Expression profiling of more than 3500 proteins of MSS-type colorectal cancer by stable isotope labeling and mass spectrometry

An efficient means of identifying protein biomarkers is essential to proper cancer management. A well-characterized proteome resource holds special promise for the discovery of novel biomarkers. However, quantification of the differences between physiological conditions together with deep down profiling has become increasingly challenging in proteomics. Here, we perform expression profiling of the colorectal cancer (CRC) proteome by stable isotope labeling and mass spectrometry. Quantitative analysis included performing mTRAQ and cICAT labeling in a pooled sample of three microsatellite stable (MSS) type CRC tissues and a pooled sample of their matched normal tissues. We identified and quantified a total of 3688 proteins. Among them, 1487 proteins were expressed differentially between normal and cancer tissues by higher than 2-fold; 1009 proteins showed increased expression in cancer tissue, whereas 478 proteins showed decreased expression. Bioinformatic analysis revealed that our data were largely consistent with known CRC relevant signaling pathways, such as the Wnt/β-catenin, caveolar-mediated endocytosis, and RAN signaling pathways. Mitochondrial dysfunction, known as the Waburg hypothesis, was also confirmed. Therefore, our data showing alterations in the proteomic profile of CRC constitutes a useful resource that may provide insights into tumor progression with later goal of identifying biologically and clinically relevant marker proteins. This article is part of a Special Issue entitled: Proteomics: The clinical link.     

Proteome profiling of human epithelial ovarian cancer cell line TOV-112D

A proteome profiling of the epithelial ovarian cancer cell line TOV-112D was initiated as a protein expression reference in the study of ovarian cancer. Two complementary proteomic approaches were used in order to maximise protein identification: two-dimensional gel electrophoresis (2DE) protein separation coupled to matrix assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) and one-dimensional gel electrophoresis (1DE) coupled to liquid-chromatography tandem mass spectrometry (LC MS/MS). One hundred and seventy-two proteins have been identified among 288 spots selected on two-dimensional gels and a total of 579 proteins were identified with the 1DE LC MS/MS approach. This proteome profiling covers a wide range of protein expression and identifies several proteins known for their oncogenic properties. Bioinformatics tools were used to mine databases in order to determine whether the identified proteins have previously been implicated in pathways associated with carcinogenesis or cell proliferation. Indeed, several of the proteins have been reported to be specific ovarian cancer markers while others are common to many tumorigenic tissues or proliferating cells. The diversity of proteins found and their association with known oncogenic pathways validate this proteomic approach. The proteome 2D map of the TOV-112D cell line will provide a valuable resource in studies on differential protein expression of human ovarian carcinomas while the 1DE LC MS/MS approach gives a picture of the actual protein profile of the TOV-112D cell line. This work represents one of the most complete ovarian protein expression analysis reports to date and the first comparative study of gene expression profiling and proteomic patterns in ovarian cancer.     

Global expression study in colorectal cancer on proteins with alkaline isoelectric point by two-dimensional difference gel electrophoresis

Colorectal cancer is one of the leading causes of cancer death worldwide. To identify candidates for biomarkers and therapeutic targets, we investigated the proteome of colorectal cancer tissues. Using 2D-DIGE in combination with our original large format electrophoresis apparatus, we compared surgically resected normal and tumor tissues from 53 patients with colorectal cancer. We focused on proteins with an alkaline pI using IPG gels for the alkaline range. We observed 1687 protein spots, and found 100 spots with statistical (p<0.01) and significant (>2-fold) differences between the normal and the tumor tissue groups. Among these 100 protein spots, five showed a different intensity between tumor tissues from the stage-II and the stage-III patients. MS experiments revealed that these 100 protein spots corresponded to 58 unique proteins. These included six proteins which had not been previously reported to be associated with colorectal cancer. Among these proteins, five were not reported in any type of malignancy. IEF/western blotting confirmed the differences in protein expression between the normal and the tumor tissues. These results may provide an insight for biomarker development and drug target discovery in colorectal cancer.     

