Identifying Mediators of Androgen-independent Prostate Cancer Using Mass Spectrometry-based Proteomics

Androgen-deprivation remains the principal therapy for advanced and metastatic prostate cancers. However, some cancer cells are able to survive this treatment and transform themselves to a more aggressive androgen-independent prostate cancer (AIPC). An understanding of the molecular alterations that occur during the progression to androgen-independence is of utmost importance in order to generate effective targeted therapies. Using two different in vitro approaches coupled to high through-put mass spectrometry, we were able to identify numerous potential mediators of AIPC and aggressive prostate cancer. Our first approach using an in vitro cell line model of androgen-independence allowed us to identify enzymes of the ketogenic pathway as elevated in cases of aggressive prostate cancer. Specifically, ACAT1, an enzyme in this pathway, was further validated and shown to be substantially elevated in clinical cases of castration-resistant metastatic prostate cancer. In addition, ACAT1 was found to be an independent tissue-based prognostic marker of biochemical recurrence-free survival. In our second approach we compared the secreted proteomes of androgen independent cell lines (PC3, DU145, PPC1, LNCaP-SF, 22Rv1) to androgen-dependent (LNCaP, VCaP) and normal prostate epithelial (RWPE) cell lines. Of the over 3000 proteins identified in the secretomes, we found more than 100 proteins that were differentially secreted in the androgen independent cell lines. Of these, Protein S (PROS1) was elevated in the secretomes of all the AIPC cell lines and was not detected in the normal or androgen-dependent prostate cancer cell lines. Next, using RT-PCR and immunohistochemistry, we observed significantly higher tissue expression levels of PROS1 in localized high-grade and castrate-resistant metastatic prostate cancer samples compared to normal and low-grade prostate cancer, further indicating its importance in prostate cancer progression. Finally, functional validation revealed that PROS1 increases cell proliferation, migration and viability and thus may play a direct role on prostate cancer biology.Taken together, the results of this research will help in not only increasing the understanding of key molecular alterations and mechanisms by which prostate cancer cells can utilize to gain androgen-independence, and more importantly, aid in the development of new targeted therapies by potentially focusing on blocking or altering specific critical signalling cascades.Ph.D

Quantitative tandem mass-spectrometry of skin tissue reveals putative psoriatic arthritis biomarkers

Abstract Background Psoriatic arthritis (PsA) is a distinct inflammatory arthritis occurring in 30% of psoriasis patients. There is a high prevalence of undiagnosed PsA in psoriasis patients; therefore, identifying soluble biomarkers for PsA could help in screening psoriasis patients for appropriate referral to a rheumatologist. Potential PsA biomarkers likely originate in sites of inflammation, such as the skin, and subsequently enter systemic circulation. Our goal was to identify candidate PsA biomarkers by comparing the proteome of skin biopsies obtained from patients with PsA to that from patients with psoriasis without PsA. Methods Skin biopsies were obtained from involved and uninvolved skin of 10 PsA and 10 age/gender-matched psoriasis patients without PsA (PsC). Using strong cation exchange chromatography, followed by label-free quantitative tandem mass spectrometry, we characterized the proteomes of pooled skin samples. Extracted ion current intensities were used to calculate protein abundance ratios, and these were utilized to identify differentially regulated proteins. Results Forty-seven proteins were elevated in PsA-derived skin compared to PsC-derived skin. Selected reaction monitoring assays were developed to quantify these potential PsA markers in individual skin samples, and 8 markers were confirmed in an independent sample set. ITGB5 and POSTN were measured in serum samples from 33 PsA and 15 PsC patients, using enzyme-linked immunosorbent assays. ITGB5 was significantly elevated in PsA serum (P < 0.01), and POSTN showed a trend. ITGB5 and POSTN correlated significantly in both patient groups (r = 0.472, P < 0.001). Conclusion Proteomic analysis of PsA and PsC skin identified eight new candidate biomarkers. These markers need to be validated with a larger and independent cohort, in order to delineate their clinical utility in PsA patients. These proteins may also uncover unknown aspects of PsA pathobiology

