Tumor suppressor gene 14-3-3sigma is down-regulated whereas the proto-oncogene translation elongation factor 1delta is up-regulated in non-small cell lung cancers as identified by proteomic profiling

Lung cancer, a leading cause of cancer deaths, consists of two major groups: small cell lung cancer (SCLC) and nonsmall cell lung cancer (NSCLC) with the NSCLC accounting for approximately 75% cases of lung cancers. It has been suggested that molecular changes including overexpression of oncogenes and decreased expression of tumor suppressor genes are responsible for lung carcinogenesis. In this study, we analyzed protein profiles of four different human NSCLC cell lines compared with normal human bronchial epithelial cells using two-dimensional PAGE and MALDI-TOF mass spectrometry. We identified 12 protein spots with different expressions between the normal and cancer cells. Of these proteins, vimentin, cytokeratin 8, YB-1, PCNA, Nm23, hnRNP A2/B1, and HSP90beta were known to be up-regulated in lung cancers, which is consistent with the current study. We also found that the expression of M-type pyruvate kinase is altered in NSCLC likely due to changes in translational control and/or differential phosphorylation of the protein. Interestingly, the expression of the tumor suppressor gene 14-3-3sigma is down-regulated while that of the proto-oncogene TEF1delta is up-regulated in NSCLC cells. On the basis of these observations and previous studies, we propose that the altered expression of 14-3-3sigma and TEF1delta may be involved in lung carcinogenesis.     

TN-C 在小细胞肺癌中的表达及STAT3 对TN-C 表达的影响

目的：探讨小细胞肺癌(SCLC)组织和小细胞肺癌细胞(H446)中肌糖蛋白-C(TN-C)的表达及STAT3 对TN-C表达的影响。 方法：应用免疫组化法检测58 例小细胞肺癌和17 例癌旁正常组织中TN-C 的表达水平,应用RT-PCR和Western blotting 法检测 STAT-siRNA和STAT3 过表达的H446 细胞中TN-C 的表达水平。结果：(1)小细胞肺癌组织中TN-C 的表达水平显著高于癌旁正 常组织(P<0.05);(2)在H446细胞中,TN-C 和STAT3 均呈现高表达;(3)STAT3-siRNA 处理的H446 细胞中STAT3 和TN-C 的表 达均显著降低(P<0.05),而STAT3 过表达的H446 细胞中STAT3 和TN-C 的表达均显著上调(P<0.05)。结论：TN-C 在小细胞肺癌 中的表达上调,可能受到STAT3 的调控。     

TN-C在小细胞肺癌中的表达及STAT3对TN-C表达的影响

目的：探讨小细胞肺癌（SCLC）组织和小细胞肺癌细胞（H446）中肌糖蛋白-C（TN-C）的表达及STAT3对TN-C表达的影响。方法：应用免疫组化法检测58例小细胞肺癌和17例癌旁正常组织中TN-C的表达水平,应用RT-PCR和Western blotting法检测STAT-siRNA和STAT3过表达的H446细胞中TN-C的表达水平。结果：（1）小细胞肺癌组织中TN-C的表达水平显著高于癌旁正常组织（P〈0.05）;（2）在H446细胞中,TN-C和STAT3均呈现高表达;（3）STAT3-siRNA处理的H446细胞中STAT3和TN-C的表达均显著降低（P〈0.05）,而STAT3过表达的H446细胞中STAT3和TN-C的表达均显著上调（P〈0.05）。结论：TN-C在小细胞肺癌中的表达上调,可能受到STAT3的调控。     

Plasma proteome profiling of a mouse model of breast cancer identifies a set of up-regulated proteins in common with human breast cancer cells