The advantage of laser‐capture microdissection over whole tissue analysis in proteomic profiling studies

Laser‐capture microdissection (LCM) offers a reliable cell population enrichment tool and has been successfully coupled to MS analysis. Despite this, most proteomic studies employ whole tissue lysate (WTL) analysis in the discovery of disease biomarkers and in profiling analyses. Furthermore, the influence of tissue heterogeneity in WTL analysis, nor its impact in biomarker discovery studies have been completely elucidated. In order to address this, we compared previously obtained high resolution MS data from a cohort of 38 breast cancer tissues, of which both LCM enriched tumor epithelial cells and WTL samples were analyzed. Label‐free quantification (LFQ) analysis through MaxQuant software showed a significantly higher number of identified and quantified proteins in LCM enriched samples (3404) compared to WTLs (2837). Furthermore, WTL samples displayed a higher amount of missing data compared to LCM both at peptide and protein levels (p‐value < 0.001). 2D analysis on co‐expressed proteins revealed discrepant expression of immune system and lipid metabolisms related proteins between LCM and WTL samples. We hereby show that LCM better dissected the biology of breast tumor epithelial cells, possibly due to lower interference from surrounding tissues and highly abundant proteins. All data have been deposited in the ProteomeXchange with the dataset identifier PXD002381 ( http://proteomecentral.proteomexchange.org/dataset/PXD002381 ).     

Temporal expression and functional analysis of long non‐coding RNAs in colorectal cancer initiation

Long non‐coding RNAs (lncRNAs) have potential applications in clinical diagnosis and targeted cancer therapies. However, the expression profile of lncRNAs in colorectal cancer (CRC) initiation is still unclear. In this study, the expression profiles of lncRNAs and mRNAs were determined by microarray at specific tumour stages in an AOM/DSS‐induced primary colon cancer model. The temporal expression of lncRNAs was analysed by K‐means clustering. Additionally, weighted correlation network analysis (WGCNA) and gene ontology analysis were performed to construct co‐expression networks and establish functions of the identified lncRNAs and mRNAs. Our results suggested that 4307 lncRNAs and 5798 mRNAs are deregulated during CRC initiation. These differential expression genes (DEGs) exhibited a clear correlation with the differential stage of tumour initiation. WGCNA results suggested that a series of hub lncRNAs are involved in regulating cell stemness, colon inflammation, oxidative stress response and cell death at each stage. Among them, lncRNA H19 was up‐regulated in colon tumours and correlated with poor patient prognosis. Collectively, we have been the first to demonstrate the temporal expression and function of lncRNAs in CRC initiation. These results provide novel diagnosis and therapy targets for CRC.     

Proteome analysis of hepatocellular carcinoma by laser capture microdissection

Hepatocellular carcinoma (HCC) is one of the most frequent visceral neoplasia worldwide and is a multifactorial and multistage pathogenesis that finally leads to the deregulation of cell homeostasis. Laser capture microdissection (LCM) may allow a more ready identification of differences in protein expression in selected cell types or areas of tissue, and microscopic regions as small as 3-5 microm in diameter can be sampled. Here we applied the LCM to the proteomic study of hepatitis B-related HCC and surrounding non-tumor tissues. Proteome alterations were observed using 2-DE and ESI-MS/MS, and alterations in the proteome were examined. Twenty protein spots were selected, of which 11 proteins were significantly altered in the HCC compared with the surrounding non-tumor tissues. Of the proteins that were selected, peroxiredoxin 2, apolipoprotein A-I precursor, 3-hydroxyacyl-CoA dehydrogenase type II, and 14.5-kDa translational inhibitor protein appear to be novel candidates as useful hepatitis B-related HCC markers. This study indicates that LCM is a useful technological method in the proteomic study of cancer tissue. The proteins revealed in this experiment can be used in the future for studies pertaining to hepatocarcinogenesis, or as diagnostic markers and therapeutic targets for HCC associated with hepatitis B virus infection.     

