MCM3 upregulation confers endocrine resistance in breast cancer and is a predictive marker of diminished tamoxifen benefit.

Resistance to endocrine therapy in estrogen receptor-positive (ER+) breast cancer is a major clinical problem with poorly understood mechanisms. There is an unmet need for prognostic and predictive biomarkers to allow appropriate therapeutic targeting. We evaluated the mechanism by which minichromosome maintenance protein 3 (MCM3) influences endocrine resistance and its predictive/prognostic potential in ER+ breast cancer. We discovered that ER+ breast cancer cells survive tamoxifen and letrozole treatments through upregulation of minichromosome maintenance proteins (MCMs), including MCM3, which are key molecules in the cell cycle and DNA replication. Lowering MCM3 expression in endocrine-resistant cells restored drug sensitivity and altered phosphorylation of cell cycle regulators, including p53(Ser315,33), CHK1(Ser317), and cdc25b(Ser323), suggesting that the interaction of MCM3 with cell cycle proteins is an important mechanism of overcoming replicative stress and anti-proliferative effects of endocrine treatments. Interestingly, the MCM3 levels did not affect the efficacy of growth inhibitory by CDK4/6 inhibitors. Evaluation of MCM3 levels in primary tumors from four independent cohorts of breast cancer patients receiving adjuvant tamoxifen mono-therapy or no adjuvant treatment, including the Stockholm tamoxifen (STO-3) trial, showed MCM3 to be an independent prognostic marker adding information beyond Ki67. In addition, MCM3 was shown to be a predictive marker of response to endocrine treatment. Our study reveals a coordinated signaling network centered around MCM3 that limits response to endocrine therapy in ER+ breast cancer and identifies MCM3 as a clinically useful prognostic and predictive biomarker that allows personalized treatment of ER+ breast cancer patients.

Increased Cholesterol Biosynthesis Is a Key Characteristic of Breast Cancer Stem Cells Influencing Patient Outcome.

Tumor eradication may be greatly improved by targeting cancer stem cells (CSCs), as they exhibit resistance to conventional therapy. To gain insight into the unique biology of CSCs, we developed patient-derived xenograft tumors (PDXs) from ER- breast cancers from which we isolated mammospheres that are enriched for CSCs. Comparative global proteomic analysis was performed on patient tumor tissues and corresponding PDXs and mammospheres. Mammospheres exhibited increased expression of proteins associated with de novo cholesterol synthesis. The clinical relevance of increased cholesterol biosynthesis was verified in a large breast cancer cohort showing correlation with shorter relapse-free survival. RNAi and chemical inhibition of the cholesterol biosynthesis pathway reduced mammosphere formation, which could be rescued by a downstream metabolite. Our findings identify the cholesterol biosynthesis pathway as central for CSC propagation and a potential therapeutic target, as well as providing a mechanistic explanation for the therapeutic benefit of statins in breast cancer.

CYPOR is a novel and independent prognostic biomarker of recurrence-free survival in triple-negative breast cancer patients.

Prognostic and predictive biomarkers of disease and treatment outcome are needed to ensure optimal treatment of patients with triple-negative breast cancer (TNBC). In a mass spectrometry-based global proteomic study of 44 formalin-fixed, paraffin-embedded (FFPE) primary TNBC tumors and 10 corresponding metastases, we found that Cytochrome P450 reductase (CYPOR) expression correlated with patient outcome. The correlation between CYPOR expression and outcome was further evaluated in a Danish cohort of 113 TNBC patients using immunohistochemistry and publicly available gene expression data from two cohorts of TNBC and basal-like breast cancer patients, respectively (N = 249 and N = 580). A significant correlation between high CYPOR gene expression and shorter recurrence-free survival (RFS), but not overall survival, was found in the cohort of 249 TNBC patients (p = 0.018, HR = 1.77, 95% CI 1.1-2.85), and this correlation was recapitulated in a cohort of 580 basal-like breast cancer patients (p = 0.018, HR = 1.4, 95% CI 1.06-1.86). High CYPOR protein expression was also associated with shorter RFS in the cohort of 113 TNBC patients (p = 0.017, HR = 2.73, 95% CI 1.20-6.19), particularly those who were lymph node tumor-negative (p = 0.029, HR = 5.22). Multivariate Cox regression analysis identified CYPOR as an independent prognostic factor for shorter RFS in TNBC patients (p = 0.032, HR = 2.19, 95% CI 1.07-4.47). Together, these data suggest high expression of CYPOR as an independent prognostic biomarker of shorter RFS, which could be used to identify patients who should receive more extensive adjuvant treatment and more aggressive surveillance.

