Hanna Lee

Identification of S100A8 and S100A9 as serological markers for colorectal cancer.

In search of novel serological protein biomarkers for human colorectal cancer (CRC), we analyzed CRC tissues using two-dimensional difference in-gel electrophoresis (2D-DIGE) on a narrow range IPG strip (pH 5.5-6.7). By comparing tumor tissues with matched normal tissues in a pairwise manner (n = 6), we identified 34 up-regulated and 17 down-regulated spots with intensity changes greater than 2-fold (Students t-test, p < 0.05). Expression of both mRNA and protein levels of four proteins, adenosylhomocysteinase, Nm23-H1, S100A8 and S100A9, in CRC tissues was further evaluated by semiquantitative RT-PCR and Western blot analysis. The results revealed that all four proteins were elevated in the tumor tissues. We also confirmed, by immunohistochemistry, that adenosylhomocysteinase and Nm23-H1 were overexpressed in tumor cell cytoplasm and that S100A8 and S100A9 proteins were strongly expressed in tumor infiltrating immune cells. Western blot analysis with fractionated plasma samples showed that S100A8 and S100A9 were significantly increased in the plasma of CRC patients (n = 77) and colorectal adenoma patients (n = 11), compared to healthy controls (n = 21). The area under a receiver operating characteristic (ROC) curve was 0.91 for S100A8 and 0.89 for S100A9, which was superior to the established tumor marker carcinoembryonic antigen with 0.78 for the area under the ROC curve. Some patients with inflammatory diseases such as pancreatitis also showed elevated levels of the proteins. Importantly, in comparison to the control group, both proteins showed a remarkable change at the early stage of cancer. Therefore, we suggest S100A8 and S100A9 as candidates for serological biomarkers in combination with other serum markers that aid CRC diagnosis.

Human plasma carboxylesterase 1, a novel serologic biomarker candidate for hepatocellular carcinoma

To identify and characterize a serologic glycoprotein biomarker for hepatocellular carcinoma (HCC), multi‐lectin affinity chromatography was used to isolate intracellular N‐linked glycoprotein fractions from five paired non‐tumor and tumor tissues. From the series of 2‐D DIGE targeted differentially expressed N‐linked glycoproteins, we identified human liver carboxylesterase 1 (hCE1), which was remarkably down‐regulated in tumor tissues, a finding confirmed by Western blot, a quantitative real‐time RT‐PCR, and immunohistochemical staining of non‐tumor and tumor tissues from total 58 HCC patients. To investigate whether hCE1 is also present in human plasma, we employed a magnetic bead‐based immunoprecipitation followed by nano‐LC‐MS/MS analysis, and we found for the first time that hCE1 is present in human plasma as opposed to that in liver tissues. That is, from normalization of hCE1 signal by the immunoprecipitation and Western blot analysis, hCE1 levels were increased in plasma specimens from HCC patients than in plasma from other disease patient groups (e.g. liver cirrhosis, chronic hepatitis, cholangiocarcinoma, stomach cancer, and pancreatic cancer). From the receiver operating characteristic analysis in HCC, both sensitivity and specificity were shown to be greater than 70.0 and 85.0%, respectively. Thus, the high‐resolution proteomic approach demonstrates that hCE1 is a good candidate for further validation as a serologic glycoprotein biomarker for HCC.

Non-histone nuclear factor HMGB1 is phosphorylated and secreted in colon cancers

The high mobility group box 1 (HMGB1) protein, a non-histone nuclear factor, is overexpressed and localizes to the cytoplasm in some cancer cells. However, the mechanism of cytoplasmic HMGB1 transport, extracellular secretion, and its role in cancer progression is not clear. To simulate the activated state of HMGB1, we mutated serine residues of nuclear localization signals (NLSs) to glutamic acid and performed transfection assays. We carried out a kinase inhibitor study and evaluated the cell migration by invasion assay. We showed that phosphorylated HMGB1 localizes in the cytoplasm of colon cancer cells and also showed the interaction of PKC and HMGB1 by immunoprecipitation analysis. Concurrent mutations at six serine residues (35, 39, 42, 46, 53, and 181) to glutamic acid induced the nuclear to cytoplasmic transport of HMGB1, which was detected in the culture medium. We also observed that the secretion of HMGB1 correlated with increased cancer cell invasiveness. Our results suggest that phosphorylated HMGB1 is transported to the cytoplasm, is subsequently secreted from the cell, and has a role in tumor progression through the activation of genes related to cell migration.