Identification and distribution of thioredoxin-like 2 as the antigen for the monoclonal antibody MC3 specific to colorectal cancer

MC3 is a colorectal cancer (CRC)-specific mAb previously prepared in our laboratory that can detect CRC with high sensitivity and specificity. However, the target antigen for MC3 had not been identified due to technological limitations. In the present study, immunocytochemistry and immunohistochemistry revealed the expression patterns of MC3 antigen (MC3-Ag) in colon cancer cell lines and CRC tissues. Western blotting analysis showed that the MC3 antibody reproducibly recognized two approximately 30 kDa proteins in the total cell lysates of human colon carcinoma cell lines SW480 and HT-29. Using a proteomic approach, we identified two MC3 immunoreactive spots as two isoforms of thioredoxin-like 2 (Txl-2) protein. Further paired immunostaining showed that Txl-2 had the same expression profile as probed by the MC3 antibody. Western blotting also showed that both antibodies could detect the same two bands, further verifying that Txl-2 is the antigen of MC3 antibody. Additionally, tissue arrays revealed the expression patterns of Txl-2 in various normal and cancer tissues. Further analysis showed that Txl-2 mRNA was elevated in 18 cases of CRC tissues compared to paracancerous tissues and adjacent normal tissues.     

蛋白质组表达图谱用于基因组功能提示的可行性研究

本文以ＥＣＯ２ＤＢＡＳＥ（Ｅｄｉｔｉｏｎ ６） 为研究材料, 探讨了利用蛋白质组表达图谱提供的生命动态活动信息提高基因组功能提示效果的可行性。在设计出一套较为完整的细胞功能簇（ＣＲＣ）聚类方案的基础上, 经考察,７９ 个蛋白质聚成４ 个不同的ＣＲＣ。结果显示出功能相关的蛋白质趋向于聚集在相同的ＣＲＣ中, 如９ 种氨酰ｔＲＮＡ 合成酶和４ 种热休克蛋白分别准确地聚合到ＣＲＣ２ 和ＣＲＣ３ 中。这些结果提示： 在蛋白质组研究资料比较充分的前提下, 通过有效的算法, 蛋白质组表达图谱可以为基因组功能提示提供非常重要的序列相似性之外的功能信息     

Proteome analysis of membrane fractions in colorectal carcinomas by using 2D-DIGE saturation labeling

Because of its high content in receptors and signaling proteins, the analysis of membrane fractions is critical for the study of neoplastic diseases as colorectal cancer. Here, we have used the new saturation labeling for 2D-DIGE analysis of the membrane proteome of colorectal cancer mucosal tissues. Samples from 6 patients (tumoral and normal paired biopsies) were included in this study. Twelve analytical gels were performed and considered for the quantitative study and statistical analysis. A spot pattern analysis, by using an unsupervised clustering algorithm, allowed the classification of the samples according to similar expression patterns in tumoral and normal samples. Those proteins whose expression changed significantly (Student's t-test, p < 0.05) were further digested and characterized by mass spectrometry. Among the differentially expressed proteins: annexin A2, annexin A4, annexin A5, annexin A7, lamin B, calponin 1 and VDAC were analyzed by immunohistochemistry using tissue microarrays. Annexin A2, annexin A4 and VDAC appear as potential markers of interest for colorectal cancer diagnosis and, presumably, therapy. In summary, saturation labeling provides a new and sensitive tool for the analysis of scarce amounts of samples, allowing sample classification and direct identification of deregulated proteins.     

Proteome Heterogeneity in Colorectal Cancer

Tumor heterogeneity is an important feature of colorectal cancer (CRC) manifested by dynamic changes in gene expression, protein expression, and availability of different tumor subtypes. Recent publications in the past 10 years have revealed proteome heterogeneity between different colorectal tumors and within the same tumor site. This paper reviews recent research works on the proteome heterogeneity in CRC, which includes the heterogeneity within a single tumor (intratumor heterogeneity), between different anatomical sites at the same organ, and between primary and metastatic sites (intertumor heterogeneity). The potential use of proteome heterogeneity in precision medicine and its implications in biomarker discovery and therapeutic outcomes will be discussed. Identification of the unique proteome landscape between and within individual tumors is imperative for understanding cancer biology and the management of CRC patients.     

