Identification of PSME3 as a novel serum tumor marker for colorectal cancer by combining two-dimensional polyacrylamide gel electrophoresis with a strictly mass spectrometry-based approach for data analysis

The purpose of this study was to identify and validate novel serological protein biomarkers of human colorectal cancer (CRC). Proteins from matched CRC and adjacent normal tissue samples were resolved by two-dimensional gel electrophoresis. From each gel all spots were excised, and enveloped proteins were identified by MS. By comparison of the resulting protein profiles, dysregulated proteins can be identified. A list of all identified proteins and validation of five exemplarily selected proteins, elevated in CRC was reported previously (Roessler, M., Rollinger, W., Palme, S., Hagmann, M. L., Berndt, P., Engel, A. M., Schneidinger, B., Pfeffer, M., Andres, H., Karl, J., Bodenmuller, H., Ruschoff, J., Henkel, T., Rohr, G., Rossol, S., Rosch, W., Langen, H., Zolg, W., and Tacke, M. (2005) Identification of nicotinamide N-methyltransferase as a novel serum tumor marker for colorectal cancer. Clin. Cancer Res. 11, 6550-6557). Here we describe identification and initial validation of another potential marker protein for CRC. Comparison of tissue protein profiles revealed strong elevation of proteasome activator complex subunit 3 (PSME3) expression in CRC tissue. This dysregulation was not detectable based on the spot pattern. The PSME3-containing spot on tumor gels showed no visible difference to the corresponding spot on matched control gels. MS analysis revealed the presence of two proteins, PSME3 and annexin 4 (ANXA4) in one and the same spot on tumor gels, whereas the matched spot contained only one protein, ANXA4, on control gels. Therefore, dysregulation of PSME3 was masked by ANXA4 and could only be recognized by MS-based analysis but not by image analysis. To validate this finding, antibody to PSME3 was developed, and up-regulation in CRC was confirmed by Western blot analysis and immunohistochemistry. Finally by developing a highly sensitive immunoassay, PSME3 could be detected in human sera and was significantly elevated in CRC patients compared with healthy donors and patients with benign bowel disease. We propose that PSME3 be considered a novel serum tumor marker for CRC that may have significance in the detection and in the management of patients with this disease. Further studies are needed to fully assess the potential clinical value of this marker candidate.     

Comparative proteomic analysis of human placenta derived from assisted reproductive technology

The aim of this study was to use proteomics-based approach to examine differences in protein expression in placenta derived from assisted reproductive technology (ART) and normal pregnancy. Using 2-DE we found that, compared with the control group, 12 spots in standard in vitro fertilization group and 18 spots in intracytoplasmic sperm injection group were identified as significantly differentially expressed proteins. Among them, six spots were differentially expressed in both standard IVF and ICSI groups with the same change tendency. Totally, 20 proteins were successfully identified by MALDI TOF/TOF MS, including proteins involved in the membrane traffic, metabolism, nucleic acid processing, stress response and cytoskeleton. Notably, five proteins detected to be differentially expressed in both ART groups were identified as annexin A3, hnRNP C1/C2, alpha-SNAP, FTL and ATP5A. Some of the proteins were confirmed by Western blot and immunohistochemistry analysis. Our study allowed for the initial identification of these proteins related to various functions in placentation with significantly altered abundance in ART groups. The present results reveal that abnormal protein profiles are involved in ART placenta and these differentially expressed proteins may be valuable for the evaluation of potential association between ART treatment and offspring outcome.     