Macrophage Migration Inhibitory Factor May Be Used as an Early Diagnostic Marker in Colorectal Carcinomas

Recent genetic studies have identified many differentially expressed genes in colorectal carcinomas. For validation of up-regulated genes in colorectal carcinomas, we performed an enzyme-linked immunosorbent assay. Candidate markers were selected from gene expression data for 40 colorectal cancers and 35 matched normal mucosal samples. Based on intensive filtering, 9 genes were selected for the further evaluations. Among them, macrophage migration inhibitory factor (MIF), inhibin betaA, and chemokine ligand 10 were screened, and the results were compared with carcinoembryonic antigen (CEA) in serum samples of 129 patients with colon cancer and 53 healthy control subjects. We found that the serum MIF level was significantly increased in patients with colorectal cancer. Compared with CEA, MIF was more sensitive in early cancer detection (47.3% vs 29.5%). However, the specificity was not as high as that of CEA (90.6% vs 100.0%). Our findings indicate that MIF may be used as a diagnostic marker in colorectal carcinomas.

Diagnostic Significance of Serum HMGB1 in Colorectal Carcinomas

High mobility group box 1 protein (HMGB1), a nuclear protein, can be translocated to the cytoplasm and secreted in colon cancer cells. However, the diagnostic significance of HMGB1 has not been evaluated in colorectal carcinomas. For this purpose, we have screened the expression and secretion of HMGB1 in 10 colon cancer cell lines and 1 control cell line and found that HMGB1 was detected in the culture medium. To evaluate the diagnostic value of HMGB1, we performed an enzyme-linked immunosorbent assay to measure HMGB1 levels and compared them to carcinoembryonic antigen (CEA) levels in the serum samples of 219 colorectal carcinoma patients and 75 healthy control subjects. We found that the serum HMGB1 level was increased by 1.5-fold in patients with colorectal carcinoma compared to those in healthy controls. When HMGB1 and CEA levels were compared, HMGB1 had similar efficacy as CEA regarding cancer detection (the sensitivity was 20.1% for HMGB1 vs. 25.6% for CEA, and the specificity was 96% for HMGB1 vs. 90.7% for CEA). Moreover, the diagnostic accuracy of HMGB1 for stage I cancer was significantly higher than that of CEA (sensitivity: 41.2% vs. 5.9%; specificity: 96% vs. 90.7). When we combined HMGB1 and CEA, the overall diagnostic sensitivity was higher than that of CEA alone (42% vs. 25.6%), and the diagnostic sensitivity for stage I was also elevated (47% vs. 5.9%). However, the prognosis of patients was not related with serum HMGB1 concentrations. Our findings indicate that serum HMGB1 levels are increased in a subset of colorectal carcinomas, suggesting their potential utility as a supportive diagnostic marker for colorectal carcinomas.

Differential Gene Expression Profiles of Metastases in Paired Primary and Metastatic Colorectal Carcinomas

Background and Methods: Despite the overwhelming clinical significance of metastases, the cellular and molecular mechanisms involved are largely unknown. In order to define significant differences between primary colon carcinomas and their metastases, we analyzed gene expression profiles of 12 sets of triple-paired tissues using 19 K human oligonucleotide microarrays. A total of 36 microarray experiments were analyzed by unsupervised two-way hierarchical clustering and multi-dimensional scaling (MDS). Results: Both methods completely distinguished normal mucosa from carcinoma, but failed to demonstrate a complete classification of primary and metastatic carcinomas. We found a separable tendency to be classified into the primary and metastatic colon carcinomas by MDS. In supervised hierarchical clustering, we identified 80 genes that were differentially expressed between paired primary and metastatic colon carcinomas. The 80 identified genes also successfully distinguished three validation sets of primary and lung-metastatic colon carcinomas. A specific set of genes was identified that distinguished the metastasis from the corresponding primary tumor in nearly half of the metastases analyzed. Conclusions: We suggest that a more accurate model of the metastatic potential is based on a global tumor expression pattern along with the appearance of distinct metastatic variants. This molecular profiling may be useful for the future study of colon cancer metastasis.

High mobility group box-1 is phosphorylated by protein kinase C zeta and secreted in colon cancer cells.

High mobility group box-1 (HMGB1), a nuclear protein, is overexpressed and secreted in cancer cells. Phosphorylation on two different nuclear localization signal regions are known to be important for the nuclear-to-cytoplasmic transport and secretion of HMGB1. However, little is known about the biochemical mechanism of HMGB1 modifications and its subsequent secretion from cancer cells. To identify the specific enzyme and important sites for HMGB1 phosphorylation, we screened the protein kinase C (PKC) family in a colon cancer cell line (HCT116) for HMGB1 binding by pull-down experiments using a 3XFLAG-HMGB1 construct. Strong interactions between atypical PKCs (PKC-ζ, λ, and ι) and cytoplasmic HMGB1 were observed in HCT116 cells. We further identified the most critical PKC isotype that regulates HMGB1 secretion is PKC-ζ by using PKC inhibitors and siRNA experiments. The serine residues at S39, S53 and S181 of HMGB1 were related to enhancing HMGB1 secretion. We also demonstrated overexpression and activation of PKC-ζ in colon cancer tissues. Our findings suggest that PKC-ζ is involved in the phosphorylation of HMGB1, and the phosphorylation of specific serine residues in the nuclear localization signal regions is related to enhanced HMGB1 secretion in colon cancer cells.