Tissue proteomics for cancer biomarker development: laser microdissection and 2D-DIGE

Novel cancer biomarkers are required to achieve early diagnosis and optimized therapy for individual patients. Cancer is a disease of the genome, and tumor tissues are a rich source of cancer biomarkers as they contain the functional translation of the genome, namely the proteome. Investigation of the tumor tissue proteome allows the identification of proteomic signatures corresponding to clinico-pathological parameters, and individual proteins in such signatures will be good biomarker candidates. Tumor tissues are also a rich source for plasma biomarkers, because proteins released from tumor tissues may be more cancer specific than those from non-tumor cells. Two-dimensional difference gel electrophoresis (2D-DIGE) with novel ultra high sensitive fluorescent dyes (CyDye DIGE Fluor satulation dye) enables the efficient protein expression profiling of laser-microdissected tissue samples. The combined use of laser microdissection allows accurate proteomic profiling of specific cells in tumor tissues. To develop clinical applications using the identified biomarkers, collaboration between research scientists, clinicians and diagnostic companies is essential, particularly in the early phases of the biomarker development projects. The proteomics modalities currently available have the potential to lead to the development of clinical applications, and channeling the wealth of produced information towards concrete and specific clinical purposes is urgent.     

The potential role of ORM2 in the development of colorectal cancer

Colorectal cancer (CRC) is the third most common malignancy in the world. The risk of death is closely correlated to the stage of CRC at the time of primary diagnosis. Therefore, there is a compelling need for the identification of blood biomarkers that can enable early detection of CRC. We used a quantitative proteomic approach with isobaric labeling (iTRAQ) to examine changes in the plasma proteome of 10 patients with CRC compared to healthy volunteers. Enzyme-Linked Immunosorbnent Assay (ELISA) and Western blot were used for further validation. In our quantitative proteomics analysis, we detected 75 human plasma proteins with more than 95% confidence using iTRAQ labeling in conjunction with microQ-TOF MS. 9 up-regulated and 4 down-regulated proteins were observed in the CRC group. The ORM2 level in plasma was confirmed to be significantly elevated in patients suffering from CRC compared with the controls. ORM2 expression in CRC tissues was significantly increased compared with that in corresponding adjacent normal mucous tissues (P<0.001). ITRAQ together with Q-TOF/MS is a sensitive and reproducible technique of quantitative proteomics. Alteration in expression of ORM2 suggests that ORM2 could be used as a potential biomarker in the diagnosis of CRC.     

Differential proteome profiling of pleural effusions from lung cancer and benign inflammatory disease patients

The pleural effusion proteome has been found containing information that directly reflects pathophysiological status and represents a potential diagnostic value for pulmonary diseases. However, the variability in protein composition between malignant and benign effusions is not well understood. Herein, we investigated the changes of proteins in pleural effusions from lung adenocarcinoma and benign inflammatory disease (pneumonia and tuberculosis) patients by two-dimensional difference gel electrophoresis (2D-DIGE). Twenty-eight protein spots displayed significantly different expression levels were positively identified by MALDI-TOF-MS representing 16 unique proteins. Five identified protein candidates were further validated and analyzed in effusions, sera or tissues. Among them, hemopexin, fibrinogen gamma and transthyretin (TTR) were up-regulated in cancer samples. The effusion concentration of serum amyloid P component (SAP) was significantly lower in lung cancer patients than in benign inflammatory patients, but no differences were found in sera samples. Moreover, a Jumonji C (JmjC)-domain-containing protein, JMJD5, was observed to be down-regulated in malignant effusions, lung cancer tissues and cancer cells. These results shed light on the altered pleural effusion proteins as a useful and important complement to plasma or other routine clinical tests for pulmonary disease diagnosis.     

An informatics-assisted label-free approach for personalized tissue membrane proteomics: case study on colorectal cancer

