Identification of breast cancer metastasis-associated proteins in an isogenic tumor metastasis model using two-dimensional gel electrophoresis and liquid chromatography-ion trap-mass spectrometry

To better understand the molecular mechanisms underlying breast cancer metastasis and search for potential markers for metastatic progression, we have developed a highly metastatic variant of human MDA-MB-435 breast cancer cell line through in vivo stepwise selection of pulmonary metastatic cells caused by parental MDA-MB-435 cells in the athymic mice. Comparative proteomic analysis using 2-DE and LC-IT-MS revealed that 102 protein spots were reproducibly altered more than three-fold between the selected variant and its parental counterpart. Eleven differentially expressed protein spots were identified with high confidence using SEQUEST with uninterpreted tandem mass raw data. Cathepsin D precursor, peroxiredoxin 6 (PDX6), heat shock protein 27 (HSP27), HSP60, tropomyosin 1 (TPM1), TPM2, TPM3, TPM4, 14-3-3 protein epsilon, and tumor protein D54 were up-regulated in the highly metastatic variant, whereas alpha B-crystalline (CRAB) was only detected in its parental counterpart. Differential expression was confirmed for four proteins including PDX6, CRAB, TPM4, and HSP60 by real-time quantitative PCR and Western blotting analysis in our model. Immunohistochemical analysis in 80 breast cancer donors demonstrated a significant association of TPM4 (p = 0.002), HSP60 (p = 0.001), PDX6 (p = 0.002) but not CRAB (p = 0.113) staining with the presence of lymph node metastasis. In addition, TPM4 staining was also associated with clinical stage (p = 0.000), but no significant association was found between TPM4, PDX6, CRAB, and HSP60 expression and tumor size, hormone receptor, and HER-2 status (p > 0.05). The functional implication of these identified proteins was also discussed. These proteomic data are valuable and informative for understanding breast cancer metastasis and searching for potential markers for metastatic progression.     

Identification and analysis of signaling networks potentially involved in breast carcinoma metastasis to the brain

Brain is a common site of breast cancer metastasis associated with significant neurologic morbidity, decreased quality of life, and greatly shortened survival. However, the molecular and cellular mechanisms underpinning brain colonization by breast carcinoma cells are poorly understood. Here, we used 2D-DIGE (Difference in Gel Electrophoresis) proteomic analysis followed by LC-tandem mass spectrometry to identify the proteins differentially expressed in brain-targeting breast carcinoma cells (MB231-Br) compared with parental MDA-MB-231 cell line. Between the two cell lines, we identified 12 proteins consistently exhibiting greater than 2-fold (p<0.05) difference in expression, which were associated by the Ingenuity Pathway Analysis (IPA) with two major signaling networks involving TNFα/TGFβ-, NFκB-, HSP-70-, TP53-, and IFNγ-associated pathways. Remarkably, highly related networks were revealed by the IPA analysis of a list of 19 brain-metastasis-associated proteins identified recently by the group of Dr. A. Sierra using MDA-MB-435-based experimental system (Martin et al., J Proteome Res 2008 7:908-20), or a 17-gene classifier associated with breast cancer brain relapse reported by the group of Dr. J. Massague based on a microarray analysis of clinically annotated breast tumors from 368 patients (Bos et al., Nature 2009 459: 1005-9). These findings, showing that different experimental systems and approaches (2D-DIGE proteomics used on brain targeting cell lines or gene expression analysis of patient samples with documented brain relapse) yield highly related signaling networks, suggest strongly that these signaling networks could be essential for a successful colonization of the brain by metastatic breast carcinoma cells.     

Expression and distribution of HSP27 in response to G418 in different human breast cancer cell lines

Heat shock proteins (HSPs) play an important role in folding, intracellular localization and degradation of cellular proteins. However, the cellular role of HSP27 is not completely understood. The conflicting results have been reported regarding stress-induced nuclear translocation of HSP27. In this study, human breast cancer cells transiently and stably expressing HSP27–EGFP chimera were utilized to observe the intracellular localization of HSP27. The data show that the transient and stable expression of HSP27–EGFP displayed distinguishingly cellular localization. The nuclear translocalization of HSP27–EGFP was correlated with the presence of G418. Experiments carried out with different human breast cancer cell lines revealed clearly different distribution patterns of endogenous HSP27. The subcellular distribution of endogenous HSP27 appeared diffuse throughout the cytoplasm in MDA435 cells. In MCF-7 and SKBR3 cells, the accumulation of the protein was distinctly seen along the cell membrane and around nucleus. Moreover, the nuclear translocation of endogenous HSP27 was stimulated by G418 only in MDA435 cells, but not in MCF-7 and SKBR3 cells. Overexpression of HSP27 has been associated with resistance to cisplatin and doxorubicin. The correlation of the expression pattern of HSP27 with the drug resistance may need to be investigated. Further studies on the intracellular function of HSP27 may take into account its interaction proteins in the cells. It may provide useful information for the identification of sensitivity of carcinoma cells to the chemotherapeutic drugs and development of more specific agents to circumvent HSP27.     