大鼠侧脑室注射瘦素对卵巢摘除给予雌激素子宫腔液2-DE图谱分析

瘦素(leptin)通过动物下丘脑-垂体轴对其生殖活动予以调节,但瘦素通过下丘脑对子宫调节作用仍不清楚.本研究在大鼠侧脑室微量注射瘦素6 h后,采用双向凝胶电泳(2-DE)结合基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)技术分离、鉴定了卵巢摘除给予雌激素的子宫腔液蛋白质表达谱.我们获得了分辨率较高、重复性较好的2-DE图谱|质谱分析结合SwissProt蛋白数据库检索,共鉴定了24个差异表达的蛋白质与生理盐水注射（对照）比较,侧脑室微量注射瘦素引起22个蛋白质分子显著上调,2个显著下调.重要的是,在上调的蛋白质分子中包括转铁蛋白、基质金属蛋白酶7、膜联蛋白A1、补体B因子、磷酸甘油酸酯变位酶1、烯醇酶、冠蛋白-1A等15个蛋白质分子,下调的2个蛋白质分子是补体C3、溶解物载体家族4成员3.这些蛋白质分子涉及细胞分化、细胞迁移、细胞凋亡,以及炎症反应等功能.实验结果提示,瘦素可能通过下丘脑-垂体轴参与免疫调节、雌性生殖及炎症反应等,从而对子宫发挥调节作用.本研究将为加深对瘦素调节子宫作用的认识,并为深入研究女性生殖系统疾病提供新的启示.     

Fine-needle aspiration of thyroid nodules: proteomic analysis to identify cancer biomarkers

At present, the clinical and pathological analysis used in the diagnosis of papillary thyroid cancer (PTC) are insufficient to discern tumor behavior, and new diagnostic and prognostic markers need to be identified. In this study, we performed a comparative proteome analysis to examine the global changes of fine needle aspiration fluid (FNA) protein patterns of two variants of malignant PTC (classical variant PTC (cPTC) and tall cell variant PTC (TCV)) with respect to the controls. Changes in protein expression were identified using two-dimensional electrophoresis (2DE) and peptide mass fingerprinting via MALDI-TOF mass spectrometry (MS), as well as Western blot analysis. A statistical significant up-regulation of 17 protein spots in cPTC and/or TCV with respect to controls was demonstrated. These proteins included transthyretin precursor (TTR), ferritin light chain (FLC), proteasome activator complex subunit 1 and 2, alpha-1-antitrypsin precursor, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), lactate dehydrogenase chain B (LDH-B), apolipoprotein A1 precursor (Apo-A1), annexin A1, DJ-1 protein and cofilin-1. In addition, 12 protein spots were found exclusively in cPTC and three exclusively in TCV. These latter proteins (ferritin heavy chain (FHC), peroxiredoxin 1 (PRX1) and 6-phosphogluconate dehydrogenase (6-PDGH)) correspond to stress response proteins and, until now, had not been described in thyroid tumors. These findings illustrate the potential use of FNA proteomics to identify protein changes associated with thyroid cancer and to advance potential protein biomarkers in the diagnostic classification of the disease.     

Minocycline binds and inhibits LYN activity to prevent STAT3-meditated metastasis of colorectal cancer

Colorectal cancer (CRC) is one of the most common malignancies worldwide. Metastasis is a major cause of CRC recurrence and mortality. Several antibiotic drugs have been reported to exert potential anticancer activities, however, whether and how the tetracycline antibiotic minocycline exhibit tumor suppressive effect on CRC remains unknown. Here, we found that minocycline markedly inhibits the epithelial-mesenchymal transition (EMT) process and metastasis of CRC cells both in vitro and in vivo. Using chemical proteomics screening combined with docking analysis and site-directed mutagenesis, we identified LYN as a direct bind target of minocycline, and Ala255 of LYN is required for minocycline binding. Mechanistically, minocycline binding inactivates LYN, leading to STAT3 inactivation and EMT suppression, thereby inhibits CRC metastasis. Tissue microarray analysis further confirmed the clinical relevance of LYN-STAT3 axis in the EMT and progression of CRC. In addition to CRC, minocycline also significantly prevents EMT process and inhibits the metastasis of several other cancer types. Our findings elucidate the mechanism of action of minocycline for the inhibition of CRC metastasis by LYN binding, and suggest that repurposing minocycline may represent a promising strategy for the treatment of advanced CRC and other cancer types.     

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.     