Research integrity among PhD students within clinical research at the University of Southern Denmark.

INTRODUCTION: Responsible conduct of research is the basis for the credibility of all research. Research misconduct is defined as the fabrication, falsification or plagiarism committed willfully or grossly negligently in the planning, performing or reporting of research. We undertook a survey of knowledge of the attitudes towards and experiences with research misconduct among PhD students in clinical research. METHODS: A questionnaire previously used in Swedish and Norwegian studies was distributed to PhD students (n = 330) affiliated with the Department of Clinical Research or Department of Regional Health Research, University of Southern Denmark. RESULTS: A total of 165 PhD students completed the questionnaire in full or in part, yielding an overall response rate of 50%. 18-34% reported to have heard (within the past year) about researchers who had plagiarised, falsified or fabricated data, or plagiarised publications. None reported this to occur in their own department. Few stated that they had felt under pressure to either falsify data (1%) or present results in a misleading way (3%). However, 22% stated to have felt an unethical pressure (within the past year) regarding the inclusion or order of authors. CONCLUSIONS: Results indicate that, albeit at a low frequency, research misconduct involving PhD students is taking place. Likewise, a high fraction of respondents reported to have been under pressure regarding authorships, which points to questionable research practices in clinical research. FUNDING: not relevant. TRIAL REGISTRATION: not relevant.

Downregulation of antigen presentation-associated pathway proteins is linked to poor outcome in triple-negative breast cancer patient tumors.

Triple-negative breast cancer (TNBC) is a heterogeneous subtype with varying disease outcomes. Tumor-infiltrating lymphocytes (TILs) are frequent in TNBC and have been shown to correlate with outcome, suggesting an immunogenic component in this subtype. However, other factors intrinsic to the cancer cells may also influence outcome. To identify proteins and molecular pathways associated with recurrence in TNBC, 34 formalin-fixed paraffin-embedded (FFPE) primary TNBC tumors were investigated by global proteomic profiling using mass spectrometry. Approximately, half of the patients were lymph node-negative and remained free of local or distant metastasis within 10 y follow-up, while the other half developed distant metastasis. Proteomic profiling identified >4,000 proteins, of which 63 exhibited altered expression in primary tumors of recurrence versus recurrence-free patients. Importantly, downregulation of proteins in the major histocompatibility complex (MHC) class I antigen presentation pathways were enriched, including TAP1, TAP2, CALR, HLA-A, ERAP1 and TAPBP, and were associated with significantly shorter recurrence-free and overall survival. In addition, proteins involved in cancer cell proliferation and growth, including GBP1, RAD23B, WARS and STAT1, also exhibited altered expression in primary tumors of recurrence versus recurrence-free patients. The association between the antigen-presentation pathway and outcome were validated in a second sample set of 10 primary TNBC tumors and corresponding metastases using proteomics and in a large public gene expression database of 249 TNBC and 580 basal-like breast cancer cases. Our study demonstrates that downregulation of antigen presentation is a key mechanism for TNBC cells to avoid immune surveillance, allowing continued growth and spread.

Elucidation of Altered Pathways in Tumor-Initiating Cells of Triple-Negative Breast Cancer: A Useful Cell Model System for Drug Screening.

