CARMA3 is overexpressed in colon cancer and regulates NF-κB activity and cyclin D1 expression

CARMA3 was recently reported to be overexpressed in cancers and associated with the malignant behavior of cancer cells. However, the expression of CARMA3 and its biological roles in colon cancer have not been reported. In the present study, we analyzed the expression pattern of CARMA3 in colon cancer tissues and found that CARMA3 was overexpressed in 30.8% of colon cancer specimens. There was a significant association between CARMA3 overexpression and TNM stage (p=0.0383), lymph node metastasis (p=0.0091) and Ki67 proliferation index (p=0.0035). Furthermore, knockdown of CARMA3 expression in HT29 and HCT116 cells with high endogenous expression decreased cell proliferation and cell cycle progression while overexpression of CARMA3 in LoVo cell line promoted cell proliferation and facilitated cell cycle transition. Further analysis showed that CARMA3 knockdown downregulated and its overexpression upregulated cyclin D1 expression and phospho-Rb levels. In addition, we found that CARMA3 depletion inhibited p-IκB levels and NF-κB activity and its overexpression increased p-IκB expression and NF-κB activity. NF-κB inhibitor BAY 11-7082 reversed the role of CARMA3 on cyclin D1 upregulation. In conclusion, our study found that CARMA3 is overexpressed in colon cancers and contributes to malignant cell growth by facilitating cell cycle progression through NF-κB mediated upregulation of cyclin D1.     

Proteins and an inflammatory network expressed in colon tumors

The adenomatous polyposis coli (APC) protein is crucial to homeostasis of normal intestinal epithelia because it suppresses the β-catenin/TCF pathway. Consequently, loss or mutation of the APC gene causes colorectal tumors in humans and mice. Here, we describe our use of multidimensional protein identification technology (MudPIT) to compare protein expression in colon tumors to that of adjacent healthy colon tissue from Apc(Min/+) mice. Twenty-seven proteins were found to be up-regulated in colon tumors and 25 were down-regulated. As an extension of the proteomic analysis, the differentially expressed proteins were used as "seeds" to search for coexpressed genes. This approach revealed a coexpression network of 45 genes that is up-regulated in colon tumors. Members of the network include the antibacterial peptide cathelicidin (CAMP), Toll-like receptors (TLRs), IL-8, and triggering receptor expressed on myeloid cells 1 (TREM1). The coexpression network is associated with innate immunity and inflammation, and there is significant concordance between its connectivity in humans versus mice (Friedman: p value = 0.0056). This study provides new insights into the proteins and networks that are likely to drive the onset and progression of colon cancer.     

Clinicopathological study of metallothionein immunohistochemical expression, in benign, borderline and malignant ovarian epithelial tumors

Metallothioneins (MTs) are a family of cystein-rich metal-binding proteins, which are expressed in normal cells during fetal and postnatal life but also in a variety of human neoplasms. MT expression in human tumors has been linked to resistance to anticancer drugs and differentiation and progression in some types of tumors. This study examined the immunohistochemical expression of MTs in benign, borderline and malignant tumors of ovarian surface epithelium and the possible correlations with clinicopathological parameters and survival. A total of 87 cases with diagnosis of ovarian surface epithelial tumors were included. Specifically, 21 cases of benign cystadenomas (11 serous and 10 mucinous), 14 borderline (low malignant potential tumors, 8 mucinous and 6 serous) and 52 cases of ovarian cancer were analysed. Immunohistochemical expression of MT (cut-off level > 10% of tumor cells) was clearly associated with malignancy. A statistically significant correlation was found between the expression of MT in cancer cases and benign tumors (p < 0.0001) and cancer cases and borderline tumors p = 0.003. In cancer cases a difference was observed between grade I and III (p = 0.002). There was no correlation of MT overexpression with survival in the small number of ovarian carcinoma patients where it was analysed. MT constitutes a marker that characterizes aggressiveness and a high malignant potential in ovarian epithelial tumors. In diagnostic problems MT may help distinguish between benign, borderline and malignant tumors.     

