Umar, A.

Prognostic protein markers for triple negative breast cancer (Miscellaneous)

2010-11-15T00:00:00Z

Breast cancer is the most commonly diagnosed malignancy in women in the Western world, with 13,000 new patients each year in the Netherlands alone. Extensive research on gene expression profiling has shown that breast cancer is a mixture of biologically different disease entities, referred to as molecular subtypes. Of all molecular subtypes, particularly the triple negative phenotype associates with poor prognosis and poor patient survival. Intriguingly, only a small subgroup of triple negative tumors (25%), which metastasize to distant organs within 3 years, accounts for this poor prognosis. Currently, no clinical markers are available to identify triple negative tumors based on positive expression, to predict disease prognosis, and to target therapy against. The aim of our project was to identify prognostic protein markers for triple negative breast cancer using a comparative tissue proteomics approach. We have subjected frozen breast cancer tissue sections to LCM and prepared tryptic digests for nLC-MS analysis. Peptide abundance levels from poor prognosis samples were compared to good prognosis samples to identify differentially abundant peptides and their corresponding proteins. A selection of 34 differentially abundant proteins appeared to significantly differentiate between the two groups. Careful validation of these proteins may lead to better prediction of disease prognosis of triple negative breast cancer patients. Furthermore, functional analysis of key proteins may help unravel the biology of triple negative breast cancer and may lead to the development of new therapies against target proteins.

Identification of a putative protein-profile associating with tamoxifen therapy-resistance in breast cancer (Article)

2009-06-08T00:00:00Z

Tamoxifen-resistance is a major cause of death in patients with recurrent breast cancer. Current clinical parameters can correctly predict therapy response in only half of the treated patients. Identification of proteins that associate with tamoxifen-resistance is a first step towards better response prediction and tailored treatment of patients. In the present study we aimed to identify putative protein biomarkers indicative of tamoxifen therapy-resistance in breast cancer, using nanoLC-FTICR MS. Comparative proteome analysis was performed on ~5,500 pooled tumor cells obtained through laser capture microdissection from two independently processed data sets (n=24 and n=27) of tamoxifen therapy-sensitive and -resistant tumors. Peptide and protein identifications were acquired by matching mass and elution time features to information in previously generated accurate mass and time tag reference data bases. A total of 17,263 unique peptides were identified that corresponded to 2,556 non-redundant proteins identified with >=2 peptides. From this total, 1,713 proteins overlapped between the two data sets, which were used for further statistical analysis. Comparative proteome analysis of the two data sets combined revealed 100 putatively differentially abundant proteins (p<0.05) between tamoxifen-sensitive and -resistant tumors. The presence and abundance of 47 of these proteins was verified by targeted nanoLC-MS/MS in the same, individual, non-microdissected tumor tissue extracts. ENPP1, EIF3E, and GNB4 significantly associated with progression-free survival upon tamoxifen treatment (p=0.005, p=0.03 and p= 0.04, respectively). Differential abundance of our top discriminating protein, EMMPRIN, was validated by tissue microarray in an independent patient cohort (n=156). EMMPRIN was not only higher expressed in PD tumors, it also significantly associated with shorter time to progression upon tamoxifen treatment (p=0.002). In summary, quantitative comparative proteomics was performed on LCM-derived breast tumor cells using ultra-sensitive nanoLC-FTICR technology; this resulted in the identification of putative biomarkers associating with tamoxifen therapy-resistance in recurrent breast cancer.

High-throughput proteomics of breast carcinoma cells: a focus on FTICR MS (Article)

2008-06-05T00:00:00Z

Discovery of better biomarkers for diagnosis, prognosis, and therapy-response prediction is the most critical task of a scientific quest aimed at developing novel, tailor-made therapies for patients with cancer. Consequently, a proteome wide analysis, in addition to genomic studies, is an absolute requirement for a complete functional understanding of tumor biology. Ultra-sensitive, high-performance Fourier transform ion cyclotron resonance (FTICR) mass spectrometry (MS) currently holds an important role in fulfilling the demands of biomarker discovery. In this review, we describe the applicability of FTICR MS for breast cancer proteomics, particularly for the analysis of complex protein mixtures obtained from a limited number of cells typically available from clinical specimens.