A limited number of cancer cells within a tumor are thought to have self-renewing and tumor-initiating capabilities that produce the remaining cancer cells in a heterogeneous tumor mass. Elucidation of central pathways preferentially used by tumor-initiating cells/cancer stem cells (CSCs) may allow their exploitation as potential cancer therapy targets. We used single cell cloning to isolate and characterize four isogenic cell clones from a triple-negative breast cancer cell line; two exhibited mesenchymal-like and two epithelial-like characteristics. Within these pairs, one, but not the other, resulted in tumors in immunodeficient NOD/Shi-scid/IL-2 Rγ null mice and efficiently formed mammospheres. Quantitative proteomics and phosphoproteomics were used to map signaling pathways associated with the tumor-initiating ability. Signaling associated with apoptosis was suppressed in tumor-initiating versus nontumorigenic counterparts with pro-apoptotic proteins, such as Bcl2-associated agonist of cell death (BAD), FAS-associated death domain protein (FADD), and myeloid differentiation primary response protein (MYD88), downregulated in tumor-initiating epithelial-like cells. Functional studies confirmed significantly lower apoptosis in tumor-initiating versus nontumorigenic cells. Moreover, central pathways, including ß-catenin and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB)-related signaling, exhibited increased activation in the tumor-initiating cells. To evaluate the CSC model as a tool for drug screening, we assessed the effect of separately blocking NF-κB and Wnt/ß-catenin signaling and found markedly reduced mammosphere formation, particularly for tumor-initiating cells. Similar reduction was also observed using patient-derived primary cancer cells. Furthermore, blocking NF-κB signaling in mice transplanted with tumor-initiating cells significantly reduced tumor outgrowth. Our study demonstrates that suppressed apoptosis, activation of pathways associated with cell viability, and CSCs are the major differences between tumor-initiating and nontumorigenic cells independent of their epithelial-like/mesenchymal-like phenotype. These altered pathways may provide targets for future drug development to eliminate CSCs, and the cell model may be a useful tool in such drug screenings. Stem Cells 2017;35:1898-1912.

NADH-Cytochrome b5 Reductase 3 Promotes Colonization and Metastasis Formation and Is a Prognostic Marker of Disease-Free and Overall Survival in Estrogen Receptor-Negative Breast Cancer.

Metastasis is the main cause of cancer-related deaths and remains the most significant challenge to management of the disease. Metastases are established through a complex multistep process involving intracellular signaling pathways. To gain insight to proteins central to specific steps in metastasis formation, we used a metastasis cell line model that allows investigation of extravasation and colonization of circulating cancer cells to lungs in mice. Using stable isotopic labeling by amino acids in cell culture and subcellular fractionation, the nuclear, cytosol, and mitochondria proteomes were analyzed by LC-MS/MS, identifying a number of proteins that exhibited altered expression in isogenic metastatic versus nonmetastatic cancer cell lines, including NADH-cytochrome b5 reductase 3 (CYB5R3), l-lactate dehydrogenase A (LDHA), Niemann-pick c1 protein (NPC1), and nucleolar RNA helicase 2 (NRH2). The altered expression levels were validated at the protein and transcriptional levels, and analysis of breast cancer biopsies from two cohorts of patients demonstrated a significant correlation between high CYB5R3 expression and poor disease-free and overall survival in patients with estrogen receptor-negative tumors (DFS: p = .02, OS: p = .04). CYB5R3 gene knock-down using siRNA in metastasizing cells led to significantly decreased tumor burden in lungs when injected intravenously in immunodeficient mice. The cellular effects of CYB5R3 knock-down showed signaling alterations associated with extravasation, TGFß and HIFα pathways, and apoptosis. The decreased apoptosis of CYB5R3 knock-down metastatic cancer cell lines was confirmed in functional assays. Our study reveals a central role of CYB5R3 in extravasation/colonization of cancer cells and demonstrates the ability of our quantitative, comparative proteomic approach to identify key proteins of specific important biological processes that may also prove useful as potential biomarkers of clinical relevance. MS data are available via ProteomeXchange with identifier PXD001391.

