS Western blotting values were related to tumor mass growth rate (p<0.001) and cell growth rate (p=0.002) but not to cell growth fraction (p=0.676). The degree of the IHC staining showed only a trend to be associated with TS protein expression measured on Western blotting, and was not related either to tumor mass growth or cell proliferation rate. Tumor xenografts were also characterized for TS promoter tandem repeat and p53 status. No relationship was observed between these variables and TS expression evaluated by both Western blotting and IHC analysis. Our results demonstrate that TS expression evaluated by Western blotting analysis is directly related to the tumor mass growth rate and question the use of the IHC approach to obtain precise quantitative information on TS expression in tumor samples.     

Prognostic significance of BAF57 expression in patients with endometrial carcinoma

This study was conducted to elucidate the prognostic significance of BAF57 in patients with endometrial carcinoma. We investigated the relationship between the immunohistochemical expression of BAF57 and various clinicopathological variables in 111 endometrial carcinomas. Both univariate and multivariate regression analyses were performed. The correlations between the BAF57 expression and the other variables including estrogen receptor (ER) and p53 were examined. The high nuclear BAF57 expression was detected in 42 (37.8%) endometrial carcinomas, and 69 (62.2%) endometrial carcinomas were defined as having low nuclear BAF57 expression. The BAF57 expression was significantly associated with the surgical stage, grade of the tumor, myometrial invasion, lympho-vascular space invasion (LVSI) and lymph node metastasis. The 10-year overall survival rates of patients with low and high BAF57 expression were 96.9% and 58.2%, respectively (p<0.001). A multivariate analysis identified BAF57 expression as an independent prognostic factor. The BAF57 expression was significantly correlated with p53 expression (r=0.312, P=0.001), but was not correlated with ER expression (r= -0.141, P=0.14). The high BAF57 expression is an independent marker of poor prognosis of the patients in endometrial carcinomas. The inhibition of BAF57 activity may be one of the candidates for endometrial cancer therapy, especially therapy for aggressive tumors showing overexpression of p53.     

Mutant p53 Attenuates the Anti-Tumorigenic Activity of Fibroblasts-Secreted Interferon Beta

Mutations in the p53 tumor suppressor protein are highly frequent in tumors and often endow cells with tumorigenic capacities. We sought to examine a possible role for mutant p53 in the cross-talk between cancer cells and their surrounding stroma, which is a crucial factor affecting tumor outcome. Here we present a novel model which enables individual monitoring of the response of cancer cells and stromal cells (fibroblasts) to co-culturing. We found that fibroblasts elicit the interferon beta (IFNβ) pathway when in contact with cancer cells, thereby inhibiting their migration. Mutant p53 in the tumor was able to alleviate this response via SOCS1 mediated inhibition of STAT1 phosphorylation. IFNβ on the other hand, reduced mutant p53 RNA levels by restricting its RNA stabilizer, WIG1. These data underscore mutant p53 oncogenic properties in the context of the tumor microenvironment and suggest that mutant p53 positive cancer patients might benefit from IFNβ treatment.     

A trisubstituted pyrazole derivative reduces DMBA-induced mammary tumor growth in rats by inhibiting estrogen receptor-α expression

P16 is the product of cyclin-dependent kinase 2 (CDKN2A) gene and plays multi-pronged roles in the cancer progression. Breast cancer (BC) is the most commonly diagnosed cancer type among females. In the current study, the potential function of P16 in the growth and metastasis of BC was investigated. Firstly, the expression statuses of P16 in different cancer types were investigated using Oncomine database and validated with corresponding cancer cell lines. Afterwards, the expression of P16 was knocked down in BC cell line BT-549 and the effect on the cell proliferation, sensitivity to paclitaxel (TAX), apoptosis, migration, and invasion abilities was assessed using CCK-8, Edu, flow cytometry, scratch, and transwell assays, respectively. The influence of P16 inhibition and P16 overexpression on the activity of IL-6/JAK/STAT3 signaling was explored. Additionally, the effect of P16 inhibition on the tumor growth was verified with a BC xenograft mice model. The abnormal expression of P16 was detected in BC cell line BT-549 as well as colorectal cancer and osteosarcoma cell lines. The inhibition of P16 suppressed the cell proliferation, invasion, and migration abilities while induced the apoptosis and sensitivity to TAX in BT-549 cells. At molecular level, P16 knockdown inhibited the expression of IL6ST and Survivin, and the phosphorylation of JAK2 and STAT3. However, the induced expression of P16 in P16-knockdown BT-549 cells restored the activity of IL-6/JAK2/STAT3 pathway. The results of in vitro assays were confirmed with BC xenograft models: the inhibition of P16 decreased the tumor growth rate. Findings outlined in the current study demonstrated that the inhibition of P16 decreased the growth and metastasis potential of BC cells by inhibiting IL-6/JAK2/STAT3 signaling.     