We have applied an in-depth quantitative proteomic approach, combining isotopic labeling extensive intact protein separation and mass spectrometry, for high confidence identification of protein changes in plasmas from a mouse model of breast cancer. We hypothesized that a wide spectrum of proteins may be up-regulated in plasma with tumor development and that comparisons with proteins expressed in human breast cancer cell lines may identify a subset of up-regulated proteins in common with proteins expressed in breast cancer cell lines that may represent candidate biomarkers for breast cancer. Plasma from PyMT transgenic tumor-bearing mice and matched controls were obtained at two time points during tumor growth. A total of 133 proteins were found to be increased by 1.5-fold or greater at one or both time points. A comparison of this set of proteins with published findings from proteomic analysis of human breast cancer cell lines yielded 49 proteins with increased levels in mouse plasma that were identified in breast cancer cell lines. Pathway analysis comparing the subset of up-regulated proteins known to be expressed in breast cancer cell lines with other up-regulated proteins indicated a cancer related function for the former and a host-response function for the latter. We conclude that integration of proteomic findings from mouse models of breast cancer and from human breast cancer cell lines may help identify a subset of proteins released by breast cancer cells into the circulation and that occur at increased levels in breast cancer.     

Identification of tumour-induced changes in endothelial cell surface protein expression: an in vitro model

The selective destruction of the supporting vasculature of tumours has been proposed as a means of therapy. Fundamental to this approach is the identification of suitable targets on tumour-endothelium. To detect proteins that may be up-regulated on the luminal (apical) surface of tumour-associated endothelium confluent endothelial cells were examined following incubation with tumour cell conditioned medium (TCM) from, or co-culture with, a range of breast carcinoma and small cell lung carcinoma (SCLC) cell lines. Exposed endothelial membrane proteins were labelled with sulpho-NHS-biotin and detected by enhanced chemiluminescence following two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and western blotting. TCM induced varying levels of proliferative activity in endothelial cells; generally breast TCM contained greater mitogenic activity than SCLC TCM. Exposure of human breast and lung microvascular, and umbilical vein endothelial cells to soluble tumour cell factors from several breast cancer and SCLC cells lines produced similar changes in luminal protein profiles: Breast cancer cells and in particular the MDA-MB-231 cell line induced the most pronounced changes. The expression of six proteins was altered consistently on endothelial cells stimulated with soluble tumour cell factors. However, similar changes were observed following incubation with ECGS suggesting that they were related to endothelial cell proliferation per se. As these proteins were altered in breast and lung microvascular, and umbilical vein endothelial cells stimulated by a variety of breast cancer and SCLC cell lines they support the potentially broad applicability of anti-vascular approaches targeted at the endothelium.     

Identification of tumor-associated plasma biomarkers using proteomic techniques: from mouse to human

In an effort to identify tumor-associated proteins from plasma of tumor-bearing mice that may be used as diagnostic biomarkers, we developed a strategy that combines a tumor xenotransplantation model in nude mice with comparative proteomic technology. Five human cancer cell lines (SC-M1, HONE-1, CC-M1, OECM1, GBM 8401) derived from stomach, nasopharyngeal, colon, oral and brain cancers were subcutaneously inoculated into nude mice and compared to control nude mice injected with phosphate-buffered saline. One month later, plasma from mice inoculated with cancer cells was collected for proteomic analysis using two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS). Comparison of plasma 2-DE maps from tumor-bearing mice with those produced from control mice revealed the overexpression of several mouse acute phase proteins (APPs) such as haptoglobin. Another APP, serum amyloid A (SAA), was found only in mice bearing tumors induced by the stomach cancer cell line SC-M1, which has not previously been demonstrated in xenotransplatation experiment. Furthermore, by using immunohistochemistry, SAA and haptoglobin were found to originate from the mouse hosts and not from the human cancer cell line donors. The protein alterations were further confirmed on patients with stomach cancers where up-regulated levels of SAA were also observed. These results indicate that APPs may be used as nonspecific tumor-associated serum markers. SAA in particular may serve as a potential marker for detecting stomach cancer. Taken together, the combination of the xenotransplatation model in nude mice and proteomics analysis provided a valuable impact for clinical applications in cancer diagnostics. In addition, our findings demonstrate that a panel of APPs might serve as screening biomarkers for early cancer detection.     