Targeting VEGF with LNA-stabilized G-rich oligonucleotide for efficient breast cancer inhibition.

In this study, we investigated the efficacy of an LNA (locked nucleic acid)-modified DNA aptamer named RNV66 targeting VEGF against various breast cancer cell lines. Our results demonstrate that RNV66 efficiently inhibits breast cancer cell proliferation both in vitro and in vivo. Introduction of LNA nucleotides were crucial for higher efficacy. Furthermore, the binding interaction of RNV66 with VEGF was investigated using molecular dynamic simulations leading to the first computational model of the LNA aptamer-VEGF complex blocking its interaction with VEGF-receptor.

S100A14 is a novel independent prognostic biomarker in the triple-negative breast cancer subtype.

Triple-negative breast cancer (TNBC) represents a heterogeneous subgroup with generally poor outcome and lack of an effective targeted therapy. Prognostic or predictive biomarkers to guide treatment decisions for this group of patients are needed. To evaluate the potential of S100A14 protein as a novel biomarker in TNBC, the protein expression of S100A14 was correlated with clinical outcomes in a Pilot Sample set and a Danish cohort of predominantly TNBC patients. Kaplan-Meier analysis identified a prognostic impact of S100A14 on disease-free survival and overall survival, showing that tumors with high S100A14 protein expression levels were significantly correlated with poor outcome in TNBC patients (p = 0.017; p = 0.038), particularly those in the basal-like subgroup (p = 0.006; p = 0.037). Importantly, TNBC patients with high S100A14 expression, but tumor-negative axillary lymph nodes (N-), had equally poor outcomes as those with tumor-positive axillary lymph nodes (N+), while TNBC/N- patients with low S100A14 expression had a significantly better disease free survival (p = 0.013). Multivariate analysis revealed that S100A14 is an independent prognostic factor for TNBC patients (p = 0.024; p = 0.05). At the cellular level, S100A14 was found to be expressed in epithelial-like, but not in mesenchymal-like, TNBC cells in vitro. S100A14 is an independent prognostic factor in TNBC and a novel potential therapeutic target in TNBC.

Elucidation of epithelial-mesenchymal transition-related pathways in a triple-negative breast cancer cell line model by multi-omics interactome analysis.

In life sciences, and particularly biomedical research, linking aberrant pathways exhibiting phenotype-specific alterations to the underlying physical condition or disease is an ongoing challenge. Computationally, a key approach for pathway identification is data enrichment, combined with generation of biological networks. This allows identification of intrinsic patterns in the data and their linkage to a specific context such as cellular compartments, diseases or functions. Identification of aberrant pathways by traditional approaches is often limited to biological networks based on either gene expression, protein expression or post-translational modifications. To overcome single omics analysis, we developed a set of computational methods that allow a combined analysis of data collections from multiple omics fields utilizing hybrid interactome networks. We apply these methods to data obtained from a triple-negative breast cancer cell line model, combining data sets of gene and protein expression as well as protein phosphorylation. We focus on alterations associated with the phenotypical differences arising from epithelial-mesenchymal transition in two breast cancer cell lines exhibiting epithelial-like and mesenchymal-like morphology, respectively. Here we identified altered protein signaling activity in a complex biologically relevant network, related to focal adhesion and migration of breast cancer cells. We found dysregulated functional network modules revealing altered phosphorylation-dependent activity in concordance with the phenotypic traits and migrating potential of the tested model. In addition, we identified Ser267 on zyxin, a protein coupled to actin filament polymerization, as a potential in vivo phosphorylation target of cyclin-dependent kinase 1.

Anti-human CD73 monoclonal antibody inhibits metastasis formation in human breast cancer by inducing clustering and internalization of CD73 expressed on the surface of cancer cells.

