Proteomic Analyses Reveal High Expression of Decorin and Endoplasmin (HSP90B1) Are Associated with Breast Cancer Metastasis and Decreased Survival

BACKGROUND: Breast cancer is the most common malignancy among women worldwide in terms of incidence and mortality. About 10% of North American women will be diagnosed with breast cancer during their lifetime and 20% of those will die of the disease. Breast cancer is a heterogeneous disease and biomarkers able to correctly classify patients into prognostic groups are needed to better tailor treatment options and improve outcomes. One powerful method used for biomarker discovery is sample screening with mass spectrometry, as it allows direct comparison of protein expression between normal and pathological states. The purpose of this study was to use a systematic and objective method to identify biomarkers with possible prognostic value in breast cancer patients, particularly in identifying cases most likely to have lymph node metastasis and to validate their prognostic ability using breast cancer tissue microarrays. METHODS AND FINDINGS: Differential proteomic analyses were employed to identify candidate biomarkers in primary breast cancer patients. These analyses identified decorin (DCN) and endoplasmin (HSP90B1) which play important roles regulating the tumour microenvironment and in pathways related to tumorigenesis. This study indicates that high expression of Decorin is associated with lymph node metastasis (p<0.001), higher number of positive lymph nodes (p<0.0001) and worse overall survival (p = 0.01). High expression of HSP90B1 is associated with distant metastasis (p<0.0001) and decreased overall survival (p<0.0001) these patients also appear to benefit significantly from hormonal treatment. CONCLUSIONS: Using quantitative proteomic profiling of primary breast cancers, two new promising prognostic and predictive markers were found to identify patients with worse survival. In addition HSP90B1 appears to identify a group of patients with distant metastasis with otherwise good prognostic features

Proteomic Analysis of Prostate Cancer Cell Line Conditioned Media for the Discovery of Candidate BIomarkers for Prostate Cancer

Early detection of prostate cancer is problematic due to the lack of a marker that has high diagnostic sensitivity and specificity. The prostate specific antigen test, in combination with digital rectal examination, is the gold standard for prostate cancer diagnosis. However, this modality suffers from low specificity. Therefore, specific markers for clinically relevant prostate cancer are needed. Our objective was to proteomically characterize the conditioned media from human prostate cancer cell lines to identify secreted proteins that could serve as novel prostate cancer biomarkers. An initial proof of principle study of the PC3 prostate cancer cell line was conducted. From this study over 200 proteins were identified in the conditioned media. Through gene ontology analysis and literature searches Mac-2 binding protein was selected as a candidate biomarker for validation in the serum of prostate cancer patients. A preliminarily validation showed that Mac-2 binding protein has discriminatory ability in prostate cancer diagnosis. However, an extended validation did not confirm this. Based on our proof of principle study we optimized our workflow and extended our analysis by culturing three different prostate cell lines [PC3 (bone metastasis), LNCaP (lymph node metastasis), and 22Rv1 (localized to prostate)]. We conducted a bottom-up analysis of each cell line by 2-dimensional liquid chromatography and tandem mass spectrometry. Of the 2124 proteins identified, 12% (329) were classified as extracellular and 18% (504) as membrane-bound. Among the identified proteins were known prostate cancer biomarkers such as PSA and KLK2. To select the most promising candidates for further investigation, tissue specificity, biological function, disease association based on literature searches, and comparison of protein overlap with the proteome of seminal plasma and serum were examined. Based on these results, several candidates were selected for validation in serum of patients with and without prostate cancer. Of these four novel candidates: follistatin, chemokine (C-X-C motif) ligand 16, pentraxin 3 and spondin 2 showed discriminatory ability. Of the four candidates, follistatin was further studied in an extended validation in serum of patients with biopsy confirmed prostate cancer and tissues of prostate cancer patients of low and high grade tumours by immunohistochemistry. In addition, follistatin was also investigated in the tissue of colon and lung cancer where intense staining was observed in one specimen of lung squamous carcinoma.Ph

Determination of the association of urine prostate specific antigen levels with anthropometric variables in children aged 5-14 years

PURPOSE: Calculation of PSA is possible in human fluids even if it presents in very low concentrations with the help of hypersensitive immunodiagnostic methods. The periurethral glands represent one of the potential sources of urine prostate specific antigen (uPSA) in both sexes but the purpose of studying PSA levels in children is still unclear in the literature. In this pilot study we studied uPSA in a small cohort of normal, pre and post pubertal children, in relation to standard anthropometric variables. MATERIALS AND METHODS: The study cohort consisted of 58 children 5-14 years old (42 boys/16 girls). Height, weight, body mass index (BMI) and the respective stature-for-age, weight-for-age and BMI-for-age percentiles of the sample were determined. uPSA levels were measured using a third generation immunodiagnostic method (DPC Immulite®) that has a lower limit of detection of 3 ng/L. When levels of PSA were above the upper limit of detection, uPSA levels were assessed using the ROCHE technique. RESULTS: uPSA levels tend to be higher in male than female children (p = 0.091, linear regression analysis). uPSA was measurable only in 3/16 girls (18.75%). Measurable uPSA was found in 18/42 boys (42.8%). The range of urine PSA in boys was 0-161000 ng/L (mean 10561.9 ± 31830.48 ng/L). Statistical analysis with linear regression showed correlation with height and age in boys. CONCLUSIONS: The use of hypersensitive assays allows calculation of uPSA in childhood. The values of this variable are measurable in both sexes and related with gender. In boys, uPSA was correlated with age and height but not with other variables tested. Further studies are required to clarify this field