Cathepsin D is secreted from M-BE cells: its potential role as a biomarker of lung cancer

The early diagnosis of lung cancer is an effective approach to reduce the mortality caused by malignancy. To explore serum biomarkers of lung cancer at early stage, M-BE, a SV40T-transformed human bronchial epithelial cell line with the phenotypic features of early tumorigenesis at high passage, was cultured in the conditioned media to collect its secretory proteins. The proteins secreted from different passage M-BE cells were extracted and then separated by two-dimensional electrophoresis (2-DE). MALDI-TOF/TOF mass spectrometry was adopted to identify the passage-dependent 2-DE spots. Totally, 47 proteins were identified, including 23 that were up-regulated and 24 that were down-regulated. Of these proteins, cathepsin D was a typical secretory protein that exhibited the increased abundance either in culture media or in cells during passaging. Furthermore, the proteomic conclusions were validated in the clinical samples of lung cancer patients. When sandwich ELISA was used, the concentrations of cathepsin D in plasma showed significant differences between lung squamous cell carcinomas (SCC, 104 cases) and normal donors (36 cases, p 相似文献   

Proteomic profiling of endothelial cells in human lung cancer

Genomic and proteomic analysis of normal and diseased tissues have yielded an abundance of molecular information for diagnostic and potential therapeutic targets. Changing the target of analysis from poorly accessible cells within tissues to easily accessible vascular endothelium has theoretical advantages in tissue-specific targeting. In this study, we sought to map a large-scale proteome of microvascular endothelium in human non-small cell lung cancer (NSCLC) and normal lung tissues, and identify lung cancer-related endothelial cell (EC)-selective proteins. Endothelial cells were isolated within NSCLC tissues and adjacent-normal lung tissue of lung cancer patients by using CD31-immunomagnetic beads. The complex proteins from the ECs were separated by one-dimensional gel electrophoresis, and the proteins in each gel band were digested by trypsin. Peptides were separated by online reverse-phase liquid-chromatography and analyzed by electrospray ionization (ESI) ion trap tandem mass spectrometry. Approximately 600-1000 proteins were identified in each individual sample. Five patient cases of paired individual data, extracted from the protein identification data sets of both normal- and cancer-derived ECs, were analyzed by subtractive proteomics. An average of 300 proteins was specifically identified from each lung cancer-derived EC isolate, compared to normal lung-derived ECs. With the use of several comparative analyses, we identified among those 300 proteins, 16 common candidate proteins that were detected in at least 3 of 5 cases specific to lung cancer-derived ECs. Proteins selectively identified in cancer-derived ECs, including coatomer protein complex, subunit gamma (COPG), and peroxiredoxin 4 (PRDX4), were validated by Western blot analysis. In an additional experiment in which 16 cancer samples were analyzed by immunohistochemistry, PRDX4, thymopoietin (TMPO), and COPG were confirmed to be abundantly expressed in lung cancer-derived ECs and in cancerous lung cells. Further ongoing analysis of these 16 candidate proteins will determine their potential applicability to NSCLC-specific diagnosis and therapeutics.     

Identification of conditioned medium proteins from human cancer cells adapted to serum-free conditions

Recent studies have shown that human cancer cell lines can be adapted to grow in serum-free, unsupplemented RPMI-1640 (RO) medium. We have developed similar techniques to rapidly identify proteins of interest in serum-free conditioned medium (CM) of human lung cancer cell lines. Classic and variant small cell lung cancer (SCLC) lines were adapted to growth in RO medium. CM from each line was concentrated and fractionated on an anion-exchange column of a fast protein liquid chromatography system. Concentrates of each fraction were loaded onto lanes of minigels of an automated electrophoresis system. Analysis of the chromatograms reveals peaks seen only in CM of the classic SCLC lines. Electrophoretic analysis of the fractions containing these peaks reveal protein bands distinguishing between the subtypes of human SCLC. One protein was purified to homogeneity with subsequent reversed-phase chromatography and identified by protein microsequencing as histone H2B. These automated techniques have general use in the rapid identification of CM proteins associated with the differentiation or progression of the many types of neoplastic cells which can be adapted to growth in RO medium.     