Recent studies have shown that Abs that target the cell-surface enzyme CD73 (ecto-5'-nucleotidase) reduce growth of primary tumors and metastasis in syngenic mice by inhibiting the catalytic activity of CD73, and thus increasing the activity of cytotoxic T lymphocytes. In this article, we report another anticancer mechanism of anti-CD73 Abs and show that an anti-CD73 mAb (AD2) inhibits metastasis formation by a mechanism independent of CD73 catalytic activity and inhibition of primary tumor growth. This mechanism involves clustering and internalization of CD73, but does not require cross-linking of CD73, because both whole IgG anti-CD73 AD2 mAb and Fab' fragments thereof exhibited this effect. Ex vivo treatment of different breast cancer cell lines with anti-CD73 AD2 mAb before i.v. injection into mice inhibited extravasation/colonization of circulating tumor cells and significantly reduced metastasis development. This effect was also observed when the cancer cell-surface expression of CD73 was significantly reduced by small interfering RNA knockdown. The antimetastatic activity is epitope specific, as another Ab that efficiently binds CD73-expressing live cancer cells did not lead to CD73 internalization and metastasis inhibition. Furthermore, anti-CD73 AD2 mAb inhibited development of metastasis in a spontaneous animal model of human metastatic breast cancer. Our study shows that some anti-CD73 mAbs cause cell-surface clustering of CD73 followed by internalization, thus inhibiting the ability of circulating tumor cells to extravasate and colonize, leading to inhibition of metastasis. Ab-based CD73 cancer therapy should include a combination of Abs that target the catalytic activity of CD73, as well as those with the characteristics described in this article.

Identification and characterization of a chitin-binding protein purified from coelomic fluid of the lugworm Arenicola marina defining a novel protein sequence family.

We have isolated a novel type of lectin named Arenicola marina lectin-1 (AML-1) from the lugworm A. marina. The lectin was purified from the coelomic fluid by affinity chromatography on a GlcNAc-derivatized column and eluted with GlcNAc. On SDS-PAGE, AML-1 showed an apparent molecular mass of 27 and 31 kDa in the reduced state. The N-terminal amino acid sequences were identical in these two bands. In the unreduced state, a complex band pattern was observed with bands from 35 kDa to more than 200 kDa. Two different full-length clones encoding polypeptides of 241 and 243 amino acids, respectively, were isolated from a coelomocyte cDNA library. The two clones, designated AML-1a and AML-1b, were 92% identical at the protein level and represent a novel type of protein sequence family. Purified AML-1 induced agglutination of rabbit erythrocytes, which could be inhibited by N-acetylated saccharides. Recombinant AML-1b showed the same band pattern as the native protein, whereas recombinant AML-1a in the reduced state lacked a 27 kDa band. AML-1b bound GlcNAc-derivatized columns and chitin, whereas AML-1a did not bind to these matrices. Immunohistochemical analysis revealed that AML-1 is expressed by coelomocytes in the nephridium and in round cells in the epidermis and in eggs. Moreover, AML-1 expression was up-regulated in response to a parasitic infection. We conclude that AML-1 purified from coelomic fluid is encoded by AML-1b and represents a novel type of protein family that binds acetylated components.

Identification of markers associated with highly aggressive metastatic phenotypes using quantitative comparative proteomics.