Functional clustering of metastasis proteins describes plastic adaptation resources of breast-cancer cells to new microenvironments

To examine the molecular mechanisms underlying breast cancer metastasis in liver and search for potential markers of metastatic progression in soft-tissue, we analyzed metastatic variants developed from the highly metastatic MDA-MB 435 cell line through in vivo stepwise selection in the athymic mice. Comparative proteomic analysis using two-dimensional electrophoresis (2DE-DIGE) revealed that 74 protein spots were reproducibly more than doubled in liver metastatic cells compared to parental counterpart. From 22 proteins identified by MALDI-TOF, belonging to intermediate filaments, intracellular transport and ATP synthesis, we generated a protein-protein interaction network containing 496 nodes, 12 of which interacted. GRP 75 was connected with four other proteins: prohibitin, HSP 27, elongin B and macropain delta chain. After functional classification, we found that pathways including hepatocyte growth factor receptor (p = 0.014), platelet-derived growth factor (p = 0.018), vascular endothelial growth factor (p = 0.021) and epidermal growth factor (p = 0.050) were predominant in liver metastatic cells, but not in lung metastatic cells. In conclusion, we suggest that GRP 75 is involved in cell proliferation, tumorigenesis and stress response in metastatic cells by recruiting signals in which the transmembrane receptor protein tyrosine kinase signaling pathway (p-value FDR = 1.71 x 10(-2)) and protein amino acid phosphorylation (p-value FDR = 3.28 x 10(-2)) might be the most significant biological process differentially increased in liver metastasis.     

Pyruvate Carboxylase Is Up-Regulated in Breast Cancer and Essential to Support Growth and Invasion of MDA-MB-231 Cells

Pyruvate carboxylase (PC) is an anaplerotic enzyme that catalyzes the carboxylation of pyruvate to oxaloacetate, which is crucial for replenishing tricarboxylic acid cycle intermediates when they are used for biosynthetic purposes. We examined the expression of PC by immunohistochemistry of paraffin-embedded breast tissue sections of 57 breast cancer patients with different stages of cancer progression. PC was expressed in the cancerous areas of breast tissue at higher levels than in the non-cancerous areas. We also found statistical association between the levels of PC expression and tumor size and tumor stage (P < 0.05). The involvement of PC with these two parameters was further studied in four breast cancer cell lines with different metastatic potentials; i.e., MCF-7, SKBR3 (low metastasis), MDA-MB-435 (moderate metastasis) and MDA-MB-231 (high metastasis). The abundance of both PC mRNA and protein in MDA-MB-231 and MDA-MB-435 cells was 2-3-fold higher than that in MCF-7 and SKBR3 cells. siRNA-mediated knockdown of PC expression in MDA-MB-231 and MDA-MB-435 cells resulted in a 50% reduction of cell proliferation, migration and in vitro invasion ability, under both glutamine-dependent and glutamine-depleted conditions. Overexpression of PC in MCF-7 cells resulted in a 2-fold increase in their proliferation rate, migration and invasion abilities. Taken together the above results suggest that anaplerosis via PC is important for breast cancer cells to support their growth and motility.     

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.     

Cluster analyses of the TCGA and a TMA dataset using the coexpression of HSP27 and CRYAB improves alignment with clinical-pathological parameters of breast cancer and suggests different epichaperome influences for each sHSP

Our cluster analysis of the Cancer Genome Atlas for co-expression of HSP27 and CRYAB in breast cancer patients identified three patient groups based on their expression level combination (high HSP27 + low CRYAB; low HSP27 + high CRYAB; similar HSP27 + CRYAB). Our analyses also suggest that there is a statistically significant inverse relationship between HSP27 and CRYAB and known clinicopathological markers in breast cancer. Screening an unbiased 248 breast cancer patient tissue microarray (TMA) for the protein expression of HSP27 and phosphorylated HSP27 (HSP27-82pS) with CRYAB also identified three patient groups based on HSP27 and CRYAB expression levels. TMA24 also had recorded clinical-pathological parameters, such as ER and PR receptor status, patient survival, and TP53 mutation status. High HSP27 protein levels were significant with ER and PR expression. HSP27-82pS associated with the best patient survival (Log Rank test). High CRYAB expression in combination with wild-type TP53 was significant for patient survival, but a different patient outcome was observed when mutant TP53 was combined with high CRYAB expression. Our data suggest that HSP27 and CRYAB have different epichaperome influences in breast cancer, but more importantly evidence the value of a cluster analysis that considers their coexpression. Our approach can deliver convergence for archival datasets as well as those from recent treatment and patient cohorts and can align HSP27 and CRYAB expression to important clinical-pathological features of breast cancer.     