Overexpression of TRIM24 correlates with tumor progression in non-small cell lung cancer

The objective of the current study was to investigate the expression pattern and clinicopathological significance of TRIM24 in patients with non-small cell lung cancer (NSCLC). The expression profile of TRIM24 in NSCLC tissues and adjacent noncancerous lung tissues was detected by immunohistochemistry. TRIM24 was found to be overexpressed in 81 of 113 (71.7%) human lung cancer samples and correlated with p-TNM stage (p = 0.0006), poor differentiation (p = 0.004), Ki67 index (p<0.0001), cyclin D1(p = 0.0096) and p-Rb expression (p = 0.0318). In addition, depleting TRIM24 expression by small interfering RNA inhibited growth and invasion in lung cell lines. Moreover, TRIM24 depletion induced cell cycle arrest at the G1/S boundary and induced apoptosis. Western blotting analysis revealed that knockdown of TRIM24 decreased the protein levels of Cyclin A, Cyclin B, Cyclin D1, cyclin E and p-Rb and increased P27 expression. These results indicate that TRIM24 plays an important role in NSCLC progression.     

nm23、EGFR和Bcl-2蛋白在肺癌组织中的表达及临床意义

目的:观察nm23、EGFR和Bcl-2在人肺癌中的表达,探讨其与肺癌临床生物学行为的关系。方法:采用免疫组化检测了49例肺癌及癌旁组织中nm23、EGFR和Bcl-2蛋白的表达情况,并对其与肺癌临床生物学行为进行相关分析。结果:在49例肺癌中,EGFR和Bel-2蛋白的阳性表达率分别为51.0%和34.7%,均高于正常肺组织17.5%和12.5%(p<0.05),nm23蛋白的阳性表达率为55.1%,则显著低于正常肺组织87.5%(p<0.01)。在病理分级的高一中分化、低分化和未分化中nm23的阳性表达率分别为82.6%、41.2%和11.1%(p<0.01)。EGFR在非小细胞肺癌(NSCLC)组为61.5%,在小细胞肺癌(SCLC)组为10.0%(p<0.01)。在NSCLC TNM分期中,Ⅰ期 Ⅱ期与Ⅲ P<0.01)期中EGFR的阳性表达率分别为39.3%和81.8%(p<0.01);病理分级的高-中分化和低分化中EGFR的阳性表达率分别为39.1%和88.2%(p<0.01);在淋巴结转移组为57.9%,无淋巴结转移组为27.3%(p<0.05)。Bcl-2的阳性表达率在NSCLC组为25.6%,在SCLC组为70%,统计学分析差异有统计学意义(p<0.01);在NSCLC组病理分级的高分化、中分化和低分化中Bcl-2阳性表达率分别为62.5%、26.7%和5.89%(p<0.01)。结论:癌基因EGFR、凋亡抑制基因Bcl-2与抑癌基因nm23在肺癌的发生、发展中存在着相互促进作用。     

Proteome analysis of human lung squamous carcinoma

Few lung cancer-specific molecular markers have been established in regard of "early-stage" diagnosis and prognosis. In this study the proteome analysis of human lung squamous carcinoma (hLSC) was carried out using two strategies to explore the carcinogenic mechanisms and identify its molecular markers more directly and comprehensively. Comparative proteome analysis on 20 hLSC tissues and paired normal bronchial epithelial tissues revealed 76 differential proteins, among which 68 proteins were identified by PMF. The identified proteins fell into three categories: oncoproteins, cell cycle regulators and signaling molecules. To validate the identified differential proteins, the expressions levels of three differential proteins mdm2, c-jun and EGFR were determined by immunohistochemical staining and immunoblots. The results verified proteome analysis results. Serological proteome analysis (SERPA) of ten hLSC tissues was performed to identify the tumor-associated antigens. The results revealed 36 +/- 8 differential proteins reactive with patients' autologous sera, of which 14 proteins were identified. Six of the 14 proteins, alpha enolase, pre-B cell-enhancing factor precursor, triosephosphate isomerase, phosphoglycerate mutase 1, fructose-bisphosphate aldolase A, and guanine nucleotide-binding protein beta subunit-like protein, were also up-regulated in hLSCs in the comparative proteomic study, which suggests potential application of these 6 hLSC-associated antigens in diagnosis and therapy of hLSC.     