Identification of platinum-resistance associated proteins through proteomic analysis of human ovarian cancer cells and their platinum-resistant sublines

Chemoresistance is a major therapeutic obstacle in cancer patients, and the mechanisms of drug resistance are not fully understood. In the present study, we established platinum-resistant human ovarian cancer cell lines and identified differentially expressed proteins related to platinum resistance. The total proteins of two sensitive (SKOV3 and A2780) and four resistant (SKOV3/CDDP, SKOV3/CBP, A2780/CDDP, and A2780/CBP) human ovarian cancer cell lines were isolated by two-dimensional gel electrophoresis (2-DE). The differentially expressed proteins were identified using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS). In total, 57 differential protein spots were identified, and five proteins, including annexin A3, destrin, cofilin 1, Glutathione-S-transferase omega 1 (GSTO1-1), and cytosolic NADP+-dependent isocitrate dehydrogenase (IDHc), were found to be co-instantaneous significance compared with their parental cells. The expression of the five proteins was validated by quantitative PCR and western blot, and the western blot results showed complete consistency with proteomic techniques. The five proteins are hopeful to become candidates for platinum resistance. These may be useful for further study of resistance mechanisms and screening of resistant biomarkers.     

LncRNA-cCSC1 modulates cancer stem cell properties in colorectal cancer via activation of the Hedgehog signaling pathway

Several long noncoding RNAs (lncRNAs) have been identified in various malignant tumors and determined to contribute to the process of tumorigenesis, including that of colorectal cancer (CRC). Cancer stem cells (CSCs) have been demonstrated to promote the expansion and maintain the invasion and metastasis of cancer cells, owing to their self-renewal capacity. However, the underlying modulation mechanism of CSC-associated lncRNAs in CRC remains largely unclear. Using integrated bioinformatic analysis, we identified a novel lncRNA (lncRNA-cCSC1) that is highly expressed in CRC and colorectal cancer stem cells (CRCSCs). The biological functions of lncRNA-cCSC1 were assessed in vitro and vivo through the silencing or upregulation of its expression. The depletion of lncRNA-cCSC1 markedly inhibited the self-renewal capacity of the CRCSCs and reduced their drug resistance to 5-fluorouracil. In contrast, lncRNA-cCSC1 overexpression increased the self-renewal effect. Furthermore, aberrant lncRNA-cCSC1 expression resulted in a concomitant alteration of smoothened (SMO) and GLI family zinc finger 1 (Gli1) expression in the Hedgehog (Hh) signaling pathway. Our study is the first to identify a novel lncRNA-cCSC1 in CRC and to indicate that it may regulate CSC-like properties via the Hh signaling pathway. Thus, lncRNA-cCSC1 could be a potential biomarker and promising therapeutic target for CRC.     

Proteomic analysis of Tiam1-mediated metastasis in colorectal cancer

Tiam1 (T lymphoma invasion and metastasis 1), a guanine nucleotide exchange factor that activates Rac, was recently identified as a novel colorectal cancer metastasis-related gene. To better understand the mechanism underlying Tiam1-mediated metastasis, we applied two-dimensional polyacrylamide gel electrophoresis (2-DE) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis to identify differentially expressed proteins between Tiam1 transfected and mock transfected colorectal cancer HT29 cells. Eleven differentially expressed proteins were identified and further validated by Western blot and/or real-time PCR. The results revealed that Tiam1 transfection in colorectal cancer cells could upregulate the expression of Fascin-1, heat shock protein 27 (HSP27), high-mobility group box 1 (HMGB1), glutathione S-transferase omega 1 (GSTO1) and downregulate the expression of annexin IV. These differentially expressed proteins may be directly or indirectly regulated by Tiam1 and be helpful in studying mechanisms that lead to the function of Tiam1. These results give some clues to elucidate the mechanism of Tiam1-mediated metastasis for colorectal cancer.     