BACKGROUND: The spread of cancer cells from a primary tumor to form metastases at distant sites is a complex process that remains poorly defined. Certain tumor cells are more aggressive and thus lead to rapid development of multiple distant metastases. Here, we identify proteins associated with these aggressive phenotypes. MATERIALS AND METHODS: To identify proteins associated with cancer cell aggressiveness, we used comparative, quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) proteome analysis of a unique metastasis model comprised of three isogenic human breast cancer cell lines that are equally tumorigenic in mice, but display different metastatic potentials ranging from non-metastatic, intermediate-metastatic and highly-metastatic. The altered expression of selected proteins was subsequently confirmed by immunocyto- and immunohistochemistry. RESULTS: The difference in metastatic capabilities was initially confirmed using live animal imaging. Comparative, quantitative proteomics identified 414 proteins, out of which 44 exhibited altered expression between the metastatic and non-metastatic cell lines. The proteins correlating with the aggressiveness of metastasis included leucine-rich repeat containing 59 (LRRC59), while CD59 and chondroitin sulfate proteoglycan 4 (CSPG4) exhibited an inverse correlation with metastatic capability. The altered expression levels of these proteins were biochemically confirmed, as well as demonstrated in xenografts generated from these cell lines. This analysis further demonstrated that the three proteins were associated with the aggressiveness of metastasis rather than metastasis colonization per se. CONCLUSION: Our study provides novel insights into key proteins associated with the metastatic potential of breast cancer cells and identified LRRC59, CD59 and CSPG4 as candidates that merit further study.

Functional heterogeneity within the CD44 high human breast cancer stem cell-like compartment reveals a gene signature predictive of distant metastasis.

The CD44(hi) compartment in human breast cancer is enriched in tumor-initiating cells; however, the functional heterogeneity within this subpopulation remains poorly defined. We used a triple-negative breast cancer cell line with a known bilineage phenotype to isolate and clone CD44(hi) single cells that exhibited mesenchymal/basal B and luminal/basal A features, respectively. Herein, we demonstrate in this and other triple-negative breast cancer cell lines that, rather than CD44(hi)/CD24(-) mesenchymal-like basal B cells, the CD44(hi)/CD24(lo) epithelioid basal A cells retained classic cancer stem cell features, such as tumor-initiating capacity in vivo, mammosphere formation and resistance to standard chemotherapy. These results complement previous findings using oncogene-transformed normal mammary cells showing that only cell clones with a mesenchymal phenotype exhibit cancer stem cell features. Further, we performed comparative quantitative proteomic and gene array analyses of these cells and identified potential novel markers of breast cancer cells with tumor-initiating features, such as lipolysis-stimulated lipoprotein receptor (LSR), RAB25, S100A14 and mucin 1 (MUC1), as well as a novel 31-gene signature capable of predicting distant metastasis in cohorts of estrogen receptor-negative human breast cancers. These findings strongly favor functional heterogeneity in the breast cancer cell compartment and hold promise for further refinements of prognostic marker profiling. Our work confirms that, in addition to cancer stem cells with mesenchymal-like morphology, those tumor-initiating cells with epithelial-like morphology should also be the focus of drug development.

Quantitative proteomics of primary tumors with varying metastatic capabilities using stable isotope-labeled proteins of multiple histogenic origins.

The development of metastasis is a complex, multistep process that remains poorly defined. To identify proteins involved in the colonization phase of the metastatic process, we compared the proteome of tumors derived from inoculation of a panel of isogenic human cancer cell lines with different metastatic capabilities into the mammary fat pad of immunodeficient mice. Using a protein standard generated by SILAC-labeling, a total of 675 proteins were identified and 30 were differentially expressed between at least two of the tumors. The protein standard contained the proteomes of seven cell lines from multiple histogenic origins and displayed superior features compared to standard super-SILAC. The expression of some proteins correlated with metastatic capabilities, such as myosin-9 (nonmuscle myosin II A) and L-lactate dehydrogenase A, while the expression of elongation factor tu correlated inversely to metastatic capabilities. The expression of these proteins was biochemically validated, and expression of myosin-9 in clinical breast cancer samples was further shown to be altered in primary tumors versus corresponding lymph node metastasis. Our study demonstrates an improved strategy for quantitative comparison of an unlimited number of tumor tissues, and provides novel insights into key proteins associated with the colonization phase of metastasis formation.

Selective enrichment of sialic acid-containing glycopeptides using titanium dioxide chromatography with analysis by HILIC and mass spectrometry.