Phosphorylation of IGFBP-1 at discrete sites elicits variable effects on IGF-I receptor autophosphorylation

Wepreviously demonstrated that hypoxia and leucine deprivation cause hyperphosphorylation of IGF-binding protein-1 (IGFBP-1) at discrete sites that markedly enhanced IGF-I affinityandinhibited IGF-I-stimulated cell growth. In this study we investigated the functional role of these phosphorylation sites using mutagenesis. We created three IGFBP-1 mutants in which individual serine (S119/S169/S98) residues were substituted with alanine and S101A was recreated for comparison. The wild-type (WT) and mutant IGFBP-1 were expressed in Chinese hamster ovary cells and IGFBP-1 in cell media was isolated using isoelectric-focusing-free-flow electrophoresis. BIACore analysis indicated that the changes in IGF-I affinity for S98A and S169A were moderate, whereas S119A greatly reduced the affinity of IGFBP-1 for IGF-I (100-fold, P\u3c.0001). Similar results were obtained with S101A. The IGF-I affinity changes of the mutants were reflected in their ability to inhibit IGF-I-induced receptor autophosphorylation. Employing receptor-stimulation assay using IGF-IRoverexpressing P6 cells, we found that WT-IGFBP-1 inhibited IGF-IRβ autophosphorylation (̃2- fold, P \u3c .001), possibly attributable to sequestration of IGF-I. Relative to WT, S98A and S169A mutants did not inhibit receptor autophosphorylation. S119A, on the other hand, greatly stimulated the receptor (2.3-fold, P\u3c.05). The data with S101A matched S119A. In summary, we show that phosphorylation at S98 and S169 resulted in milder changes in IGF-I action; nonetheless most dramatic inhibitory effects on the biological activity of IGF-I were due to IGFBP-1 phosphorylation at S119. Our resultsprovidenoveldemonstrationthatIGFBP- 1phosphorylationatS119canenhanceaffinityforIGF-I possibly through stabilization of the IGF-IGFBP-1 complex. These data also propose that the synergistic interaction of distinct phosphorylation sites may be important in eliciting more pronounced effects on IGF-I affinity that needs further investigation. Copyright © 2013 by The Endocrine Society

Confirmation of peptide identity using SID SRM-MS.

<p>List of peptides for which SID SRM-MS was used to confirm their identification. UniProtKB accession number and amino acid sequence are shown.</p><p>SID: Stable isotope dilution.</p><p>SRM-MS: selected reaction monitoring mass spectrometry.</p

Overall survival curves based on DE/HE expression and hormone treatment.

<p>A, Survival curves for cases with high and low DE that did not receive hormone treatment. B, Survival curves for cases with high and low DE that received hormone treatment. C, Survival curves for cases with high and low HE that did not receive hormone treatment. D, Survival curves for cases with high and low HE that received hormone treatment. Univariate Cox regression used to determine HR and 95% CI. DE: Decorin staining in malignant epithelial tissue. HE: HSP90B1 staining in malignant epithelial tissue.</p

Univariate and multivariate analysis of predictors of overall survival.

<p> <b>Univariate and Multivariable Cox proportional hazards regression to determine predictors of overall survival. Note: Molecular subtype was used in place of ER and HER2 status; Decorin Iwavg was used in place of Decorin Iavg.</b></p

Scoring system used for DCN and HSP90B1 immunohistochemistry.

<p>A, Strong DCN positivity in stroma (3+) and negative in carcinoma (0) (magnification 200×). B, Strong DCN positivity in carcinoma (3+), weak stromal positivity (1+) (200×). C, Moderate HSP90B1 positivity in carcinoma (1+) (200×). D, Strong HSP90B1 positivity in carcinoma (3+) (200×). Decorin antibody (Sigma-Aldrich, St. Louis, MO) used at a dilution of 1∶400. HSP90B1 antibody (Sigma-Aldrich, St. Louis, MO) used at a dilution of 1∶4000.</p

Overall survival curves using combinations of DE and HE expression levels.

<p>Univariate Cox regression used to determine HR; logrank p-values reported; Bonferroni multiple testing adjustment for pairwise comparisons p = 0.05/5 = 0.01. DE: Decorin staining in malignant epithelial tissue. HE: HSP90B1 staining in malignant epithelial tissue. HR: Hazard ratio.</p