Expression of pim-1 in Tumors,Tumor Stroma and Tumor-Adjacent Mucosa Co-Determines the Prognosis of Colon Cancer Patients

Provirus integration site for Moloney murine leukemia virus (pim-1) is a proto-oncogene that is linked to the development and progression of several cancers. In this study, we evaluated pim-1 expression in tumors, tumor stroma and tumor-adjacent mucosa together as an independent prognostic factor for colon cancer patients. The study included 343 colon cancer patients. Immunohistochemical staining was used to detect pim-1. Multivariate cox regression for disease-free survival (DFS) were used to identify independent prognostic factors. Analytic hierarchy process (AHP) was used to calculate the weight of pim-1 in tumors, tumor stroma and tumor-adjacent mucosa in order to obtain a Pim-1 total score (PTS) for recurrence and survival. Kaplan–Meier DFS curves and OS curves for patients with different pim-1 expression levels were compared using the log-rank test. In this study, four independent prognostic factors were identified for colon cancer patients: pim-1 expression in tumors, tumor stroma, tumor-adjacent mucosa, as well as tumor stage. It has been established that clinical stage is an important prognostic factor for colon cancer patients. However, PTS can identify the patients who are likely to recur not only in the whole radical excision group but also within each stage of this group. Based on the results of this study we can conclude that the PTS combined with clinical staging system may be a better predictor of colon cancer patients’ prognosis than using the clinical stage system alone. Clinical Trials Gov. Number: ChiCTR-PRCH-12002842     

Functional activity of ABC transporters (markers of multidrug resistance) in human colon adenocarcinoma and normal colonic mucosa]

Functional activity of multidrug resistance (MDR) markers (total activity of ABC-transporters, P-glycoprotein (Pgp) and multidrug resistance-associated protein (MRP) activities) in human colon adenocarcinoma and normal mucosa was examined. Functional activity of ABC-transporters was revealed in all colon tumors and in 70% of normal mucosa samples investigated. Expression of Pgp and MRP functional activity was determined in about 50% and 70% of colon tumors respectively. Pgp+MRP+ phenotype was determined in 36% of normal mucosa and adenocarcinoma samples. Expression of Pgp+MRP- phenotype was practically the same in normal mucosa and tumors (in 10 and 18% of samples respectively). Pgp-MRP+ phenotype was revealed two times more often in tumors than in mucosa--in 36 and 18% respectively. On the contrary, Pgp-MRP- phenotype was detected more rarely in tumors than in mucosa (in 10 and 36% of samples respectively). Transporters different from Pgp and MRP were also determined in some tumors and normal mucosa. At the patients with expression of Pgp function in normal mucosa the activity of the transporter was revealed in 25% of tumor samples only. On the contrary, at the patients with expression of MRP function in normal mucosa the activity of the transporter was revealed in 70% of tumor samples. At the patients with no expression of Pgp or MRP activity in normal mucosa the function of the transporters in tumors was determined in 60% and 70% of samples respectively. It is concluded that functional activity of various ABC-transporters (Pgp, MRP and other different from Pgp and MRP) is expressed in human colon adenocarcinoma; expression of ABC-transporters functional activity in normal mucosa does not predict MDR phenotype of the tumor.     

A Proteomics Analysis Reveals 9 Up-Regulated Proteins Associated with Altered Cell Signaling in Colon Cancer Patients