The terminal monosaccharide of cell surface glycoconjugates is typically a sialic acid (SA), and aberrant sialylation is involved in several diseases. Several methodological approaches in sample preparation and subsequent analysis using mass spectrometry (MS) have enabled the identification of glycosylation sites and the characterization of glycan structures. In this paper, we describe a protocol for the selective enrichment of SA-containing glycopeptides using a combination of titanium dioxide (TiO(2)) and hydrophilic interaction liquid chromatography (HILIC). The selectivity of TiO(2) toward SA-containing glycopeptides is achieved by using a low-pH buffer that contains a substituted acid such as glycolic acid to improve the binding efficiency and selectivity of SA-containing glycopeptides to the TiO(2) resin. By combining TiO(2) enrichment of sialylated glycopeptides with HILIC separation of deglycosylated peptides, a more comprehensive analysis of formerly sialylated glycopeptides by MS can be achieved. Here we illustrate the efficiency of the method by the identification of 1,632 unique formerly sialylated glycopeptides from 817 sialylated glycoproteins. The TiO(2)/HILIC protocol requires 2 d and the entire procedure from protein isolation can be performed in <5 d, depending on the time taken to analyze data.

Plasma membrane proteomics and its application in clinical cancer biomarker discovery.

Plasma membrane proteins that are exposed on the cell surface have important biological functions, such as signaling into and out of the cells, ion transport, and cell-cell and cell-matrix interactions. The expression level of many of the plasma membrane proteins involved in these key functions is altered on cancer cells, and these proteins may also be subject to post-translational modification, such as altered phosphorylation and glycosylation. Additional protein alterations on cancer cells confer metastatic capacities, and some of these cell surface proteins have already been successfully targeted by protein drugs, such as human antibodies, that have enhanced survival of several groups of cancer patients. The combination of novel analytical approaches and subcellular fractionation procedures has made it possible to study the plasma membrane proteome in more detail, which will elucidate cancer biology, particularly metastasis, and guide future development of novel drug targets. The technical advances in plasma membrane proteomics and the consequent biological revelations will be discussed herein. Many of the advances have been made using cancer cell lines, but because the main goal of this research is to improve individualized treatment and increase cancer patient survival, further development is crucial to direct analysis of clinically relevant patient samples. These efforts include optimized specimen handling and preparation as well as improved proteomics platforms. Identification of potentially useful proteomics-based biomarkers must be validated in larger, well defined retrospective and prospective clinical studies, and these combined efforts should result in identification of biomarkers that will greatly improve early detection, prognosis, and prediction of treatment response.

Multimeric and trimeric subunit SP-D are interconvertible structures with distinct ligand interaction.

Surfactant protein-D (SP-D) is a calcium dependent lectin in the innate immune system that facilitates clearance of microbes. The protein is associated with mucosal surfaces, and also found in bronchoalveolar lavage, serum and amniotic fluid. Human SP-D includes trimeric subunits and multimeric assemblies of trimeric subunits, which are stabilized by N-terminal interchain disulfide crosslinks. An N-terminal structural polymorphism (Met11Thr) and associated O-glycosylation are previously shown accompanied by incomplete multimerization and with a relative low proportion of multimeric Thr11 SP-D compared to Met11 SP-D. Multimerization has proven important for enhancement of microbial phagocytosis. In the present study defined multimeric forms of Met11Thr SP-D were isolated from human amniotic fluid. Implementation of ManNAc-affinity chromatography allowed high recovery of natural trimeric SP-D subunits. However, affinity chromatography increased the relative proportion of multimers at the expense of natural trimeric subunits. Multimeric SP-D partially disassembled to form trimeric subunits. The resulting distribution of structural forms was independent of the Met11Thr genotype. Trimeric and multimeric SP-D appeared with distinct patterns of disulphide crosslinking, which partly changed according to interconversion between the structural forms. Solid phase assays demonstrated that trimeric SP-D subunits showed greater binding to LPS and PGN, but lower binding to mannan and LTA, than SP-D multimers. Trimeric SP-D subunits also showed greater binding to endogenous lipoproteins: LDL, oxLDL, and HDL, than multimeric SP-D. In conclusion, purified trimeric and multimeric SP-D represent separate and only partly interconvertible molecular populations with distinct biochemical properties.