Increased Expression and Aberrant Localization of Mucin 13 in Metastatic Colon Cancer

MUC13 is a newly identified transmembrane mucin. Although MUC13 is known to be overexpressed in ovarian and gastric cancers, limited information is available regarding the expression of MUC13 in metastatic colon cancer. Herein, we investigated the expression profile of MUC13 in colon cancer using a novel anti-MUC13 monoclonal antibody (MAb, clone ppz0020) by immunohistochemical (IHC) analysis. A cohort of colon cancer samples and tissue microarrays containing adjacent normal, non-metastatic colon cancer, metastatic colon cancer, and liver metastasis tissues was used in this study to investigate the expression pattern of MUC13. IHC analysis revealed significantly higher (p<0.001) MUC13 expression in non-metastatic colon cancer samples compared with faint or very low expression in adjacent normal tissues. Interestingly, metastatic colon cancer and liver metastasis tissue samples demonstrated significantly (p<0.05) higher cytoplasmic and nuclear MUC13 expression compared with non-metastatic colon cancer and adjacent normal colon samples. Moreover, cytoplasmic and nuclear MUC13 expression correlated with larger and poorly differentiated tumors. Four of six tested colon cancer cell lines also expressed MUC13 at RNA and protein levels. These studies demonstrate a significant increase in MUC13 expression in metastatic colon cancer and suggest a correlation between aberrant MUC13 localization (cytoplasmic and nuclear expression) and metastatic colon cancer.     

Individual variation of scavenger receptor expression in human macrophages with oxidized low-density lipoprotein is associated with a differential inflammatory response

Atherosclerosis is an inflammatory disease in which oxidized low-density lipoprotein (oxLDL) plays important roles. Scavenger receptors (SR) CD36, SR-A, and LOX-1 uptake over 90% of the oxLDL leading to foam cell formation and secretion of inflammatory cytokines. To investigate whether the interindividual differences in macrophage SR gene expression could determine the inflammatory variability in response to oxLDL, we quantified the gene and protein expression of SR and inflammatory molecules from macrophages isolated from 18 volunteer subjects and incubated with oxLDL for 1, 3, 6, and 18 h. The individual gene expression profile of the studied SR at 1 h of incubation was highly variable, showing a wide fold-change range: CD36: -3.57-4.22, SR-A: -5.0-4.43, and LOX-1: -1.56-75.32. We identified subjects as high and low responders depending on whether their SR gene expression was above or below the median, showing a different inflammation response pattern. CD36 and LOX-1 gene expression correlated positively with IL-1beta; SR-A correlated negatively with IL-8 and positively with PPARgamma and NF-kappaBIotaA. These results were confirmed in the same subjects 3 mo after the first sampling. Furthermore, a negative correlation existed between CD36 and SR-A at protein level after 18 h of oxLDL incubation (R = -0.926, p = 0.024). These data would suggest that the type of SR could determine the macrophage activation: more proinflammatory when associated to CD36 and LOX-1 than when associated with SR-A.     

Allelic expression of the putative tumor suppressor gene p73 in human fetal tissues and tumor specimens

p73, a proposed tumor suppressor, shares significant amino acid sequence homology with p53. However, p73 is rarely mutated in tumors but it has been suggested that p73 is monoallelically expressed in some tissues. This latter feature would predispose p73 to gene inactivation because a single genetic 'hit' or the loss of the expressed parental allele would leave the cell without p73 activity. We examined the allelic expression of p73 in normal fetal tissues and in ovarian cancer and Wilms' tumor. We found that p73 was biallelically expressed in all fetal tissues, except in brain, where differential expression of the two parental alleles was observed. Biallelic expression of p73 was also observed in paired samples of ovary cancer and Wilms' tumor. Loss of heterozygosity of p73 occurred at relatively low rates in tumors: one of 11 informative samples (9.1%) of ovarian cancer and two of 19 (10.1%) Wilms' tumors. These data demonstrate that p73 is biallelically expressed in most tissues, thus excluding genomic imprinting as a molecular mechanism to predispose to allelic inactivation of p73 in human tumors.     

Long interspersed element-1 open reading frame 1 protein expression profiles in ovarian cancers

Long interspersed element-1 (LINE-1) retrotransposons are autonomous mobile DNA elements with unique activity that account for about one-fifth of the human genome. Recently, it has been reported that the expression of LINE-1 is closely related to cancer prognosis, and LINE-1 hypomethylation might contribute to the acquisition of aggressive tumor behavior. Despite the importance of LINE-1 expression in cancers, research on the expression of LINE-1 open reading frame (ORF) proteins is very limited. Here, we investigated the expression profiles of LINE-1 ORF1p in ovarian cancer tissue microarrays containing 100 surgical specimens including adjacent normal ovary tissue, primary ovarian cancers, and metastatic ovarian cancers in lymph node. The tissue microarray was stained with mouse monoclonal antibody to LINE-1 ORFp1 for immunofluorescence analysis, and expression levels were evaluated by image analysis. LINE-1 ORFp was significantly overexpressed in ovarian cancers compared with normal tissues and especially upregulated in metastatic ovarian cancers. In addition, the expression of LINE-1 ORF1p was significantly higher in older ovarian cancer patients compared with young patients. These results indicate that expression of LINE-1 ORF1p is related to the progression of ovarian cancers and, in particular, to the age of the patient and the metastatic potential of the cancer.     