Identification of annexin A1 as a novel substrate for E6AP‐mediated ubiquitylation

E6‐associated protein (E6AP) is a cellular ubiquitin protein ligase that mediates ubiquitylation and degradation of p53 in conjunction with the high‐risk human papillomavirus E6 proteins. However, the physiological functions of E6AP are poorly understood. To identify a novel biological function of E6AP, we screened for binding partners of E6AP using GST pull‐down and mass spectrometry. Here we identified annexin A1, a member of the annexin superfamily, as an E6AP‐binding protein. Ectopic expression of E6AP enhanced the degradation of annexin A1 in vivo. RNAi‐mediated downregulation of endogenous E6AP increased the levels of endogenous annexin A1 protein. E6AP interacted with annexin A1 and induced its ubiquitylation in a Ca²⁺‐dependent manner. GST pull‐down assay revealed that the annexin repeat domain III of annexin A1 is important for the E6AP binding. Taken together, our data suggest that annexin A1 is a novel substrate for E6AP‐mediated ubiquitylation. Our findings raise the possibility that E6AP may play a role in controlling the diverse functions of annexin A1 through the ubiquitin‐proteasome pathway. J. Cell. Biochem. 106: 1123–1135, 2009. © 2009 Wiley‐Liss, Inc.     

2D differential in-gel electrophoresis for the identification of esophageal scans cell cancer-specific protein markers

The reproducibility of conventional two-dimensional (2D) gel electrophoresis can be improved using differential in-gel electrophoresis (DIGE), a new emerging technology for proteomic analysis. In DIGE, two pools of proteins are labeled with 1-(5-carboxypentyl)-1'-propylindocarbocyanine halide (Cy3) N-hydroxy-succinimidyl ester and 1-(5-carboxypentyl)-1'-methylindodi-carbocyanine halide (Cy5) N-hydroxysuccinimidyl ester fluorescent dyes, respectively. The labeled proteins are mixed and separated in the same 2D gel. 2D DIGE was applied to quantify the differences in protein expression between laser capture microdissection-procured esophageal carcinoma cells and normal epithelial cells and to define cancer-specific and normal-specific protein markers. Analysis of the 2D images from protein lysates of approximately 250,000 cancer cells and normal cells identified 1038 protein spots in cancer cell lysates and 1088 protein spots in normal cell lysates. Of the detected proteins, 58 spots were up-regulated by >3-fold and 107 were down-regulated by >3-fold in cancer cells. In addition to previously identified down-regulated protein annexin I, tumor rejection antigen (gp96) was found up-regulated in esophageal squamous cell cancer. Global quantification of protein expression between laser capture-microdissected patient-matched cancer cells and normal cells using 2D DIGE in combination with mass spectrometry is a powerful tool for the molecular characterization of cancer progression and identification of cancer-specific protein markers.     

Differential intestinal mucosal protein expression in hypercholesterolemic mice fed a phytosterol-enriched diet

The molecular mechanisms involved in the phytosterol-induced decrease in intestinal cholesterol absorption remain unclear. Further, other biological properties such as immunomodulatory activity and protection against cancer have also been ascribed to these plant compounds. To gain insight into the mechanisms underlying phytosterol actions, we conducted a proteomic study in the intestinal mucosa of phytosterol-fed apolipoprotein E-deficient hypercholesterolemic (apoE-/-) mice. With respect to control-fed apoE-/- mice, nine differentially expressed proteins were identified in whole-enterocyte homogenates using 2-D DIGE and MALDI-TOF MS. These proteins are involved in plasma membrane stabilization, cytoskeleton assembly network, and cholesterol metabolism. Four of these proteins were selected for further study since they showed the highest abundance change or had a potential functional relationship with known effects of phytosterols. Annexin A2 (ANXA2) and beta-actin decrease and annexin A4 (ANXA4) and annexin A5 (ANXA5) increase were confirmed by Western blot analysis. Intestinal gene expression of ANXA2 and A5 and beta-actin was reduced, whereas that of ANXA4 was unchanged. The main results were retested in normocholesterolemic C57BL/6J mice. ANXA4 and ANXA5 protein upregulation and ANXA2 and beta-actin downregulation were reproduced in these animals. However, no changes in gene expression were found in C57BL/6J mice in either of the four proteins selected. ANXA2, A4, and A5 and beta-actin are proteins of special interest given their pleiotropic functions that include cholesterol-ester transport from caveolae, apoptosis, and anti-inflammatory properties. Therefore, the protein expression changes identified in this study might be involved in the biological effects of phytosterols.     

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.     