Colorectal cancer is the second most common cancer in women and third most common cancer in men. Cell signaling alterations in colon cancer, especially in aggressive metastatic tumors, require further investigations. The present study aims to compare the expression pattern of proteins associated with cell signaling in paired tumor and non-tumor samples of patients with colon cancer, as well as to define the cluster of proteins to differentiate patients with non-metastatic (Dukes’ grade B) and metastatic (Dukes’ grade C&D) colon cancer. Frozen tumor and non-tumor samples were collected after tumor resection from 19 patients with colon cancer. The Panorama? Antibody Microarray-Cell Signaling kits were used for the analyses. The expression ratios of paired tumor/non-tumor samples were calculated for the each protein. We employed R packages ‘samr’, ‘gplots’, ‘supclust’ (pelora, wilma algorithms), ‘glmnet’ for the differential expression analysis, supervised clustering and penalized logistic regression. Significance analysis of microarrays revealed 9 significantly up-regulated proteins, including protein kinase C gamma, c-Myc, MDM2, pan cytokeratin, and 1 significantly down-regulated protein (GAP1) in tumoral mucosa. Pan-cytokeratin and APP were up-regulated in tumor versus non-tumor tissue, and were selected in the predictive cluster to discriminate colon cancer type. Higher levels of S-100b and phospho-Tau-pSer199/202 were confirmed as the predictors of non-metastatic colon cancer by all employed regression/clustering methods. Deregulated proteins in colon cancer are involved in oncogenic signal transduction, cell cycle control, and regulation of cytoskeleton/transport. Further studies are needed to validate potential protein markers of colon cancer development and metastatic progression.     

CHAF1A和ERα在宫颈鳞状细胞癌中的表达和临床意义

该研究探讨染色质组装因子1亚基(CHAF1A)和雌激素受体1(ERα)在宫颈鳞状细胞癌(CSCC)组织中的表达,探究CHAF1A和ERα的关系及其对CSCC潜在的临床意义,采用了免疫组织化学染色法检测CHAF1A和ERα在CSCC组织及对应的癌旁组织中的表达情况;从CSCC组织及对应癌旁组织中提取总RNA,RT-PCR...     

Cytosolic levels of an estrogen-induced breast cancer-associated peptide (TFF1/pS2) in colorectal cancer: clinical significance and relationship with steroid receptors

BACKGROUND: The Trefoil Factor 1 (TFF1/pS2), a peptide consisting of 60 amino acids, is the most abundant estrogen-induced messenger RNA in MCF-7 breast cancer cells and is also expressed by colorectal carcinomas. The objective of this work was to evaluate the cytosolic TFF1 content in colorectal carcinomas, its possible relationship with estrogen and progesterone receptors as well as with clinicopathological tumor parameters, and its potential prognostic significance. METHODS: Cytosolic TFF1 levels were examined by immunoradiometric assay in 178 patients with resectable colorectal cancer. The mean follow-up period was 32 months. RESULTS: There was a wide variability of cytosolic TFF1 levels in tumor-surrounding mucosa samples (0.09-42.5 ng/mg protein) as well as in tumors (0.01-270 ng/mg protein). Comparison of paired mucosa and carcinoma samples showed significantly higher TFF1 levels in tumors (mean: 17.1 ng/mg protein) than in mucosa samples (10 ng/mg protein) (p = 0.027). TFF1 levels were significantly higher in mucosa samples surrounding distal colon and rectal tumors (p = 0.0001) and in tumor samples obtained from older patients (p = 0.007). However, there were no significant differences in tumor TFF1 levels with respect to clinicopathological parameters such as the patient's sex, tumor location, stage, histological grade, ploidy, S-phase, or tumor estrogen and progesterone receptors. In addition, there was no significant relationship between tumor TFF1 levels and disease outcome. CONCLUSIONS:TFF1 may play an as yet undetermined role in the tumorigenesis of colorectal carcinomas. However, cytosolic levels of TFF1 do not seem to have any prognostic significance in colorectal carcinomas.     

Fascin在结肠癌中表达的临床病理学研究

目的:Fascin是一种肌动蛋白结合蛋白,在多种上皮性肿瘤中高表达并与肿瘤侵袭有关。在本研究中观察fascin在结肠癌中表达及探讨其临床病理意义,为其在结肠癌早期诊断和预后预测方面的应用提供依据。方法:应用免疫组织化学技术检测Fascin在结肠肿瘤中的表达,并分析其表达的结肠癌临床病理意义。结果:Fascin在癌旁肠粘膜、腺瘤、腺癌中表达有显著性差异,其中癌旁肠粘膜组和腺瘤组阳性表达率无显著性差异(X2=0.344,P0.05),腺癌组阳性表达率显著高于癌旁粘膜组(X2=8.492,P0.0,1),腺癌组阳性表达率显著高于腺瘤组(X2=7.450,P0.01)。Fascin表达与结肠癌患者的年龄、性别、肿瘤浸润肠壁深度、淋巴结转移、分化程度无显著相关性,而在中-晚期病例(III/IV期)中的表达率显著高于早期病例(P0.05)。Fascin表达阳性病例的生存期显著高于Fascin表达阴性病例(P0.022)。结论:Fascin表达与结肠癌发生和预后密切相关,可能作为结肠癌早期诊断和预后的标志物。     