Bradykinin-related compounds as new drugs for cancer and inflammation

Bradykinin (BK) (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg) is an important growth factor for small-cell lung cancer (SCLC) and prostate cancer (PC). These cancers have cells of neuroendocrine origin and express receptors for a variety of neuropeptides. BK receptors are expressed on almost all lung cancer cell lines and on many PC cells. Our very potent BK antagonist B9430 (D-Arg-Arg-Pro-Hyp-Gly-lgl-Ser-D-Igl-Oic-Arg) (Hyp, trans-4-hydroxy-L-proline; Ig1, alpha-2-indanylglycine; Oic, octahydroindole-2-carboxylic acid) is a candidate anti-inflammatory drug but does not inhibit growth of SCLC or PC. When B9430 is dimerized by N-terminal cross-linking with a suberimide linker, the product B9870 is a potent growth inhibitor for SCLC both in vitro and in vivo in athymic nude mice. Daily i.p. injection at 5 mg x kg(-1) day(-1) beginning on day 8 after SCLC SHP-77 cell implantation gave 65% inhibition of tumor growth. B9870 stimulates apoptosis in SCLC by a novel "biased agonist" action. We have also developed new small mimetic antagonists. BKM-570 (F5C-OC2Y-Atmp) (F5C, pentafluorocinnamic acid; OC2Y, O-2,6-dichlorobenzyl tyrosine; Atmp, 4-amino-2,2,6,6-tetramethylpiperidine) is very potent for inhibition of SHP-77 growth in nude mice. When injected daily i.p. at 5 mg x kg(-1), M-570 gave 90% suppression of tumor growth. M-570 is more potent than the well-known anticancer drug cisPlatin (60% inhibition) or the recently developed SU5416 (40% inhibition) in this model. M-570 also showed activity against various other cancer cell lines in vitro (SCLC, non-SCLC, lung, prostate, colon, cervix) and inhibited growth of prostate cell line PC3 in nude mice. M-570 and related compounds evidently act in vivo through pathways other than BK receptors. These compounds have clinical potential for treatment of human lung and prostate cancers.     

The Function and Significance of SELENBP1 Downregulation in Human Bronchial Epithelial Carcinogenic Process

Background

Our quantitative proteomic study showed that selenium-binding protein 1 (SELENBP1) was progressively decreased in human bronchial epithelial carcinogenic process. However, there is little information on expression and function of SELENBP1 during human lung squamous cell cancer (LSCC) carcinogenesis.

Methods

iTRAQ-tagging combined with 2D LC-MS/MS analysis was used to identify differentially expressed proteins in the human bronchial epithelial carcinogenic process. SELENBP1, member of selenoproteins family and progressively downregulated in this process, was selected to further study. Both Western blotting and immunohistochemistry were performed to detect SELENBP1 expression in independent sets of tissues of bronchial epithelial carcinogenesis, and ability of SELENBP1 for discriminating NBE (normal bronchial epithelium) from preneoplastic lesions from invasive LSCC was evaluated. The effects of SELENBP1 downregulation on the susceptibility of benzo(a)pyrene (B[a]P)-induced human bronchial epithelial cell transformation were determined.

Results

102 differentially expressed proteins were identified by quantitative proteomics, and SELENBP1 was found and confirmed being progressively decreased in the human bronchial epithelial carcinogenic process. The sensitivity and specificity of SELENBP1 were 80% and 79% in discriminating NBE from preneoplastic lesions, 79% and 82% in discriminating NBE from invasive LSCC, and 77% and 71% in discriminating preneoplastic lesions from invasive LSCC, respectively. Furthermore, knockdown of SELENBP1 in immortalized human bronchial epithelial cell line 16HBE cells significantly increased the efficiency of B[a]P-induced cell transformation.