UCH-L1在乳腺癌中的表达及其与转移关系的研究

目的研究泛素羧基末端水解酶L1(UCH-L1)与磷酸化p38(p-p38)在乳腺癌组织、细胞系中的表达情况、两种蛋白的表达与临床病理特征的关系以及UCH-L1与乳腺癌侵袭转移的关系。方法用免疫组织化学方法检测乳腺癌组织中UCH-L1与p-p38蛋白的表达情况,用Western Blot方法检测乳腺癌组织以及细胞系中UCH-L1与p-p38蛋白的表达情况。应用UCH-L1特异性抑制剂作用于乳腺癌高侵袭高转移细胞系MDA-MB-435s后,用Western Blot观察UCH-L1与p-p38蛋白表达改变的情况,用Transwell实验检测MDA-MB-435s细胞侵袭潜能的改变。结果 UCH-L1和p-p38蛋白在乳腺浸润性导管癌中的表达高于其在癌旁正常乳腺组织中的表达(P=0.012,P=0.001),二者呈正相关(r=0.397,P=0.000),并与乳腺癌的TNM分期(P=0.017,P=0.010)、淋巴结转移情况(P=0.033,P=0.021)相关。乳腺上皮细胞系MCF-10A、乳腺癌低侵袭低转移细胞系MCF-7和乳腺癌高侵袭高转移细胞系MDA-MB-435s中两种蛋白表达水平呈递增趋势(P均<0.05)。UCH-L1特异性抑制剂可以浓度依赖性地下调MDA-MB-435s细胞系中p-p38蛋白的表达水平(P均<0.05),并能抑制乳腺癌细胞的侵袭转移潜能。结论 UCH-L1、p-p38过表达与乳腺癌的TMN分期、淋巴结转移有关。UCH-L1可能通过上调p-p38介导乳腺癌转移。     

Purification and characterisation of the breast cancer metastasis suppressor, BRMS1

The breast cancer metastasis suppressor 1 (BRMS1) is a member of a family of proteins that actively suppress tumour metastasis. Understanding BRMS1 mediated metastasis suppression is critical to the development of new therapies designed to prevent and treat patients with late stage breast cancer. To aid research into the functional aspects that underpin BRMS1 mediated metastasis suppression we have expressed and purified recombinant BRMS1 and produced BRMS1 polyclonal antibodies. Using these antibodies to immunoprecipitate endogenous BRMS1 containing complexes from MCF7 breast cancer cell lines we have identified, by mass spectrometry, the small heat shock protein Hsp27 in complex with BRMS1. We also show that the expression of both BRMS1 and Hsp27 are inversely correlated with metastatic potential.     

Inhibition of heat shock protein (Hsp) 27 potentiates the suppressive effect of Hsp90 inhibitors in targeting breast cancer stem-like cells

Heat shock protein (Hsp) 90 is an ATP-dependent chaperone and its expression has been reported to be associated with poor prognosis of breast cancer. Cancer stem cells (CSCs) are particular subtypes of cells in cancer which have been demonstrated to be important to tumor initiation, drug resistance and metastasis. In breast cancer, breast CSCs (BCSCs) are identified as CD24-CD44 + cells or cells with high intracellular aldehyde dehydrogenase activity (ALDH+). Although the clinical trials of Hsp90 inhibitors in breast cancer therapy are ongoing, the BCSC targeting effect of them remains unclear. In the present study, we discovered that the expression of Hsp90α was increased in ALDH + human breast cancer cells. Geldanamycin (GA), a Hsp90 inhibitor, could suppress ALDH + breast cancer cells in a dose dependent manner. We are interesting in the insufficiently inhibitory effect of low dose GA treatment. It was correlated with the upregulation of Hsp27 and Hsp70. By co-treatment with HSP inhibitors, quercetin or KNK437 potentiated BCSCs, which determined with ALDH+ population or mammosphere cells, toward GA inhibition, as well as anti-proliferation and anti-migration effects of GA. With siRNA mediated gene silencing, we found that knockdown of Hsp27 could mimic the effect of HSP inhibitors to potentiate the BCSC targeting effect of GA. In conclusion, combination of HSP inhibitors with Hsp90 inhibitors could serve as a potential solution to prevent the drug resistance and avoid the toxicity of high dose of Hsp90 inhibitors in clinical application. Furthermore, Hsp27 may play a role in chemoresistant character of BCSCs.     