We developed a multiplexed label-free quantification strategy, which integrates an efficient gel-assisted digestion protocol, high-performance liquid chromatography tandem MS analysis, and a bioinformatics alignment method to determine personalized proteomic profiles for membrane proteins in human tissues. This strategy provided accurate (6% error) and reproducible (34% relative S.D.) quantification of three independently purified membrane fractions from the same human colorectal cancer (CRC) tissue. Using CRC as a model, we constructed the personalized membrane protein atlas of paired tumor and adjacent normal tissues from 28 patients with different stages of CRC. Without fractionation, this strategy confidently quantified 856 proteins (≥2 unique peptides) across different patients, including the first and robust detection (Mascot score: 22,074) of the well-documented CRC marker, carcinoembryonic antigen 5 by a discovery-type proteomics approach. Further validation of a panel of proteins, annexin A4, neutrophils defensin A1, and claudin 3, confirmed differential expression levels and high occurrences (48-70%) in 60 CRC patients. The most significant discovery is the overexpression of stomatin-like 2 (STOML2) for early diagnostic and prognostic potential. Increased expression of STOML2 was associated with decreased CRC-related survival; the mean survival period was 34.77 ± 2.03 months in patients with high STOML2 expression, whereas 53.67 ± 3.46 months was obtained for patients with low STOML2 expression. Further analysis by ELISA verified that plasma concentrations of STOML2 in early-stage CRC patients were elevated as compared with those of healthy individuals (p < 0.001), suggesting that STOML2 may be a noninvasive serological biomarker for early CRC diagnosis. The overall sensitivity of STOML2 for CRC detection was 71%, which increased to 87% when combined with CEA measurements. This study demonstrated a sensitive, label-free strategy for differential analysis of tissue membrane proteome, which may provide a roadmap for the subsequent identification of molecular target candidates of multiple cancer types.     

Proteomic-based identification of the APC-binding protein EB1 as a candidate of novel tissue biomarker and therapeutic target for colorectal cancer

Novel candidates of biomarker and therapeutic target in colorectal cancer (CRC) were investigated using a proteomic approach. The proteome of normal colorectal epithelial tissues was compared with that of the tumor ones in 59 CRC patients using two-dimensional difference gel electrophoresis. Of 3458 protein spots, 110 exhibited statistically significant (p<0.01) differences in intensity (more than 2.5-folds) between the normal and tumor tissue groups. Of 67 unique gene products that were identified for 105 of the 110 protein spots, we focused on the higher expression of the adenoma polyposis coli-binding protein EB1 (EB1). EB1 was originally discovered as a binding protein of APC, which is a tumor suppressor gene product, and the expression of EB1 has been associated with poor prognosis in several malignancies but not in CRC. Immunohistochemical analysis of the 132 CRC cases revealed that EB1 was overexpressed in tumor cells in correlation with poor prognosis. Suppression of EB1 by RNAi inhibited CRC cell proliferation and invasion. In this study, the overexpression of EB1 in CRC tissues correlating with prognosis, and its functional contribution to the malignant phenotypes of CRC cells are described. The present findings indicate that EB1 is a potential biomarker and therapeutic target in CRC.     

CircPLCE1 facilitates the malignant progression of colorectal cancer by repressing the SRSF2-dependent PLCE1 pre-RNA splicing

Studies have demonstrated that circular RNAs (circRNAs) play important roles in various types of cancer; however, the mechanisms of circRNAs located in the nucleus have rarely been explored. Here, we report a novel circular RNA circPLCE1 (hsa_circ_0019230) that facilitates the malignant progression of colorectal cancer (CRC) by repressing serine/arginine-rich splicing factor 2 (SRSF2)-dependent phospholipase C epsilon 1 (PLCE1) pre-RNA splicing. Quantitative real-time polymerase chain reaction was used to determine the expression of circPLCE1 in CRC tissues and cells. Cell Counting Kit-8, Transwell and flow cytometric assays were used to assess the role of circPLE1 in CRC cell proliferation, migration and apoptosis, respectively. An animal study was conducted to test the role of circPLCE1 in vivo. Furthermore, catRAPID and RPISeq were used to predict the possible binding proteins of circPLCE1. RNA fractionation and RNA immunoprecipitation assays were used to confirm the RNA-protein interaction. In this study, we found that circPLCE1 was more significantly down-regulated in CRC tissues compared with that in adjacent normal tissues. However, circPLCE1 knockdown suppressed CRC cell proliferation, migration and invasion and increased apoptosis. Nude mouse experiments showed that ectopic expression of circPLCE1 dramatically increased tumour growth in vivo. Mechanistically, circPLCE1 directly bound to the SRSF2 protein, repressing SRSF2-dependent PLCE1 pre-RNA splicing, resulting in the progression of CRC. Individually mutating the binding sites of circPLCE1 abolished the inhibition of PLCE1 mRNA production. Our study revealed a novel molecular mechanism in the regulation of PLCE1 and suggested a new function of circular RNA.