Conclusions

The present data shows for the first time that decreased SELENBP1 is an early event in LSCC, increases B[a]P-induced human bronchial epithelial cell transformation, and might serve as a novel potential biomarker for early detection of LSCC.     

Variations of chromosome 2 gene expressions among patients with lung cancer or non-cancer

Lung cancer is one of the most common malignancies worldwide. The present study aimed to investigate specific genotypes of different subtypes or stages of lung cancer through gene expression variations of chromosome 2 genes, trying to identify predictors for diagnosis or prognosis of lung cancer. About 537 patients with lung adenocarcinoma (ADC), 140 patients with lung squamous carcinoma (SQC), 9 patients with lung large cell carcinoma (LCC), 56 patients with small cell lung cancer (SCLC), and 590 patients without cancer were analyzed in present study. Co-expressed, subtype-specific, and stage-specific chromosome 2 genes were identified and further analyzed by bioinformatic methods. As a result, 15 or 10 genes were significantly up- or down-regulated in all four subtypes of lung cancer. GKN1, LOC100131510, prominin-2 (PROM2), IL37, and SNORA41 were identified as ADC-specific up-regulated genes; SQC-specific up-regulated genes included HOXD family (HOXD1, HOXD3, HOXD4, HOXD8, and HOXD9) and UGT1A family (UGT1A1, UGT1A3, UGT1A4, UGT1A5, UGT1A7, UGT1A8, UGT1A9, and UGT1A10); and LCC- or SCLC-specific genes were also identified. Nine genes were significantly up-expressed at all four stages of ADC while 230 genes at all three stages of SQC. MFSD2B, CCL20 and STAT1, or STARD7 and ZNF512 genes may be risk or protect factors in prognosis of ADC, while HTR2B, DPP4, and TGFBRAP1 genes may be risk factors in prognosis of SQC. Our results suggested that a number of altered chromosome 2 genes have the subtype or stage specificities of lung cancer and may be considered as diagnostic and prognostic biomarkers.     

Proteomic expression analysis of surgical human colorectal cancer tissues: up-regulation of PSB7, PRDX1, and SRP9 and hypoxic adaptation in cancer

Colorectal adenocarcinoma is one of the worldwide leading causes of cancer deaths. Discovery of specific biomarkers for early detection of cancer progression and the identification of underlying pathogenetic mechanisms are important tasks. Global proteomic approaches have thus far been limited by the large dynamic range of molecule concentrations in tissues and the lack of selective enrichment of the low-abundance proteome. We studied paired cancerous and normal clinical tissue specimens from patients with colorectal adenocarcinomas by heparin affinity fractionation enrichment (HAFE) followed by 2-D PAGE and tandem mass spectrometric (MS/MS) identification. Fifty-six proteins were found to be differentially expressed, of which 32 low-abundance proteins were only detectable after heparin affinity enrichment. MS/MS was used to identify 5 selected differentially expressed proteins as proteasome subunit beta type 7 (PSB7), hemoglobin alpha subunit (HBA), peroxiredoxin-1 (PRDX1), argininosuccinate synthase (ASSY), and signal recognition particle 9 kDa protein (SRP9). This is the first proteomic study detecting the differential expression of these proteins in human colorectal cancer tissue. Several of the proteins are functionally related to tissue hypoxia and hypoxic adaptation. The relative specificities of PSB7, PRDX1, and SRP9 overexpression in colon cancer were investigated by Western blot analysis of patients with colon adenocarcinomas and comparison with a control cohort of patients with lung adenocarcinomas. Furthermore, immunohistochemistry on tissue sections was used to define the specific locations of PSB7, PRDX1, and SRP9 up-regulation within heterogeneous primary human tumor tissue. Overexpression of the three proteins was restricted to the neoplastic cancer cell population within the tumors, demonstrating both cytoplasmic and nuclear localization of PSB7 and predominantly cytoplasmic localization of PRDX1 and SRP9. In summary, we describe heparin affinity fractionation enrichment (HAFE) as a prefractionation tool for the study of the human primary tissue proteome and the discovery of PSB7, PRDX1, and SRP9 up-regulation as candidate biomarkers of colon cancer.     