Nocodazole-induced p53-dependent c-Jun N-terminal kinase activation reduces apoptosis in human colon carcinoma HCT116 cells

Microtubule-interfering agents are widely used in cancer chemotherapy, and prognostic results vary significantly from tumor to tumor, depending on the p53 status. In preliminary experiments, we compared the expression and phosphorylation profiles of more than 100 protein kinases and protein phosphatases in human colorectal carcinoma cell line HCT116 between p53+/+ and p53-/- cells in response to short term nocodazole treatment through application of Kinetworks immunoblotting screens. Among the proteins tracked, the regulation of the phosphorylation of c-Jun N-terminal kinase (JNK)1/2 at Thr-183/Tyr-185 was the major difference between p53+/+ and p53-/- cells. With the loss of the p53 gene, the levels of phosphorylation of Ser-63 of c-Jun and Thr-183/Tyr-185 of JNK1/2 in p53-/- cells did not increase as markedly as in p53+/+ cells in response to a 1-h treatment with nocodazole or other microtubule-disrupting drugs such as vinblastine and colchicine. Similar observations were also made in MCF-7 and A549 tumor cells, which were rendered p53-deficient by E6 oncoprotein expression. However, arsenate-induced JNK activation in p53-/- cells was preserved. Inhibition of p53 expression by its antisense oligonucleotide also attenuated nocodazole-induced JNK activation in p53+/+ cells. Surprisingly, cotransfection of p53+/+ cells with dominant negative mutants of JNK isoforms and treatment of p53+/+ cells with the JNK inhibitor SP600125 actually further enhanced apoptosis in p53+/+ cells by up to 2-fold in response to nocodazole. These findings indicate that inhibition of p53-mediated JNK1/2 activity in certain tumor cells could serve to enhance the apoptosis-inducing actions of cancer chemotherapeutic agents that disrupt mitotic spindle function.     

Lipocalin2 Expressions Correlate Significantly With Tumor Differentiation in Epithelial Ovarian Cancer

We recently identified lipocalin2 (LCN2) as being upregulated in ovarian cancer cell lines. The purpose of this study was to validate LCN2 upregulation in ovarian cancers and to investigate its potential as a serum biomarker. We assayed LCN2 expression in ovarian cancers using real-time PCR and IHC. To evaluate the potential of LCN2 as a biomarker, we measured serum LCN2 levels in 54 ovarian cancers, 15 borderline and 53 benign ovarian tumors, and 90 healthy controls. SYBR green PCR and IHC showed LCN2 overexpression in ovarian cancers. LCN2 immunoreactivity was significantly associated with tumor differentiation (p=0.009), as well-differentiated tumors showed the highest LCN2 expression. Serum LCN2 level in ovarian cancer was significantly higher than in the other study groups (p<0.001), and in accordance with IHC results, it also correlated with tumor differentiation, with well-differentiated tumors having the highest value. The sensitivity and specificity of LCN2 in detecting ovarian cancer was 72.2% and 50.4%, respectively. By Cox univariate analysis, LCN2 positivity was an independent prognostic factor for overall survival (hazard ratio = 1.47, p=0.012). In conclusion, LCN2 expressions are upregulated and related to tumor differentiation in ovarian cancers and should be included in future research assessing potential biomarkers for ovarian cancer. (J Histochem Cytochem 57:513–521, 2009)     

Breast cancer stem cell-derived extracellular vesicles transfer ARRDC1-AS1 to promote breast carcinogenesis via a miR-4731-5p/AKT1 axis-dependent mechanism

ObjectivesDeregulation of long non-coding RNAs (lncRNAs) has been frequently reported in breast cancer (BC). This goes to show the importance of understanding its significant contribution towards breast carcinogenesis. In the present study, we clarified a carcinogenic mechanism based on the ARRDC1-AS1 delivered by breast cancer stem cells-derived extracellular vesicles (BCSCs-EVs) in BC.MethodsThe isolated and well characterized BCSCs-EVs were co-cultured with BC cells. The expression of ARRDC1-AS1, miR-4731-5p, and AKT1 was determined in BC cell lines. BC cells were assayed for their viability, invasion, migration and apoptosis in vitro by CCK-8, Transwell and flow cytometry, as well as tumor growth in vivo after loss- and gain-of function assays. Dual-luciferase reporter gene, RIP and RNA pull-down assays were performed to determine the interactions among ARRDC1-AS1, miR-4731-5p, and AKT1.ResultsElevation of ARRDC1-AS1 and AKT1 as well as miR-4731-5p downregulation were observed in BC cells. ARRDC1-AS1 was enriched in BCSCs-EVs. Furthermore, EVs containing ARRDC1-AS1 enhanced the BC cell viability, invasion and migration and glutamate concentration. Mechanistically, ARRDC1-AS1 elevated the expression of AKT1 by competitively binding to miR-4731-5p. ARRDC1-AS1-containing EVs were also found to enhance tumor growth in vivo.ConclusionCollectively, BCSCs-EVs-mediated delivery of ARRDC1-AS1 may promote the malignant phenotypes of BC cells via the miR-4731-5p/AKT1 axis.     

Glyoxalase-I is a novel prognosis factor associated with gastric cancer progression

Glyoxalase I (GLO1), a methylglyoxal detoxification enzyme, is implicated in the progression of human malignancies. The role of GLO1 in gastric cancer development or progression is currently unclear. The expression of GLO1 was determined in primary gastric cancer specimens using quantitative polymerase chain reaction, immunohistochemistry (IHC), and western blotting analyses. GLO1 expression was higher in gastric cancer tissues, compared with that in adjacent noncancerous tissues. Elevated expression of GLO1 was significantly associated with gastric wall invasion, lymph node metastasis, and pathological stage, suggesting a novel role of GLO1 in gastric cancer development and progression. The 5-year survival rate of the lower GLO1 expression groups was significantly greater than that of the higher expression groups (log rank P = 0.0373) in IHC experiments. Over-expression of GLO1 in gastric cancer cell lines increases cell proliferation, migration and invasiveness. Conversely, down-regulation of GLO1 with shRNA led to a marked reduction in the migration and invasion abilities. Our data strongly suggest that high expression of GLO1 in gastric cancer enhances the metastasis ability of tumor cells in vitro and in vivo, and support its efficacy as a potential marker for the detection and prognosis of gastric cancer.