NanoLC-FTICR MS improves proteome coverage attainable for ~3,000 laser microdissected breast carcinoma cells (Article)

2007-01-01T00:00:00Z

Proteomics assays hold great promise for unrevealing molecular events that underlie human disease. Effective analysis of clinical samples is essential, but this task is considerably complicated by tissue heterogeneity. Laser capture microdissection (LCM) can be used to selectively isolate target cells from their native tissue environment. However, the small number of cells that is typically procured by LCM severely limits proteome coverage and biomarker discovery potential achievable by conventional proteomics platforms. Herein, we describe the use of nano liquid chromatography-Fourier transform ion cyclotron resonance mass spectrometry for analyzing protein digests of ~3,000 LCM-derived tumor cells from breast carcinoma tissue, corresponding to ~300 ng of total protein. A total of 2,282 peptides were identified by matching LC-MS data to accurate mass and time tag databases that were previously established for human breast (cancer) cell lines. 1,003 unique proteins were confidently identified with 2 or more peptides. Based on Gene Ontology categorization, identified proteins appear to cover a wide variety of biological functions and cellular compartments. This work demonstrates that a substantial number of proteins can be detected and identified from limited number of cells using the AMT tag approach, and opens doors for high throughput in-depth proteomics analysis of clinical samples.

Functional analysis of a novel androgen receptor mutation, Q902K, in an individual with partial androgen insensitivity. (Article)

2005-01-01T00:00:00Z

Androgen insensitivity syndrome (AIS) is caused by defects in the androgen receptor (AR) that render the AR partially or completely inactive. As a result, embryonic sex differentiation is impaired. Here, we describe a novel mutation in the AR found in a patient with partial AIS. The mutation results in a substitution of a glutamine (Q) by a lysine (K) residue at position 902, Q902K. The AR Q902K mutation was investigated in vitro with respect to its functional properties. The equilibrium dissociation constants (K(d)s) of AR Q902K in the presence of either the synthetic androgen R1881 or the natural ligand DHT were slightly elevated. The R1881 dissociation rate (t(1/2)) was increased 3-fold for AR Q902K compared with wild type. Transcriptional activity was decreased to 85% of wild type, and the dose-response curve revealed that the sensitivity to hormone was decreased due to the mutation. Furthermore, the 114-kDa androgen-induced phosphorylated AR protein band was not detectable in genital skin fibroblasts. However, it could be detected in transfected CHO cells expressing the mutant receptor in the presence of 10 and 100 nm R1881. Functional interaction assays and a GST pull-down assay showed that the interaction between the NH2 and COOH terminus of AR Q902K was reduced to 50% of wild type. Furthermore, the transactivation by the coactivator TIF2 (transcriptional intermediary factor 2) was decreased 2- to 3-fold. The half-maximal response in both assays was shifted to a higher hormone concentration compared with wild type. These results indicate that residue Q902 is involved in TIF2 and NH2/COOH interaction and that the Q to K mutation results in a mild impairment of AR function, which can explain the partial AIS phenotype of the patient.

Differential modulation of androgen receptor transcriptional activity by the nuclear receptor co-repressor (N-CoR). (Article)