MT1F mRNA在结肠癌组织中的表达及其临床病理意义

目的:探讨MT1F mRNA在结肠癌组织中的表达及其临床病理意义。方法:采用Taq Man探针实时荧光定量Real-time PCR检测40例结肠癌及对应正常粘膜组织中MT1F mRNA的表达,并通过免疫组化检测Metallothionein(MT)的蛋白表达。结果:82.5%(33/40例)的结肠癌组织MT1F mRNA表达较对应正常组织明显下调,降低2.4倍至113倍不等。MT1F mRNA水平与结肠癌患者的年龄、性别、肿瘤部位、肉眼形态、直径、分化及Dukes分期无关。在MT1F mRNA低表达的病例中,癌组织MT蛋白表达低于对应正常组织(P0.05)。结论:结肠癌组织MT1F mRNA水平显著下调,但与结肠癌的临床病理特征无关。     

朊蛋白在结肠癌及结肠炎组织中的表达及其临床意义

目的：观察朊蛋白（PrPc）在正常结肠粘膜、结肠炎及结肠癌组织中的表达,并探讨其临床意义。方法：采用免疫组织化学法分别检测PrPc在正常结肠组织、结肠炎及结肠癌组织（各40例）中的表达,并分析其在结肠癌及结肠炎组织中的表达与患者性别、分化程度、TNM分期、淋巴结转移及炎症程度等临床特征间的关系。结果：PrPc在正常结肠及结肠癌组织中均有表达,在结肠炎中表达较低,在结肠癌组织中阳性表达率为60％（24／40）,明显高于正常结肠组织的35％（14／40）及结肠炎组织的30％（12／40）（P〈0．05）。轻度结肠炎中PrPc的阳性表达率明显高于重度结肠炎,结肠癌中PrPc的表达与患者的肿瘤分级、分期显著相关（P〈0．05）。结论：PrPc在结肠癌中的表达增高,在结肠炎中表达较低,可作为临床判断结肠癌恶性程度及结肠炎炎症程度的重要参考指标。     

Five candidate biomarkers associated with the diagnosis and prognosis of cervical cancer

Purpose: Cervical cancer (CC) is one of the most general gynecological malignancies and is associated with high morbidity and mortality. We aimed to select candidate genes related to the diagnosis and prognosis of CC.Methods: The mRNA expression profile datasets were downloaded. We also downloaded RNA-sequencing gene expression data and related clinical materials from TCGA, which included 307 CC samples and 3 normal samples. Differentially expressed genes (DEGs) were obtained by R software. GO function analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of DEGs were performed in the DAVID dataset. Using machine learning, the optimal diagnostic mRNA biomarkers for CC were identified. We used qRT-PCR and Human Protein Atlas (HPA) database to exhibit the differences in gene and protein levels of candidate genes.Results: A total of 313 DEGs were screened from the microarray expression profile datasets. DNA methyltransferase 1 (DNMT1), Chromatin Assembly Factor 1, subunit B (CHAF1B), Chromatin Assembly Factor 1, subunit A (CHAF1A), MCM2, CDKN2A were identified as optimal diagnostic mRNA biomarkers for CC. Additionally, the GEPIA database showed that the DNMT1, CHAF1B, CHAF1A, MCM2 and CDKN2A were associated with the poor survival of CC patients. HPA database and qRT-PCR confirmed that these genes were highly expressed in CC tissues.Conclusion: The present study identified five DEmRNAs, including DNMT1, CHAF1B, CHAF1A, MCM2 and Kinetochore-related protein 1 (KNTC1), as potential diagnostic and prognostic biomarkers of CC.     