Proteomic search for potential diagnostic markers and therapeutic targets for ovarian clear cell adenocarcinoma

Clear cell adenocarcinoma (CCA) has a highly malignant potential in human epithelial ovarian cancer. The serum CA-125 is widely used as a marker for ovarian cancer, but the level is relatively low in CCA. Therefore, new sensitive biomarkers are required. In this report, we describe a promising proteomic analysis that is differentially expressed in CCA when compared to mucinous adenocarcinoma, using the ovarian cultured cell lines OVISE, OVTOKO, and MCAS. The disease-associated proteins were identified by 2-D differential gel electrophoresis (2-D DIGE) and MS. In this analysis, 18 up-regulated and 31 down-regulated spots were observed that had at least two-fold differences in the two CCA cell lines than in MCAS as control cells. Some of the proteins differentially expressed in CCA were previously observed as alternative expression levels in ovarian and/or other cancers in clinical samples. In a subsequent preliminary differential study using surgical specimens from patients with CCA, it was demonstrated that the identified proteins were expressed differentially in actual tissues, as well as in the CCA culture cells. The results from this investigation show the potentiality of a proteomic approach for identifying disease-associated proteins, which may eventually serve as diagnostic markers or therapeutic targets in CCA.     

Identification of the heterogeneous nuclear ribonucleoprotein A2/B1 as the antigen for the gastrointestinal cancer specific monoclonal antibody MG7

MG7 is an early gastrointestinal cancer specific monoclonal antibody. It can detect gastric cancer with high sensitivity and specificity. However, the target antigen for MG7 has not been identified. Western blot analysis revealed that the MG7 antibody reproducibly recognized two approximately 35 kDa proteins in the total cell lysates of human gastric carcinoma cell lines KATO III and MKN-45. Using a proteomic approach, we identified these MG7 immunoreactive proteins as the human heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1). Western blot analysis of nuclear and cytosolic fraction of KATO III cells using either MG7 or hnRNP A2/B1 antibodies confirmed that the target antigen is located exclusively in the nucleus. With the use of archival samples, we also found that the level of hnRNP A2/B1 protein was increased in gastric cancer tissues (4 out of 5 patients), when compared to their corresponding matching normal stomach tissue.     

A quantitative proteomic approach of the different stages of colorectal cancer establishes OLFM4 as a new nonmetastatic tumor marker

Expression profiles represent new molecular tools that are useful to characterize the successive steps of tumor progression and the prediction of recurrence or chemotherapy response. In this study, we have used quantitative proteomic analysis to compare different stages of colorectal cancer. A combination of laser microdissection, OFFGEL separation, iTRAQ labeling, and MALDI-TOF/TOF MS was used to explore the proteome of 28 colorectal cancer tissues. Two software packages were used for identification and quantification of differentially expressed proteins: Protein Pilot and iQuantitator. Based on ～1,190,702 MS/MS spectra, a total of 3138 proteins were identified, which represents the largest database of colorectal cancer realized to date and demonstrates the value of our quantitative proteomic approach. In this way, individual protein expression and variation have been identified for each patient and for each colorectal dysplasia and cancer stage (stages I-IV). A total of 555 proteins presenting a significant fold change were quantified in the different stages, and this differential expression correlated with immunohistochemistry results reported in the Human Protein Atlas database. To identify a candidate biomarker of the early stages of colorectal cancer, we focused our study on secreted proteins. In this way, we identified olfactomedin-4, which was overexpressed in adenomas and in early stages of colorectal tumors. This early stage overexpression was confirmed by immunohistochemistry in 126 paraffin-embedded tissues. Our results also indicate that OLFM4 is regulated by the Ras-NF-κB2 pathway, one of the main oncogenic pathways deregulated in colorectal tumors.