2004-05-01T00:00:00Z

Antiandrogens are widely used agents in the treatment of prostate cancer, as inhibitors of AR (androgen receptor) action. Although the precise mechanism of antiandrogen action is not yet elucidated, recent studies indicate the involvement of nuclear receptor co-repressors. In the present study, the regulation of AR transcriptional activity by N-CoR (nuclear receptor co-repressor), in the presence of different ligands, has been investigated. Increasing levels of N-CoR differentially affected the transcriptional activity of AR occupied with either agonistic or antagonistic ligands. Small amounts of co-transfected N-CoR repressed CPA (cyproterone acetate)- and mifepristone (RU486)-mediated AR activity, but did not affect agonist (R1881)-induced AR activity. Larger amounts of co-transfected N-CoR repressed AR activity for all ligands, and converted the partial agonists CPA and RU486 into strong AR antagonists. In the presence of the agonist R1881, co-expression of the p160 co-activator TIF2 (transcriptional intermediary factor 2) relieved N-CoR repression up to control levels. However, in the presence of RU486 and CPA, TIF2 did not functionally compete with N-CoR, suggesting that antagonist-bound AR has a preference for N-CoR. The AR mutation T877A (Thr877-->Ala), which is frequently found in prostate cancer and affects the ligand-induced conformational change of the AR, considerably reduced the repressive action of N-CoR. The agonistic activities of CPA- and hydroxyflutamide-occupied T877A-AR were hardly affected by N-CoR, whereas TIF2 strongly enhanced their activities. These results indicate that lack of N-CoR action allows these antiandrogens to act as strong agonists on the mutant AR.

Proteomic profiling of epididymis and vas deferens: identification of proteins regulated during rat genital tract development. (Article)

2003-10-01T00:00:00Z

Epididymis and vas deferens form part of the male internal genital tract and are dependent on androgens for their growth and development. To better understand the molecular action of androgens during male genital tract development, protein expression profiles were generated using two-dimensional gels, for rat epididymides and vasa deferentia isolated on embryonic days (E) 17-21. Proteins that were differentially expressed between E17 and E21 were cut from the gels, digested into tryptic peptides and analyzed on a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer. Using this approach, 20 proteins could be identified that were regulated in time and were categorized into cytoskeletal proteins, nuclear proteins, transport proteins, chaperones, and enzymes (mainly glycolytic). Furthermore, epididymides and vasa deferentia isolated on E19 were cultured in vitro in the absence or presence of 10 nm of the synthetic androgen R1881, for 9, 24, and 48 h. Under these conditions, regulation and posttranslational modification were observed for glyceraldehyde 3-phosphate dehydrogenase, triosephosphate isomerase, heterogeneous nuclear ribonucleoprotein A2/B1 and heterogeneous nuclear ribonucleoprotein A3, similar to the observed changes in vivo. In addition, posttranslational modification of RhoGDI1 (also named RhoGDIalpha) was found in response to androgen. Androgen-induced posttranslational modification of RhoGDI1 and glycolytic enzymes may be an important functional link between signaling pathways and cytoskeletal rearrangements in control of growth and development of the male internal genital tract.

Proteomic analysis of androgen-regulated protein expression in a mouse fetal vas deferens cell line (Article)

2003-04-01T00:00:00Z

During sex differentiation, androgens are essential for development of the male genital tract. The Wolffian duct is an androgen-sensitive target tissue that develops into the epididymis, vas deferens, and seminal vesicle. The present study aimed to identify androgen-regulated proteins that are involved in development of Wolffian duct-derived structures. We have used male mouse embryos transgenic for temperature-sensitive simian virus 40 large tumor antigen at 18 d of gestation, to generate immortalized mouse fetal vas deferens (MFVD) parental and clonal cell lines. The MFVD parental and clonal cell lines express androgen receptor protein and show features of Wolffian duct mesenchymal cells. Clonal cell line MFVD A6 was selected for proteomic analysis and cultured in the absence or presence of androgens. Subsequently, two-dimensional gel electrophoresis was performed on total cell lysates. Differentially expressed proteins were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and two androgen-regulated proteins were identified as mElfin and CArG-binding factor-A (CBF-A). CBF-A and mElfin are known to bind to cytoskeletal F-actin. Both proteins appeared to be regulated by androgens at the posttranslational level, possibly involving phosphorylation. Posttranslational modification of mElfin and CBF-A by androgens may be associated with a cytoskeletal change that is involved in androgen-regulated gene expression.

Proteomic analysis of androgen-regulated protein expression in a mouse fetal vas deferens cell line (Article)

2003-01-01T00:00:00Z