Increased expression of calcineurin in human colorectal adenocarcinomas

Colorectal cancer (CRC) is the third most common cause of cancer death in the Western world. Calcineurin (CaN), a Ca2+/calmodulin (CaM)-dependent protein phosphatase, is important for Ca2+-mediated signal transduction. The main objective of this study is to examine the potential role of Ca2+/CaM-dependent protein phosphatase in both normal and in invasive tumor components of human samples. In this study, we carried out 45 cases of CaN activity, 13 cases of CaN protein expression by Western blot analysis, and 6 cases for immunohistochemical analysis in both normal and invasive tumor components of human samples. Immunohistochemical analysis revealed that strong cytoplasmic staining of varying intensity was observed in colon tumors of all patients compared to normal mucosa. In addition, Western blot analysis revealed a prominent overexpressed immunoreactive band with an apparent molecular mass of 60 kDa catalytic alpha subunit (CaN A) as well as CaN Aalpha and beta in colon tumor samples. Elevated CaN protein expression appears to be a possible link between Ca2+ signaling and oncogenic processes.     

Tumor-specific expression of the novel cytochrome P450 enzyme, CYP2W1

A novel human cytochrome P450, CYP2W1, was cloned and expressed heterologously. No or very low CYP2W1 mRNA levels were detected in fetal and adult human tissues, expression was however seen in 54% of human tumor samples investigated (n=37), in particular colon and adrenal tumors. Western blotting also revealed high expression of CYP2W1 in some human colon tumors. In rat tissues, CYP2W1 mRNA was expressed preferentially in fetal but also in adult colon. The CYP2W1 gene was shown to encompass one functional CpG island in the exon 1-intron 1 region which was methylated in cell lines lacking CYP2W1 expression, but unmethylated in cells expressing CYP2W1. Re-expression of CYP2W1 was seen following demethylation by 5-Aza-2'-deoxycytidine. Transfection of HEK293 cells with CYP2W1 caused the formation of a properly folded enzyme, which was catalytically active with arachidonic acid as a substrate. It is concluded that CYP2W1 represents a tumor-specific P450 isoform with potential importance as a drug target in cancer therapy.     

Arf6, RalA and BIRC5 protein expression in non small cell lung cancer

Evaluation of tumor markers expression pattern which determines individual progression parameters is one of the major topics in molecular oncopathology research. This work presents research on expression analysis of several Ras-Ral associated signal transduction pathway proteins (Arf6, RalA and BIRC5) in accordance with clinical criteria in non small cell lung cancer patients. Using Western-blot analysis and RT-PCR Arf6, RalA and BIRC5 expression has been analyzed in parallel in 53 non small cell lung cancer samples of different origin. Arf6 protein expression was elevated in 55% non small cell lung cancer tumor samples in comparison with normal tissue. In the group of squamous cell lung cancer Arf6 expression elevation was observed more often. RalA protein expression was decreased in comparison to normal tissue samples in 64% of non small cell lung cancer regardless to morphological structure. Correlation between RalA protein expression decrease and absence of regional metastases was revealed for squamous cell lung cancer. BIRC5 protein expression in tumor samples versus corresponding normal tissue was 1.3 times more often elevated in the squamous cell lung cancer group (in 76% tumor samples). At the same time elevation of BIRC5 expression was fixed only in 63% of adenocarcinoma tumor samples. A statistically significant decrease (p = 0.0158) of RalA protein expression and increase (p = 0.0498) of Arf6 protein expression in comparison with normal tissue was found for T1-2N0M0 and T1-2N1-2M0 groups of squamous cell lung cancer correspondingly.     