Profiling of differentially expressed proteins in stage IV colorectal cancers with good and poor outcomes.

Screening patients at high risk of recurrence of cancer would allow for more accurate and personalized treatment. In this study, we tried to identify the prognosis-related protein profile by applying two different quantitative proteomic techniques, difference in-gel electrophoresis and cleavable isotope-coded affinity tag method. Six tumor tissues were obtained from stage IV colorectal cancer (CRC) patients, of which three have survived more than five years (good prognostic group, GPG) and the other three died within 25 months (poor prognostic group, PPG) after palliative surgery and subsequent chemotherapy treatment. From the two independent quantitative analyses, we identified 175 proteins with abundance ratios greater than 2-fold. Gene ontology analysis revealed that proteins related to cellular assembly/organization and movement were generally increased in the PPG. Twenty-two proteins, including caveolin-1, were chosen for confirmatory studies by Western blot and immunohistochemistry. The Western blot data for each individual protein were analyzed with Mann-Whitney tests, and a multi-marker panel was generated by logistic regression analysis. Five proteins, fatty acid binding protein 1, intelectin 1, transitional endoplasmic reticulum ATPase, transgelin and tropomyosin 2, were significantly different between the two prognostic groups within 95% confidence. No single protein could completely distinguish the two groups from each other. However, a combination of the five proteins effectively distinguished PPG from GPG patients (AUC=1). Our study suggests a multi-marker panel composed of proteome signatures that provide accurate predictive information and potentially assist personalized therapy. This article is part of a Special Issue entitled: Proteomics: The clinical link.

MicroRNA expression profile of gastrointestinal stromal tumors is distinguished by 14q loss and anatomic site

MicroRNAs are known to regulate gene expression. Although unique microRNA expression profiles have been reported in several tumors, little is known about microRNA expression profiles in GISTs. To evaluate the relationship between microRNA expression and clinicopathologic findings of GISTs, we analyzed the microRNA expression profiles of GISTs. We used fresh frozen tissues from 20 GISTs and analyzed KIT and PDGFRA mutations and chromosomal loss status. MicroRNA expression was analyzed using a microRNA chip containing 470 microRNAs. Using unsupervised hierarchical clustering analysis, we found four distinct microRNA expression patterns in our 20 GISTs. Six GISTs that did not have 14q loss formed a separate cluster. In the 14 GISTs with 14q loss, 5 small bowel GISTs formed a separate cluster and the remaining 9 GISTs could be divided into two groups according to frequent chromosomal losses and tumor risk. We found 73 microRNAs that were significantly down‐regulated in the GISTs with 14q loss; 38 of these microRNAs are encoded on 14q. We also found many microRNAs that were down‐regulated in small bowel and high‐risk group GISTs. Most of the microRNAs down‐regulated in the high‐risk group and small bowel GISTs are known to be involved in tumor progression, specifically by stimulating mitogen‐activated protein kinase (MAPK) and the cell cycle. The microRNA expression patterns of GISTs are closely related to the status of 14q loss, anatomic site, and tumor risk. These findings suggest that microRNA expression patterns can differentiate several subsets of GISTs.

Analysis of Nuclear High Mobility Group Box 1 (HMGB1)-Binding Proteins in Colon Cancer Cells: Clustering with Proteins Involved in Secretion and Extranuclear Function

HMGB1 is a nuclear protein that is overexpressed and secreted in cancer cells. However, little is known about the roles of HMGB1 in the cytoplasm and secretory pathway in cancer cells. To clarify this aspect of HMGB1 function, we fractionated the cytoplasm of HCT116 colon cancer cells and used a proteomic approach to analyze cytoplasmic HMGB1-binding proteins. Pull-down experiments using recombinant HMGB1 protein as bait, followed by mass spectrometry analysis identified 162 interacting proteins. Among them were 74 proteins known to be localized exclusively to the extra-nuclear region, and 60 proteins known to be localized to both nuclear and extranuclear regions. The functions of these binding proteins include involvement in cell-cycle progression, cell proliferation, anti-apoptosis, and angiogenesis. In addition, nine of the identified proteins are related to protein translocation and secretion. These include annexin A2, myosin IC isoform a, myosin-9, and Ras-related protein Rab10, which are involved in unconventional protein secretion. Cytoplasmic HMGB1 was primarily associated with the lysosomal cytosol fraction and was colocalized with the lysosomal marker LAMP1. Our findings suggest that cytoplasmic HMGB1 binds to a number of molecules related to cancer progression and the unconventional secretory pathway.