The Lin28/let-7a/c-Myc pathway plays a role in non-muscle invasive bladder cancer

We investigate the role of the Lin28/let-7a/c-Myc pathway in non-muscle invasive bladder cancer (NMIBC). Using RT-PCR, western blot and immunohistochemistry techniques, the levels of pre-let-7a, let-7a, Lin28 and c-Myc RNA and/or proteins were determined in samples of normal bladder tissue and bladder cancer. Expression of pre-let-7a was found to be negatively correlated with the pathological grade of bladder cancer, while let-7a showed a positive correlation with bladder cancer pathological grade. Expression of Lin28 RNA and protein was not significantly different between normal bladder tissue and low-grade transitional cell carcinoma of bladder (TCC) but the expression levels in high-grade TCC were remarkably increased. Expression of c-Myc RNA and protein was significantly higher in bladder cancer samples in comparison to normal bladder tissue without correlation with cancer differentiation. Expression of all the above RNAs and proteins showed no significant difference in Ta and T1 stages. The Lin28/let-7a/c-Myc pathway plays an important role in NMIBC. In particular, expression levels of let-7a correlate with the degree of cancer differentiation but not cancer stage.     

Overexpression of annexin a1 induced by terephthalic acid calculi in rat bladder cancer

Prolonged cell proliferation in response to irritation by bladder calculi can evoke malignant transformation of the urothelium. However, the molecular mechanisms responsible for calculi-associated bladder carcinogenesis are unknown. We compared the protein expression pattern of rat bladder transitional cell carcinomas (TCCs) induced by terephthalic acid with that of normal bladder tissues using 2-DE. Comparative analysis of the respective spot patterns on 2-DE showed 146 spots that were markedly changed in TCC samples. Subsequently, 56 of the variant protein spots were identified by MALDI-TOF MS. Among them, overexpression of annexin a1 (ANNA1) in rat TCCs was confirmed by Western blotting and real-time RT-PCR analysis. Immunohistochemical staining revealed that ANNA1, usually a cytoplasmic protein in normal urothelium, was translocated to the nucleus in rat bladder cancer cells. In contrast to the animal studies, examination of human clinical specimens showed that ANNA1 expression was reduced in TCC compared to normal urothelium. The expression of ANNA1 was inversely related to the level of differentiation of TCC. Our data suggest that overexpression of ANNA1 is involved in bladder carcinogenesis induced by bladder calculi and that translocation of the protein may be partly responsible for the effect. ANNA1 may serve as a new marker of differentiation for the histopathological grading of human TCC.     

Proteome analysis of human androgen-independent prostate cancer cell lines: variable metastatic potentials correlated with vimentin expression

To better understand the molecular mechanisms of prostate cancer (PCA) dissemination and to develop new anti-metastasis therapies, key regulatory molecules involved in PCA metastasis were identified in two human androgen-independent PCA cell lines, highly metastatic 1E8-H and lowly metastatic 2B4-L cells. Through 2-DE and MS analyses, 12 proteins with different expression levels in the two cell lines were identified. The following proteins were found to be significantly up-regulated in 1E8-H cells compared with 2B4-L cells: gp96 precursor, calreticulin precursor, vimentin (VIM), Hsp90alpha, peroxiredoxin 2, HNRPH1, ezrin, T-complex protein 1, alpha subunit, and hypothetical protein mln2339. In contrast, heart L-lactate dehydrogenase H chain, annexin I, and protein disulfide isomerase were notably down-regulated in 1E8-H cells compared with 2B4-L cells. To our knowledge, this study is the first to demonstrate that up-regulation of VIM expression positively correlates with the invasion and metastasis of androgen-independent PCA.     