Tumor-infiltrating lymphocytes in colorectal tumors display a diversity of T cell receptor sequences that differ from the T cells in adjacent mucosal tissue

Tumors from colorectal cancer (CRC) are generally immunogenic and commonly infiltrated with T lymphocytes. However, the details of the adaptive immune reaction to these tumors are poorly understood. We have accrued both colon tumor samples and adjacent healthy mucosal samples from 15 CRC patients to study lymphocytes infiltrating these tissues. We apply a method for detailed sequencing of T-cell receptor (TCR) sequences from tumor-infiltrating lymphocytes (TILs) in CRC tumors at high throughput to probe T-cell clones in comparison with the TCRs from adjacent healthy mucosal tissue. In parallel, we captured TIL counts using standard immunohistochemistry. The variation in diversity of the TIL repertoire was far wider than the variation of T-cell clones in the healthy mucosa, and the oligoclonality was higher on average in the tumors. However, the diversity of the T-cell repertoire in both CRC tumors and healthy mucosa was on average 100-fold lower than in peripheral blood. Using the TCR sequences to identify and track clones between mucosal and tumor samples, we determined that the immune response in the tumor is different than in the adjacent mucosal tissue, and the number of shared clones is not dependent on distance between the samples. Together, these data imply that CRC tumors induce a specific adaptive immune response, but that this response differs widely in strength and breadth between patients.     

The mitochondrial protein C1qbp promotes cell proliferation,migration and resistance to cell death

Complement 1q-binding protein (C1qbp) is a mitochondrial protein reported to be upregulated in cancer. However, whether C1qbp plays a tumor suppressive or tumorigenic role in the progression of cancer is controversial. Moreover, the exact effects of C1qbp on cell proliferation, migration and death/survival have not been definitely proven. To this end, we comprehensively examined the effects of C1qbp on mitochondrial-dependent cell death, proliferation and migration in both normal and breast cancer cells using genetic gain- and loss-of-function approaches. In normal fibroblasts, overexpression of C1qbp protected the cells against staurosporine-induce apoptosis, increased proliferation, decreased cellular ATP and increased cell migration in a wound-healing assay. In contrast, the opposite effects were observed in fibroblasts depleted of C1qbp by RNA interference. C1qbp expression was found to be markedly elevated in 4 different human breast cancer cell lines as well as in ductal and adenocarcinoma tumors from breast cancer patients. Stable knockdown of C1qbp by shRNA in the aggressive MDA-MB-231 breast cancer cell line greatly reduced cell proliferation, increased ATP levels and decreased cell migration compared with control shRNA-transfected cells. Moreover, C1qbp knockdown elicited a significant increase in doxorubicin-induced apoptosis in the MDA-MB-231 cells. Finally, C1qbp upregulation was not restricted to breast cancer cells and tumors, as levels of C1qbp were also found to be significantly elevated in both human lung and colon cancer cell lines and carcinomas. Together, these results establish a pro-tumor, rather than antitumor, role for C1qbp and indicate that C1qbp could serve as a molecular target for cancer therapeutics.Key words: mitochondria, cell proliferation, cell migration, cell death, tumor cells     

Down-regulation of tumor suppressor in lung cancer 1 (TSLC1) expression correlates with poor prognosis in patients with colon cancer

Tumor suppressor in lung cancer 1 (TSLC1), a novel tumor suppressor gene, has been reported to be frequently inactivated in a variety of human malignant tumors. The aim of this study was to detect TSLC1 expression in human colon cancer and to analyze its association with prognosis of patients with colon cancer. Using quantitative real-time PCR and Western blot analysis, we found significantly decreased expression of TSLC1 in primary colon tumor tissues (n?=?30) compared with adjacent normal tissues. Immunohistochemistry analysis also found decreased TSLC1 expression in 41.3?% (33/80) colon tumor tissues. In clinicopathological analysis, loss of TSLC1 expression significantly correlated with female gender and lymph node metastasis of colon cancer patients (P?<?0.05). In addition, decreased expression of TSLC1 in tumors was found to be closely associated with a poor prognosis (P?=?0.037, log-rank test), and multivariate analysis showed that lower TSLC1 protein expression was an independent prognostic factor for colon cancer patients. Our study suggests that down-regulated expression of TSLC1 may play an important role in the progression of colon cancer and TSLC1 expression may serve as a useful marker for the prognostic evaluation of patients with colon cancer.     