MAGE-F1, a novel ubiquitously expressed member of the MAGE superfamily

Most known members of the MAGE superfamily are expressed in tumors, testis and fetal tissues, which has been described as a cancer/testis or "CT" expression pattern. We have identified a novel member of this superfamily, MAGE-F1, which is expressed in all adult and fetal tissues tested. In addition to normal tissues, MAGE-F1 is expressed in many tumor types including ovarian, breast, cervical, melanoma and leukemia. MAGE-F1 is encoded on chromosome 3, identifying a sixth chromosomal location for a MAGE superfamily gene. The coding region of MAGE-F1 is contained within a single exon and includes a microsatellite repeat. Sequence analysis and expression profiles define a new class of ubiquitously expressed MAGE superfamily genes that includes MAGE-F1, MAGE-D1, MAGE-D2/JCL-1 and NDN. The finding that several MAGE genes are ubiquitously expressed suggests a role for MAGE encoded proteins in normal cell physiology. Furthermore, potential cross-reactivity to these ubiquitously expressed MAGE gene products should be considered in the design of MAGE-targeted immunotherapies for cancer.     

Stage-specific alterations of DNA methyltransferase expression, DNA hypermethylation, and DNA hypomethylation during prostate cancer progression in the transgenic adenocarcinoma of mouse prostate model

We analyzed DNA methyltransferase (Dnmt) protein expression and DNA methylation patterns during four progressive stages of prostate cancer in the transgenic adenocarcinoma of mouse prostate (TRAMP) model, including prostatic intraepithelial neoplasia, well-differentiated tumors, early poorly differentiated tumors, and late poorly differentiated tumors. Dnmt1, Dnmt3a, and Dnmt3b protein expression were increased in all stages; however, after normalization to cyclin A to account for cell cycle regulation, Dnmt proteins remained overexpressed in prostatic intraepithelial neoplasia and well-differentiated tumors, but not in poorly differentiated tumors. Restriction landmark genomic scanning analysis of locus-specific methylation revealed a high incidence of hypermethylation only in poorly differentiated (early and late) tumors. Several genes identified by restriction landmark genomic scanning showed hypermethylation of downstream regions correlating with mRNA overexpression, including p16INK4a, p19ARF, and Cacna1a. Parallel gene expression and DNA methylation analyses suggests that gene overexpression precedes downstream hypermethylation during prostate tumor progression. In contrast to gene hypermethylation, genomic DNA hypomethylation, including hypomethylation of repetitive elements and loss of genomic 5-methyldeoxycytidine, occurred in both early and late stages of prostate cancer. DNA hypermethylation and DNA hypomethylation did not correlate in TRAMP, and Dnmt protein expression did not correlate with either variable, with the exception of a borderline significant association between Dnmt1 expression and DNA hypermethylation. In summary, our data reveal the relative timing of and relationship between key alterations of the DNA methylation pathway occurring during prostate tumor progression in an in vivo model system.     

miR-145 promotes miR-133b expression through c-myc and DNMT3A-mediated methylation in ovarian cancer cells

Ovarian cancer presents as malignant tumors in the female reproductive system with high mortality. MicroRNAs are involved in the progression of ovarian cancer; however, the regulatory relationship among miRs remains unclear. In our study, we verified that both miR-145 and miR-133b messenger RNA (mRNA) levels in ovarian cancer tissues were lower than in normal ovarian tissues, and their mRNA level in serum of patients with ovarian cancer was reduced. We demonstrated miR-145 targeted c-myc, and c-myc interacted physically with DNMT3A in ovarian cancer cells. We confirmed that c-myc recruited DNMT3A to the miR-133b promoter. miR-133b overexpression also inhibited target gene PKM2 expression along with the Warburg effect. Our results indicate that miR-145 inhibited the Warburg effect through miR-133b/PKM2 pathways, which may improve approaches to ovarian cancer diagnosis and treatment.     