Inactivation of the hMSH3 mismatch repair gene in bladder cancer

Deficiency in the DNA mismatch repair (MMR) is frequently involved in various cancers. The hMSH3 gene is one of the human MMR genes whose role in bladder cancer is not known. We hypothesized that down-regulation of the hMSH3 gene might be involved in bladder cancer. In this study we analyzed this gene with regard to frame-shift mutation, single nucleotide polymorphism (SNP), a 9bp repeat in exon 1, loss of heterozygosity (LOH), immunohistochemistry, and methylation status in 102 bladder cancer samples. Immunohistochemistry revealed that hMSH3 expression in bladder cancer was significant decreased compared to normal epithelium (p<0.0001). An inverse correlation with pathological grade was found. The frame-shift mutation in the (A) 8 tract was lacking in bladder cancer. There was no significantly difference between bladder cancer samples and healthy controls' with regard to SNP and the 9bp repeat. In bladder cancer, presence of the codon 222 polymorphism, LOH, and the 9bp repeats in exon 1 had a correlation with either pathological stage or pathological grade. Presence of the codon 1036 polymorphism had significant correlation with pathological stage and a trend to correlation with pathological grade. After 5-aza-dC treatment, MSH3 expression was significantly enhanced in TCC and UMUC bladder cancer cells when compared to untreated cells. This is the first report suggesting that genetic and epigenetic alterations in the human MSH3 gene might play a significant role in the progression of bladder tumors.     

Identification of Gene-Expression Signatures and Protein Markers for Breast Cancer Grading and Staging

The grade of a cancer is a measure of the cancer''s malignancy level, and the stage of a cancer refers to the size and the extent that the cancer has spread. Here we present a computational method for prediction of gene signatures and blood/urine protein markers for breast cancer grades and stages based on RNA-seq data, which are retrieved from the TCGA breast cancer dataset and cover 111 pairs of disease and matching adjacent noncancerous tissues with pathologists-assigned stages and grades. By applying a differential expression and an SVM-based classification approach, we found that 324 and 227 genes in cancer have their expression levels consistently up-regulated vs. their matching controls in a grade- and stage-dependent manner, respectively. By using these genes, we predicted a 9-gene panel as a gene signature for distinguishing poorly differentiated from moderately and well differentiated breast cancers, and a 19-gene panel as a gene signature for discriminating between the moderately and well differentiated breast cancers. Similarly, a 30-gene panel and a 21-gene panel are predicted as gene signatures for distinguishing advanced stage (stages III-IV) from early stage (stages I-II) cancer samples and for distinguishing stage II from stage I samples, respectively. We expect these gene panels can be used as gene-expression signatures for cancer grade and stage classification. In addition, of the 324 grade-dependent genes, 188 and 66 encode proteins that are predicted to be blood-secretory and urine-excretory, respectively; and of the 227 stage-dependent genes, 123 and 51 encode proteins predicted to be blood-secretory and urine-excretory, respectively. We anticipate that some combinations of these blood and urine proteins could serve as markers for monitoring breast cancer at specific grades and stages through blood and urine tests.     

DNA content and p53 protein expression in ductal breast cancer

DNA content and p53 protein expression in ductal breast cancer
The DNA content of 85 ductal breast cancers of different histological grades was evaluated using static cytometry and correlated with immunocytochemical expression of p53 protein in tumour cells in cytological material. A statistically significant difference was observed between p53 protein expression and grade of malignancy ( P <0.001). The percentage of euploid tumours significantly decreased from grade I through grade II to grade III tumours ( P <0.001). Clonal DNA heterogeneity was observed in 26.6% of cases analysed and was correlated with p53 protein expression ( P <0.001). These changes probably reflect genomic alterations which may affect potential malignancy of breast cancer.     

Bladder cancer associated glycoprotein signatures revealed by urinary proteomic profiling

Current methods in the noninvasive detection and surveillance of bladder cancer via urine analysis include voided urine cytology (VUC) and some diagnostic urinary protein biomarkers; however, due to the poor sensitivity of VUC and high false-positive rates of currently available protein assays, detection of bladder cancer via urinalysis remains a challenge. In the study presented here, a rapid, high-sensitivity technique was developed to profile the N-linked glycoprotein component in naturally micturated human urine specimens. Concanavalin A (Con A) affinity chromatography coupled to nanoflow liquid chromatography was utilized to separate the complex peptide mixture prior to a linear ion trap MS analysis. Of 186 proteins identified with high confidence by multiple analyses, 40% were secreted proteins, 18% membrane proteins, and 14% extracellular proteins. In this study, the presence of several proteins appeared to be associated with the presence of bladder cancer, including alpha-1B-glycoprotein that was detected in all tumor-bearing patient samples but in none of the samples obtained from non-tumor-bearing individuals. The combination of Con A affinity chromatography and nano-LC/MS/MS provides an initial investigation of N-glycoproteins in complex biological samples and facilitates the identification of potential biomarkers of bladder cancer in noninvasively obtained human urine.     