Efficient isolation and quantitative proteomic analysis of cancer cell plasma membrane proteins for identification of metastasis-associated cell surface markers.

Cell surface membrane proteins are involved in central processes such as cell signaling, cell-cell interactions, ion and solute transport, and they seem to play a pivotal role in several steps of the metastatic process of cancer cells. The low abundance and hydrophobic nature of cell surface membrane proteins complicate their purification and identification by MS. We used two isogenic cell lines with opposite metastatic capabilities in nude mice to optimize cell surface membrane protein purification and to identify potential novel markers of metastatic cancer. The cell surface membrane proteins were isolated by centrifugation/ultracentrifugation steps, followed by membrane separation using a Percoll/sucrose density gradient. The gradient fractions containing the cell surface membrane proteins were identified by enzymatic assays. Stable isotope labeling of the proteome of the metastatic cell line by SILAC followed by mass spectrometry analysis enabled identification and quantification of proteins that were differentially expressed in the two cell lines. Dual stable isotopic labels ((13)C-arginine and (13)C-lysine) instead of a single label ((13)C-arginine) increased the percentage of proteins that could be quantified from 40 to 93%. Repeated LC-MS/MS analyses (3-4 times) of each sample increased the number of identified proteins by 60%. The use of Percoll/sucrose density separation allowed subfractionation of membranes leading to enrichment of membrane proteins (66%) and reduction from 33% to only 16% of protein from other membranous organelles such as endoplasmatic reticulum, Golgi, and mitochondria. In total, our optimized methods resulted in 1919 protein identifications (corresponding to 826 at similarity level 80% (SL80); 1145 (509 at SL80) were identified by two or more peptides of which 622 (300 at SL80) were membrane proteins. The quantitative proteomic analysis identified 16 cell surface proteins as potential markers of the ability of breast cancer cells to form distant metastases.

Metastasis-related plasma membrane proteins of human breast cancer cells identified by comparative quantitative mass spectrometry.

The spread of cancer cells from a primary tumor to form metastasis at distant sites is a complex multistep process. The cancer cell proteins and plasma membrane proteins in particular involved in this process are poorly defined, and a study of the very early events of the metastatic process using clinical samples or in vitro assays is not feasible. We have used a unique model system consisting of two isogenic human breast cancer cell lines that are equally tumorigenic in mice; but although one gives rise to metastasis, the other disseminates single cells that remain dormant at distant organs. Membrane purification and comparative quantitative LC-MS/MS proteomics identified 13 membrane proteins that were expressed at higher levels and three that were underexpressed in the metastatic compared with the non-metastatic cell line from a total of 1919 identified protein entries. Among the proteins were ecto-5'-nucleotidase (CD73), NDRG1, integrin beta1, CD44, CD74, and major histocompatibility complex class II proteins. The altered expression levels of proteins identified by LC-MS/MS were validated using flow cytometry, Western blotting, and immunocyto- and immunohistochemistry. Analysis of clinical breast cancer biopsies demonstrated a significant correlation between high ecto-5'-nucleotidase and integrin beta1 expression and poor outcome, measured as tumor spread or distant recurrence within a 10-year follow-up. Further the tissue analysis suggested that NDRG1, HLA-DRalpha, HLA-DRbeta, and CD74 were associated with the ER(-)/PR(-) phenotype represented by the two cell lines. The study demonstrates a quantitative and comparative proteomics strategy to identify clinically relevant key molecules in the early events of metastasis, some of which may prove to be potential targets for cancer therapy.