一种新的抑癌候选基因——NDR2在人类正常组织及相应肿瘤中的表达

为观察NDR2基因在人类正常组织及相应肿瘤组织中的表达分布特点,收集人脑与胶质瘤、肺与肺癌、胃与胃癌、结肠与结肠癌组织标本,分别进行组织石蜡切片和总RNA提取,应用免疫组化方法和RT-PCR技术检测NDR2蛋白质及mRNA表达水平,并通过DNA测序验证PCR产物的正确性.免疫组化结果表明,在上述各组织均有NDR2蛋白不同程度的表达.RT-PCR结果显示,在人脑和胶质瘤组织、肺与肺癌组织、胃与胃癌组织、结肠与结肠癌组织中均有NDR2 mRNA表达,其表达水平以脑组织为最高.NDR2 mRNA在正常脑和肺组织的表达水平分别显著高于胶质瘤与肺癌组织,而结肠与结肠癌组织,胃与胃癌组织NDR2 mRNA表达水平则无显著差别.以上结果表明,NDR2基因可能广泛表达于人体正常组织内,而在胶质瘤与肺癌中的表达较相应正常组织减低,提示该基因可能与神经系统及呼吸系统肿瘤的发生发展有关,为进一步探讨该基因功能提供了线索.     

Selective gene expression profiling of mTOR-associated tumor suppressor and oncogenes in ovarian cancer

The aim of this study was to selectively profile the activation status of mammalian target of rapamycin (mTOR)-associated oncogenes and tumor suppressor genes (TSGs) in ovarian cancer specimens, healthy ovaries and benign ovarian tumors, including endometrial cysts. We used a novel type of microfluidic gene array to examine the expression of 15 human tumor suppressors and oncogenes in ovarian cancer specimens of 53 patients, benign ovarian cysts of 29 women (endometrial and simple) and 11 healthy ovaries of individuals in whom the material was obtained during total hysterectomies performed because of fibroid changes. The array was custom-designed to include the following genes: NF1, RHEB, mTOR1, AKT-1, PTEN, TSC1, TSC2, KRAS, RPS6KB1, 4EBP1, TP53, EIF4E, STK11, PIK3CA and BECN1. Confirmatory immunohistochemical detection was performed for a group of selected proteins. Particularly significant differences were observed as to the expression of PTEN (p < 0.0001), TP53 (p = 0.0003), PIK3CA (p = 0.0003) and BECN1 (p = 0.0014) which were shown to be downregulated in cancer patients when compared to healthy ovaries and benign ovarian cysts (endometrial and simple). These markers did not show association with grade or stage of the tumor. Immunohistochemistry showed that PTEN, TP53, PIK3CA and BECN1 proteins are expressed in ovarian cancer. Our results indicate that there are significant differences in the expression of some of the mTOR-related tumor suppressors and oncogenes which could be associated with the pathogenesis of ovarian cancer.     

Identification of a scavenger receptor homologue on nonspecific cytotoxic cells and evidence for binding to oligodeoxyguanosine

In mammals scavenger receptors (SR) are expressed by monocytic-macrophage lineage cells and B-cells. Studies of various teleost species have indirectly demonstrated the presence of SR receptors on phagocytic or endothelial cells by showing the uptake of SR ligands (i.e. derivatised (acetylated) lipoproteins) by these cells. In the present study, nonspecific cytotoxic cells (NCC) were examined for membrane expression of an SR-like protein. Approximately 15-25% of purified NCC expressed scavenger receptor class A (SR-A) demonstrated by binding by a monoclonal (2F8) specific for mouse SR-A (types I, II). Flow cytometric analysis determined that SR binding cells had the same size and 'side scatter' characteristics as NCC. Two colour flow analysis of NCC demonstrated that only a subset of NCC expressed the SR-A-like protein and non-NCC were SR-A negative. Membrane expression of SR on NCC was confirmed by fluorescence microscopy. Analysis of the tissue distribution of SR bearing cells demonstrated that in both catfish and tilapia, SR-A was expressed by NCC in the peripheral blood, spleen and anterior kidney. Experiments were also done to determine if the ligands known to bind mammalian SR-A had a similar specificity for the teleost receptor. Cold competition binding experiments determined that anti-SR-A antibody competed with and reduced biotinylated polyguanosine 20-mer binding to NCC by approximately 40%. Two other types of ligands known to bind (mammalian) SR-A (i.e. polyvinyl sulphate and dextran sulphate) likewise decreased anti-SR-A antibody binding to NCC by 40%. These studies for the first time demonstrated that NCC express the teleost orthologue of mammalian SR-A, suggesting that NCC may participate in physiologic regulation of lipid metabolism in addition to functions of innate immunity.