血清SELDI蛋白质指纹图谱在乳腺癌术前分级中的应用

应用SELDI技术和生物信息学方法从血清中筛选能反映乳腺癌术前分期的蛋白质峰并构建检测模型。采用CM10芯片,对34例Ⅰ～Ⅱ期乳腺癌患者和31例Ⅲ～Ⅳ期乳腺癌的血清进行了检测,发现11个蛋白质峰在两组患者之间表达量有显著性差异（P<0.05）,M/Z为 M2042.87、M2459.83、M3881.37、M4804.47、M6683.24和 M6706.06的6个蛋白质峰被选为分类变量构成决策树分类模型,该模型的交叉验证（测试组）总准确率为80.0%,Ⅰ～Ⅱ期乳腺癌检出率为82.4%,Ⅲ～Ⅳ期检出率为77.4%。SELDI技术在乳腺癌患者术前分级的判断方面具有一定的应用价值。     

Polymorphic methyl group metabolism genes in patients with transitional cell carcinoma of the urinary bladder.

Because polymorphisms in the methyl group metabolism genes methylene-tetrahydrofolate reductase (MTHFR), methionine synthase (MS), and cystathione beta-synthetase (CBS) affect plasma homocysteine levels and intracellular concentrations of S-adenosylmethionine (SAM), they modify the susceptibility to cardiovascular diseases and cancer. Specifically, genome-wide decreased DNA methylation ('hypomethylation') in human cancers might be a consequence of decreased SAM levels. Because hypomethylation is particularly prevalent in transitional cell carcinoma of the urinary bladder (TCC), the genotype distributions for the two each most prevalent MTHFR, MS, and CBS alleles were compared between 165 TCC patients and 150 population controls. The distributions of the MTHFR 677A/V and the MS 919G/D alleles were not significantly different between cancer patients and controls, even after stratification according to age, gender, tumor stage or grade. The CBS 844INS68 allele was slightly less frequent in TCC patients than in controls (q=0.07 versus 0.10), but was rarer among males in both groups. Among the TCC patients, this gender difference was highly significant (Mantel-Haenszel and chi(2)-test P=0.007). No significant difference between TCC patients and controls was found for any combined genotype. Likewise, the extent of DNA hypomethylation determined in 62 carcinoma specimens was not related to the respective genotypes. Thus, on their own, the MTHFR, MS and CBS genotypes do not appear to act upon susceptibility to TCC or influence the extent of DNA hypomethylation in this cancer.     

Identification and analysis of altered alpha1,6-fucosylated glycoproteins associated with hepatocellular carcinoma metastasis

Tumor metastasis might be associated with the expression levels of cellular glycoproteins and the alteration of their glycan parts. In order to screen the aberrantly alpha1,6-fucosylated glycoproteins related to hepatocellular carcinoma (HCC) metastasis, a high-throughput glycomic approach which consisted of 2-DE, electronic transfer of proteins, lectin affinity blot and precipitation, and MALDI-TOF-MS/MS, was established. Lens culinaris agglutinin (LCA) affinity glycoprotein profiles of higher and lower metastatic HCC cell lines were compared and analyzed. Seven out of 34 identified glycoproteins were differentially displayed; they were cytokeratin 8 (CK8), annexin I, annexin II, heterogeneous nuclear ribonucleoprotein A/B, PDZ and LIM domain 1, RNA-binding motif protein 4, and poly(rC)-binding protein 1. On comparison with Hep3B, CK8 showed a higher affinity to Ricinus communis agglutinin 1 (RCA-I) and LCA, and annexin I presented a higher affinity to LCA and Con A by the lectin-binding assay. Furthermore, the up-regulation of CK8, annexin I, and annexin II were found by Western blot and immunofluorescence analysis in higher metastatic HCC cell lines. This implied that the alteration of CK8, annexin I, and annexin II both in their expression levels and their glycan parts might be related to metastatic ability, and play a critical role in the process of HCC metastasis.     