Identification of psoriatic arthritis mediators in synovial fluid by quantitative mass spectrometry

Abstract Background Synovial fluid (SF) is a dynamic reservoir for proteins originating from the synovial membrane, cartilage, and plasma, and may therefore reflect the pathophysiological conditions that give rise to arthritis. Our goal was to identify and quantify protein mediators of psoriatic arthritis (PsA) in SF. Methods Age and gender-matched pooled SF samples from 10 PsA and 10 controls [early osteoarthritis (OA)], were subjected to label-free quantitative proteomics using liquid chromatography coupled to mass spectrometry (LC-MS/MS), to identify differentially expressed proteins based on the ratios of the extracted ion current of each protein between the two groups. Pathway analysis and public database searches were conducted to ensure these proteins held relevance to PsA. Multiplexed selected reaction monitoring (SRM) assays were then utilized to confirm the elevated proteins in the discovery samples and in an independent set of samples from patients with PsA and controls. Results We determined that 137 proteins were differentially expressed between PsA and control SF, and 44 were upregulated. The pathways associated with these proteins were acute-phase response signalling, granulocyte adhesion and diapedesis, and production of nitric oxide and reactive oxygen species in macrophages. The expression of 12 proteins was subsequently quantified using SRM assays. Conclusions Our in-depth proteomic analysis of the PSA SF proteome identified 12 proteins which were significantly elevated in PsA SF compared to early OA SF. These proteins may be linked to the pathogenesis of PsA, as well serve as putative biomarkers and/or therapeutic targets for this disease

The Fowler Syndrome-Associated Protein FLVCR2 Is an Importer of Heme ▿

Mutations in FLVCR2, a cell surface protein related by homology and membrane topology to the heme exporter/retroviral receptor FLVCR1, have recently been associated with Fowler syndrome, a vascular disorder of the brain. We previously identified FLVCR2 to function as a receptor for FY981 feline leukemia virus (FeLV). However, the cellular function of FLVCR2 remains unresolved. Here, we report the cellular function of FLVCR2 as an importer of heme, based on the following observations. First, FLVCR2 binds to hemin-conjugated agarose, and binding is competed by free hemin. Second, mammalian cells and Xenopus laevis oocytes expressing FLVCR2 display enhanced heme uptake. Third, heme import is reduced after the expression of FLVCR2-specific small interfering RNA (siRNA) or after the binding of the FY981 FeLV envelope protein to the FLVCR2 receptor. Finally, cells overexpressing FLVCR2 are more sensitive to heme toxicity, a finding most likely attributable to enhanced heme uptake. Tissue expression analysis indicates that FLVCR2 is expressed in a broad range of human tissues, including liver, placenta, brain, and kidney. The identification of a cellular function for FLVCR2 will have important implications in elucidating the pathogenic mechanisms of Fowler syndrome and of phenotypically associated disorders

A drug discovery platform to identify compounds that inhibit EGFR triple mutants.

Receptor tyrosine kinases (RTKs) are transmembrane receptors of great clinical interest due to their role in disease. Historically, therapeutics targeting RTKs have been identified using in vitro kinase assays. Due to frequent development of drug resistance, however, there is a need to identify more diverse compounds that inhibit mutated but not wild-type RTKs. Here, we describe MaMTH-DS (mammalian membrane two-hybrid drug screening), a live-cell platform for high-throughput identification of small molecules targeting functional protein-protein interactions of RTKs. We applied MaMTH-DS to an oncogenic epidermal growth factor receptor (EGFR) mutant resistant to the latest generation of clinically approved tyrosine kinase inhibitors (TKIs). We identified four mutant-specific compounds, including two that would not have been detected by conventional in vitro kinase assays. One of these targets mutant EGFR via a new mechanism of action, distinct from classical TKI inhibition. Our results demonstrate how MaMTH-DS is a powerful complement to traditional drug screening approaches