Identification of isocitrate dehydrogenase 1 as a potential diagnostic and prognostic biomarker for non-small cell lung cancer by proteomic analysis

Lung cancer is the leading cause of cancer-related death in the world. To explore tumor biomarkers for clinical application, two-dimensional fluorescence difference gel electrophoresis and subsequent MALDI-TOF/TOF mass spectrometry were performed to identify proteins differentially expressed in 12 pairs of lung squamous cell tumors and their corresponding normal tissues. A total of 28 nonredundant proteins were identified with significant alteration in lung tumors. The up-regulation of isocitrate dehydrogenase 1 (IDH1), superoxide dismutase 2, 14-3-3ε, and receptor of activated protein kinase C1 and the down-regulation of peroxiredoxin 2 in tumors were validated by RT-PCR and Western blot analysis in independent 15 pairs of samples. Increased IDH1 expression was further verified by the immunohistochemical study in extended 73 squamous cell carcinoma and 64 adenocarcinoma clinical samples. A correlation between IDH1 expression and poor overall survival of non-small cell lung cancer (NSCLC) patients was observed. Furthermore, ELISA analysis showed that the plasma level of IDH1 was significantly elevated in NSCLC patients compared with benign lung disease patients and healthy individuals. In addition, knockdown of IDH1 by RNA interference suppressed the proliferation of NSCLC cell line and decreased the growth of xenograft tumors in vivo. These observations suggested that IDH1, as a protein promoting tumor growth, could be used as a plasma biomarker for diagnosis and a histochemical biomarker for prognosis prediction of NSCLC.     

MEK1 mutations,but not ERK2 mutations,occur in melanomas and colon carcinomas,but none in thyroid carcinomas

Mitogen-activated protein kinase (MAPK) signaling pathway plays an important role in the pathogenesis of melanoma, colon cancer and thyroid cancer, which commonly harbor RAS and BRAF mutations. However, mutations in exon 2 of MEK1 and exon 7 of ERK2 have not been investigated in these cancers although they are occasionally found in some other cancers or cell lines. In this study, we performed mutational analysis to search for these mutations in 185 samples, including 167 tumor samples and 18 cell lines of melanoma, colon cancer, and thyroid cancer. We found one MEK1 mutation in 1 of 37(3%) melanoma tumor samples and another MEK1 mutation in 1 of 45 (2.2%) colon cancer samples. We did not find any MEK1 mutation in 99 thyroid tumor samples and 12 thyroid cancer cell lines. We also did not find any ERK2 mutation in melanoma, colon cancer, and thyroid cancer. We thus report for the first time a low prevalence of MEK1 mutations in melanoma and colon cancer. Both of the two mutants have been demonstrated to be activating in the MAPK signaling pathway and may therefore provide potential target for effective therapy in cases of melanomas and colon cancer harboring these mutations.     

GNAS Mutations Identify a Set of Right-Sided,RAS Mutant,Villous Colon Cancers

The purpose of this study is to determine the genetic frequency of GNAS activating mutations in colorectal cancer and the corresponding pathology of GNAS mutant tumors. Oncogenic mutations in GNAS have been described in a number of neoplasms including those of the pituitary, kidney, pancreas, and, more recently, in colon cancer. To ascertain the frequency in colon cancer we employed a sensitive pyrosequencing platform for mutation detection of the R201C and R201H GNAS hotspots in tumor samples representing all clinical stages. We additionally assayed for KRAS and BRAF mutations as previous reports have shown that these often co-occur with activating GNAS mutations. Of the 428 colon tumors assayed, mutations in GNAS were present in 10 of the samples (2.3%), indicating this is a significant, albeit infrequent, mutation in colorectal tumors. Nine GNAS mutant tumors (90%) harbored concomitant activating mutations in either the KRAS or BRAF oncogene, which was significantly greater than the mutation frequency of these genes in the tumor population (56%, p<0.0305). All ten of the GNAS mutant tumors arose in the right (proximal) colon (p<0.007), and 7 of 8 reviewed cases exhibited a marked villous morphology. Taken together, these data indicate that GNAS mutant colon tumors commonly have synchronous mutations in KRAS or BRAF, are right-sided in location, and are associated with a villous morphology.