Identification of annexin II as a novel secretory biomarker for breast cancer

Early prediction of metastatic breast cancer is important for improvement of prognosis and survival rate. The present study aimed to identify secreted protein biomarkers for detection of invasive breast cancer. To this end, we performed a comparative proteomic analysis by a combination of 2DE and MALDI‐TOF MS analysis of conditioned media from invasive H‐Ras MCF10A human breast epithelial cells and noninvasive MCF10A and N‐Ras MCF10A cells. We identified a list of 25 proteins that were strongly detected in media of H‐Ras MCF10A and focused on annexin II, which was shown to be involved in cell motility. Invasive triple‐negative human breast carcinoma cells, Hs578T, and MDA‐MB‐231, showed increased levels of annexin II in media, demonstrating that secretion of annexin II correlated well with the invasive phenotype of cells. We demonstrated a crucial role of annexin II in breast cell invasion/migration and actin cytoskeleton reorganization required for filopodia formation. Annexin II levels in the plasma samples and breast cancer tissues of breast cancer patients were significantly higher than those of normal groups, providing a clinical relevance to our in vitro findings. Taken together, we identified annexin II as a novel secretory biomarker candidate for invasive breast cancer, especially estrogen receptor‐negative breast cancer.     

Proteomic profiling identifies breast tumor metastasis-associated factors in an isogenic model

A combination of LC and MS was applied to an isogenic breast tumor metastasis model to identify proteins associated with a cellular phenotype. Chromatofocusing followed by nonporous-RP-HPLC/ESI-TOF MS was applied to cell lysates of a pair of monoclonal cell lines from the human breast carcinoma cell line MDA-MB-435 that have different metastatic phenotypes in immune-compromised mice. This method was developed to separate proteins based on pI and hydrophobicity. The high resolution and mass accuracy of ESI-TOF measurements provided a good correlation of theoretical MW and experimental Mr values of intact proteins measured in mass maps obtained in the pH range 3.8-6.4. The isolated proteins were digested by trypsin and analyzed by MALDI-TOF MS, MALDI-QIT-TOF MS, and monolith-based HPLC/MS/MS. The unique combination of the techniques provided valuable information including quantitation and modification of proteins. We identified 89 selected proteins, of which 43 were confirmed as differentially expressed. Metastasis-associated proteins included galectin-1, whereas annexin I and annexin II were associated with the nonmetastatic phenotype. In this study, we demonstrate that combining a variety of MS tools with a multidimensional liquid-phase separation provides the ability to map cellular protein content, to search for modified proteins, and to correlate protein expression with cellular phenotype.     

Surgical site infections following colorectal cancer surgery: a randomized prospective trial comparing common and advanced antimicrobial dressing containing ionic silver

Background

The role of annexin II in the development and progression of gastric cancer was explored.

Methods

Real-time PCR was conducted to detect annexin II and S100A6 mRNA expression. Protein expressions of annexin II and S100A6 were also examined by immunohistochemistry in 436 clinicopathologically characterized gastric cancer cases.

Results

The expression of annexin II and S100A6 mRNA differ significantly among gastric tumor tissue and matched non-cancerous gastric mucosa. Protein levels of annexin II and S100A6 were up-regulated in gastric cancer compared with adjacent non-cancerous tissues. High expression of annexin II correlated with age, location of tumor, size of tumor, differentiation, histological type, depth of invasion, vessel invasion, lymph node metastasis, distant metastasis and Tumor, Node, Metastasis (TNM) stage, and also with expression of S100A6. Further multivariate analysis suggested that expression of annexin II and S100A6 were independent prognostic indicators for gastric cancer. Cumulative five-year survival rates of patients with high expression of both annexin II and S100A6 was significantly lower than those with low expression of both.

Conclusion

Expression of annexin II in gastric cancer was significantly associated with depth of invasion, lymph node metastasis and distant metastasis, TNM stage, high S100A6 expression, and poor prognosis. Annexin II and S100A6 proteins could be useful prognostic marker to predict tumor progression and prognosis in gastric cancer.     