Expression and targeting of the tight junction protein CLDN1 in CLDN1-negative human breast tumor cells

Claudins and occludin constitute the major transmembrane proteins of tight junctions (TJs). We have previously identified the human homologue of the murine Cldn1, CLDN1 (SEMP1) that is expressed in normal, mammary gland-derived epithelial cells but is absent in most human breast cancer cell lines. To investigate the potential functions of CLDN1 protein in tumor and normal epithelial cells, we developed an I-NGFR retroviral vector and monoclonal anti-CLDN1 antibody. In subconfluent and confluent breast cancer cells, MDA-MB-435 and MDA-MB-361, endogenous CLDN1 expression was not detected by an anti-CLDN1 monoclonal antibody by Western blot analysis or quantitative RT-PCR. When CLDN1-negative breast cancer cell lines were transduced with a CLDN1 retrovirus the cells express CLDN1 mRNA constitutively as shown by quantitative RT-PCR. Immunofluorescence analyses of the CLDN1 retroviral transduced breast tumor cells using monoclonal antibodies against CLDN1 reveals a subcellular distribution at cell-cell contact sites similar to the CLDN1 homing pattern in T47-D cells, which express endogenous CLDN1. This cell-cell contact co-localization of CLDN1 was evident in CLDN1-transduced breast tumor cells which fail to express occludin protein (MDA-MB-361 and MDA-MB-435) and express relatively little ZO-1 protein (MDA-MB-435), suggesting that other proteins may be responsible for targeting of CLDN1 to cell-cell contact sites. The re-expression of CLDN1 decreases the paracellular flux of 3 and 40 kDa dextran despite the absence of occludin in the MDA-MB-361 tumor cells. Our findings indicate that in CLDN1-negative breast tumor cells, the basal protein partner requirements for physiological homing of the CLDN1 protein are intact, and that CLDN1 gene transfer and protein expression itself might be sufficient to exert a TJ-mediate gate function in metastatic tumor cells even in the absence of other TJ-associated proteins, such as occludin.     

Proteomics-based strategy to delineate the molecular mechanisms of the metastasis suppressor gene BRMS1

The breast cancer metastasis suppressor 1 (BRMS1) gene has been shown to suppress metastasis without affecting the growth of the primary tumor in mouse models. It has also been shown to suppress the metastasis of tumors derived from breast, melanoma, and, more recently, ovarian carcinoma (see ref 1). However, how BRMS1 exerts its metastasis suppressor function remains unknown. To shed light into its metastatic mechanism of action, the sensitive 2D-DIGE analysis coupled with MS has been used to identify proteins differentially expressed by either overexpressing (Mel-BRMS1) or silencing BRMS1 (sh635) in a melanoma cell line. After comparison of the protein profiles from WT, Mel-BRMS1, and sh635 cells, 79 spots were found to be differentially expressed. Mass spectrometry analysis allowed the unambiguous identification of 55 polypeptides, corresponding to 43 different proteins. Interestingly, more than 75% of the identified proteins were down-regulated in Mel-BRMS1 cells compared to WT. In contrast, all the identified proteins in sh635 cells extracts were up-regulated compared to WT. Most of the deregulated proteins are involved in cell growth/maintenance and signal transduction among other cell processes. Six differentially expressed proteins (Hsp27, Alpha1 protease inhibitor, Cofilin1, Cathepsin D, Bone morphogenetic protein receptor2, and Annexin2) were confirmed by immunoblot and functional assays. Excellent correlation was found between DIGE analysis and immunoblot results, indicating the reliability of the analysis. Available evidence on the reported functions of the identified proteins supports the emerging role of BRMS1 as negative regulator of the metastasis development. This work opens an avenue for the molecular mechanisms' characterization of metastasis suppressor genes with the aim to understand their roles.     