NSC666715 and Its Analogs Inhibit Strand-Displacement Activity of DNA Polymerase β and Potentiate Temozolomide-Induced DNA Damage,Senescence and Apoptosis in Colorectal Cancer Cells

Recently approved chemotherapeutic agents to treat colorectal cancer (CRC) have made some impact; however, there is an urgent need for newer targeted agents and strategies to circumvent CRC growth and metastasis. CRC frequently exhibits natural resistance to chemotherapy and those who do respond initially later acquire drug resistance. A mechanism to potentially sensitize CRC cells is by blocking the DNA polymerase β (Pol-β) activity. Temozolomide (TMZ), an alkylating agent, and other DNA-interacting agents exert DNA damage primarily repaired by a Pol-β-directed base excision repair (BER) pathway. In previous studies, we used structure-based molecular docking of Pol-β and identified a potent small molecule inhibitor (NSC666715). In the present study, we have determined the mechanism by which NSC666715 and its analogs block Fen1-induced strand-displacement activity of Pol-β-directed LP-BER, cause apurinic/apyrimidinic (AP) site accumulation and induce S-phase cell cycle arrest. Induction of S-phase cell cycle arrest leads to senescence and apoptosis of CRC cells through the p53/p21 pathway. Our initial findings also show a 10-fold reduction of the IC₅₀ of TMZ when combined with NSC666715. These results provide a guide for the development of a target-defined strategy for CRC chemotherapy that will be based on the mechanisms of action of NSC666715 and TMZ. This combination strategy can be used as a framework to further reduce the TMZ dosages and resistance in CRC patients.     

Comparative proteomic analysis of apomictic monosomic addition line of <Emphasis Type="Italic">Beta corolliflora</Emphasis> and <Emphasis Type="Italic">Beta vulgaris</Emphasis> L. in sugar beet

Apomixis refers to a process in which plants produce seed without fertilization through female syngamy that produces embryos genetically identical to the maternal parent. In sugar beet, interspecific hybrids between diploid Beta vulgaris and tetraploid Beta corolliflora were established and monosomic addition line M14 was selected because of the apomictic phenotype. By using two-dimensional electrophoresis gels we identified the proteins which were differently expressed between the M14 and B. vulgaris. A total of 27 protein spots which varied expressed between lines were isolated and successfully identified with MALDI-TOF MS. Among them five protein spots were found to be only presented in M14 and two protein spots only expressed in Beta. According to their functional annotations described in Swissprot database, these proteins were, respectively, involved in important biological pathways, such as cell division, functionally classified using the KEGG functional classification system. The result may be useful for us to better understand the genetic mechanism of apomixes.     

Secretion of annexin A3 from ovarian cancer cells and its association with platinum resistance in ovarian cancer patients

Early detection of resistance to platinum-based therapy is critical for improving the treatment of ovarian cancers. We have previously found that increased expression of annexin A3 is a mechanism for platinum resistance in ovarian cancer cells. Here we demonstrate that annexin A3 can be detected in the culture medium of ovarian cancer cells, particularly these cells that express high levels of annexin A3. Levels of annexin A3 were then determined in sera from ovarian cancer patients using an enzyme-linked immunosorbent assay. Compared with those from normal donors, sera from ovarian cancer patients contain significantly higher levels of annexin A3. Furthermore, serum levels of annexin A3 were significantly higher in platinum-resistant patients than in platinum-sensitive patients. To gain insight into the mechanism of secretion, the ovarian cancer cell lines were examined using both transmission electron microscopy and immunoelectron microscopy. Compared with parent cells, there are significantly more vesicles in the cytoplasm of ovarian cancer cells that express high levels of annexin A3, and at least some vesicles are annexin A3-positive. Moreover, some vesicles appear to be fused with the cell membrane, suggesting that annexin A3 secretion may be associated with exocytosis and the release of exosomes. This is supported by our observation that ovarian cancer cells expressing higher levels of annexin A3 released increased numbers of exosomes. Furthermore, annexin A3 can be detected in exosomes released from cisplatin-resistant cells (SKOV3/Cis) by immunoblotting and immunoelectron microscopy.