Proteomic and transcriptomic analyses of differential stress/inflammatory responses in mandibular lymph nodes and oropharyngeal tonsils of European wild boars naturally infected with Mycobacterium bovis

Differential stress/inflammatory responses were characterized at the mRNA and protein levels in mandibular lymph nodes (MLN) and oropharyngeal tonsils of European wild boars (Sus scrofa), naturally infected with Mycobacterium bovis. Suppression-subtractive hybridization combined with immunohistochemistry and/or quantitative real-time RT-PCR were used to identify and characterize abundant stress/inflammatory gene sequences differentially expressed in tuberculous (TB+) wild boars. Genes identified in MLN and tonsils corresponded to serum amyloid A, arginase I, osteopontin, lysozyme, annexin I, and heat shock proteins, respectively. Global protein patterns in MLN and tonsils were compared between TB+ and nontuberculous (TB-) boars by 2-DE and MALDI-TOF MS. Five proteins, including stress/inflammatory proteins annexin V, serum albumin, and apolipoprotein A1 were found at lower levels in MLN of TB+ boars. Manganese superoxide dismutase was found up-regulated in MLN of TB+ boars. Five proteins, including creatine kinase and MHC class II antigens were found up-regulated in tonsils of TB+ boars. These results demonstrated differential stress/inflammatory responses in wild boars naturally infected with M. bovis and suggest possible markers of tuberculosis in this species that may prove useful for future studies of host-pathogen interactions and for diagnostics and vaccine development.     

Overexpression of annexin A2 is associated with abnormal ubiquitination in breast cancer

Abnormal expression of annexin A2 contributes to metastasis and infiltration of cancer cells.To elucidate the cause of abnormal expression of annexin A2,Western blotting,immunoproteomics and immunohistochemical staining were performed to analyze differentially ubiquitinated proteins between fresh breast cancer tissue and its adjacent normal breast tissue from five female volunteers.We detected an ubiquitinated protein that was up-regulated in the cancer tissue,which was further identified as annexin A2 by mass spectrometry.These results suggest that abnormal ubiquitination and/or degradation of annexin A2 may lead to presence of annexin A2 at high level,which may further promote metastasis and infiltration of the breast cancer cells.     

Analysis of AGE modified proteins and RAGE expression in HER2/neu negative invasive ductal carcinoma

Cancer is associated with increased glycolysis and carbonyl stress. In view of this, AGE modified proteins were identified from clinical breast cancer tissue using 2DE-immunoblot and mass-spectrometry. These proteins were identified to be serotransferrin, fibrinogen gamma chain, glycerol-3-phosphate dehydrogenase, lactate dehydrogenase, annexin II, prohibitin and peroxiredoxin 6, which have established role in cancer. Further, RAGE expression and its downstream signaling proteins NADPH oxidase and NF-kB were studied. Role of these AGE modified proteins and RAGE signaling in breast cancer is discussed.     

Decorin antagonizes IGF receptor I (IGF-IR) function by interfering with IGF-IR activity and attenuating downstream signaling

We have recently discovered that the insulin-like growth factor receptor I (IGF-IR) is up-regulated in human invasive bladder cancer and promotes migration and invasion of transformed urothelial cells. The proteoglycan decorin, a key component of the tumor stroma, can positively regulate the IGF-IR system in normal cells. However, there are no available data on the role of decorin in modulating IGF-IR activity in transformed cells or in tumor models. Here we show that the expression of decorin inversely correlated with IGF-IR expression in low and high grade bladder cancers (n = 20 each). Decorin bound with high affinity IGF-IR and IGF-I at distinct sites and negatively regulated IGF-IR activity in urothelial cancer cells. Nanomolar concentrations of decorin promoted down-regulation of IRS-1, one of the critical proteins of the IGF-IR pathway, and attenuated IGF-I-dependent activation of Akt and MAPK. This led to decorin-evoked inhibition of migration and invasion upon IGF-I stimulation. Notably, decorin did not cause down-regulation of the IGF-IR in bladder, breast, and squamous carcinoma cells. This indicates that decorin action on the IGF-IR differs from its known activity on other receptor tyrosine kinases such as the EGF receptor and Met. Our results provide a novel mechanism for decorin in negatively modulating both IGF-I and its receptor. Thus, decorin loss may contribute to increased IGF-IR activity in the progression of bladder cancer and perhaps other forms of cancer where IGF-IR plays a role.     