Identification of a potential tumor differentiation factor receptor candidate in prostate cancer cells

Tumor differentiation factor (TDF) is a pituitary protein that is secreted into the bloodstream and has an endocrine function. TDF and TDF-P1, a 20-residue peptide selected from the ORF of TDF, induce differentiation in human breast and prostate cancer cells, but not in other cells. TDF has no known mechanism of action. In our recent study, we identified heat shock 70 kDa proteins (HSP70s) as TDF receptors (TDF-Rs) in breast cancer cells. Therefore, we sought to investigate whether TDF-R candidates from prostate cancer cells are the same as those identified in breast cancer cells. Here, we used TDF-P1 to purify the potential TDF-R candidates by affinity purification chromatography from DU145 and PC3 steroid-resistant prostate cancer cells, LNCaP steroid-responsive prostate cancer cells, and nonprostate NG108 neuroblastoma and BLK CL.4 fibroblast-like cells. We identified the purified proteins by MS, and validated them by western blotting, immunofluorescence microscopy, immunoaffinity purification chromatography, and structural biology. We identified seven candidate proteins, of which three were from the HSP70 family. These three proteins were validated as potential TDF-R candidates in LNCaP steroid-responsive and in DU145 and PC3 steroid-resistant prostate cancer cells, but not in NG108 neuroblastoma and BLK CL.4 fibroblast-like cells. Our previous study and the current study suggest that GRP78, and perhaps HSP70s, are strong TDF-R candidates, and further suggest that TDF interacts with its receptors exclusively in breast and prostate cells, inducing cell differentiation through a novel, steroid-independent pathway.     

Identification of potential tumor differentiation factor (TDF) receptor from steroid-responsive and steroid-resistant breast cancer cells

Tumor differentiation factor (TDF) is a recently discovered protein, produced by the pituitary gland and secreted into the bloodstream. TDF and TDF-P1, a 20-amino acid peptide selected from the open reading frame of TDF, induce differentiation in human breast and prostate cancer cells but not in other cells. TDF protein has no identified site of action or receptor, and its mechanism of action is unknown. Here, we used TDF-P1 to purify and identify potential TDF receptor (TDF-R) candidates from MCF7 steroid-responsive breast cancer cells and non-breast HeLa cancerous cells using affinity purification chromatography (AP), and mass spectrometry (MS). We identified four candidate proteins from the 70-kDa heat shock protein (HSP70) family in MCF7 cells. Experiments in non-breast HeLa cancerous cells did not identify any TDF-R candidates. AP and MS experiments were validated by AP and Western blotting (WB). We additionally looked for TDF-R in steroid-resistant BT-549 cells and human dermal fibroblasts (HDF-a) using AP and WB. TDF-P1 interacts with potential TDF-R candidates from MCF7 and BT-549 breast cells but not from HeLa or HDF-a cells. Immunofluorescence (IF) experiments identified GRP78, a TDF-R candidate, at the cell surface of MCF7, BT-549 breast cells, and HeLa cells but not HDF-a cells. IF of other HSP70 proteins demonstrated labeling on all four cell types. These results point toward GRP78 and HSP70 proteins as strong TDF-R candidates and suggest that TDF interacts with its receptor, exclusively on breast cells, through a steroid-independent pathway.     

应用蛋白质组学技术筛选胃癌耐药相关蛋白质

胃癌多药耐药性是临床胃癌化疗失败最主要的原因之一,但其分子机制仍然不太清楚.为了寻找新的胃癌耐药相关的蛋白质,揭示胃癌多药耐药的分子机制,以胃癌细胞SGC7901和长春新碱诱导的耐药胃癌细胞SGC7901/VCR为研究对象,应用二维凝胶电泳(two-dimensionalelectrophoresis,2-DE)技术分离两种细胞的总蛋白质,图像分析识别差异表达的蛋白质点,基质辅助激光解吸电离飞行时间质谱(matrix-assistedlaserdesorption/ionizationtimeofflightmassspectrometry,MALDI-TOF-MS)及电喷雾电离串联质谱(electrosprayionizationtandemmassspectrometry,ESI-Q-TOF)对差异表达的蛋白质点进行鉴定,蛋白质印迹和实时RT-PCR验证部分差异蛋白质在两株细胞中的表达水平,反义核酸转染技术分析HSP27(heatshockprotein27,HSP27)高表达与SGC7901/VCR耐药的相关性.得到了分辨率较高、重复性较好的两株细胞系的二维凝胶电泳图谱,质谱分析共鉴定了24个差异蛋白质点,蛋白质印迹和实时RT-PCR验证了部分差异蛋白的表达水平,反义寡核苷酸抑制HSP27表达能增加SGC7901/VCR对长春新碱的敏感性.研究结果不仅提示这些差异蛋白质如HSP27,Sorcin等可能与胃癌的多药耐药相关,而且为揭示胃癌细胞的多药耐药性产生机制提供了线索.