Differential expression of annexins I and II in normal and malignant human mammary epithelial cells

Abstract. Collagen-binding proteins ( CBPs ) of rat mammary tumors are identical to Ca²⁺-binding annexins [49]. We have now isolated a protein of 38 kDa from the human mammary tumor cell line ALAB by collagen type I affinity chromatography as well as by extraction of calcium-binding proteins. The 38-kDa band of both preparations was identified as annexin II (calpactin I) by its reaction with an annexin II-specific monoclonal antibody in Western blot analysis. Annexin I (lipocortin I) was not detectable in these cells. Two other human cell lines, the SV40-transformed cell line SV3 and cell line HBL-100, both established from normal mammary glands, were also positive for annexin II and negative for annexin I.
In vivo expression of annexins was investigated by immunohistological staining of normal and malignant human mammary tissue. The annexin II-specific mAb reacted with normal and tumor parenchyme whereas the annexin I-specific mAb reacted with acini and ductal myoepithelium of the normal mammary gland but showed no reaction with tumor tissue. Immunolocalization studies also showed annexin II expression in both normal and tumor stroma while only tumor stromal cells were found to be reactive with the antibody against annexin I. The differential expression of annexins in normal and malignant human mammary tissue suggests special functions of these proteins in the mammary gland.     

Proteomic tracking of serum protein isoforms as screening biomarkers of ovarian cancer

Epithelial ovarian cancer is the fourth leading cause of cancer death among women. Due to the asymptomatic nature and poor survival characteristic of the disease, screening for specific biomarkers for ovarian cancer is a major health priority. Differentially expressed proteins in the serum of ovarian cancer patients have the potential to be used as cancer-specific biomarkers. In this study, proteomic methods were used to screen 24 serum samples from women with high-grade ovarian cancer and compared to a control group of 11 healthy women. Affigel-Blue treated serum samples were processed either by linear (pH 4-7) or narrow range (pH 5.5-6.7) IEF strips for the first dimension. Proteins separated in first dimension were resolved by 8-16% gradient SDS-PAGE. Protein spots were visualized by SYPRO Ruby staining, imaged by FX-imager and compared and analyzed by PDQuest software. Twenty-two protein spots were consistently differentially expressed between normal and ovarian cancer patients by resolving proteins in a linear pH strip of 4-7 for the first dimension. Six of the protein spots, significantly up-regulated in grade 3 ovarian cancer patients (p < 0.05), were identified by MALDI-TOF MS and Western blotting as the isoforms of haptoglobin precursor. When serum proteins were resolved on narrow pH range strips (5.5-6.7), 23 spots were consistently differentially expressed between normal and grade 3 ovarian cancer patients. Of these, 4 protein spots significantly down regulated in grade 3 ovarian cancer patients (p < 0.05) were identified by MALDI-TOF MS and Western blotting, as isoforms of transferrin precursor. Increased expression of serum haptoglobin and transferrin was also identified in peritoneal tumor fluid obtained from women diagnosed with grade 2/3 ovarian cancer (n = 7). Changes in the expression of haptoglobin and transferrin in the serum of women with different pathological grades of ovarian cancer was examined by one-dimensional Western blotting method. Serum samples collected from women suffering from benign, borderline, grade 1, grade 2 and grade 3 cancer (n = 4 for haptoglobin and n = 5 for transferrin in each group) were analyzed and compared to the serum of normal healthy women. The mean serum haptoglobin expression in grade 3 ovarian cancer patients was fourfold higher than in the control subjects (p < 0.05). On the other hand, transferrin expression in grade 3 ovarian cancer patients was decreased by twofold than in normal healthy women (p < 0.05). Haptoglobin expression in the serum of cancer patients (n = 7) decreased following chemotherapy (six cycles of taxol/carboplatin). Concomitant with the decrease of haptoglobin, transferrin expression remained constant in four patients, but increased in three out of seven patients included in the study. Changes in serum expression of haptoglobin correlated with the change of CA 125 levels before and after chemotherapy. In conclusion, proteomic profiling of differentially expressed proteins in the sera of normal women compared to women with ovarian cancer can greatly facilitate the discovery of a panel of biomarkers that may aid in the detection of ovarian cancer with greater specificity.