Gergana Metodieva

Proteome profiling of breast tumors by gel electrophoresis and nanoscale electrospray ionization mass spectrometry.

We have conducted proteome-wide analysis of fresh surgery specimens derived from breast cancer patients, using an approach that integrates size-based intact protein fractionation, nanoscale liquid separation of peptides, electrospray ion trap mass spectrometry, and bioinformatics. Through this approach, we have acquired a large amount of peptide fragmentation spectra from size-resolved fractions of the proteomes of several breast tumors, tissue peripheral to the tumor, and samples from patients undergoing noncancer surgery. Label-free quantitation was used to generate protein abundance maps for each proteome and perform comparative analyses. The mass spectrometry data revealed distinct qualitative and quantitative patterns distinguishing the tumors from healthy tissue as well as differences between metastatic and non-metastatic human breast cancers including many established and potential novel candidate protein biomarkers. Selected proteins were evaluated by Western blotting using tumors grouped according to histological grade, size, and receptor expression but differing in nodal status. Immunohistochemical analysis of a wide panel of breast tumors was conducted to assess expression in different types of breast cancers and the cellular distribution of the candidate proteins. These experiments provided further insights and an independent validation of the data obtained by mass spectrometry and revealed the potential of this approach for establishing multimodal markers for early metastasis, therapy outcomes, prognosis, and diagnosis in the future.

Stat1 and CD74 overexpression is co-dependent and linked to increased invasion and lymph node metastasis in triple-negative breast cancer.

Triple-negative breast cancer is difficult to treat because of the lack of rationale-based therapies. There are no established markers and targets that can be used for stratification of patients and targeted therapy. Here we report the identification of novel molecular features, which appear to augment metastasis of triple negative breast tumors. We found that triple-negative breast tumors can be segregated into 2 phenotypes based on their genome-wide protein abundance profiles. The first is characterized by high expression of Stat1, Mx1, and CD74. Seven out of 9 tumors from this group had invaded at least 2 lymph nodes while only 1 out of 10 tumors in group 2 was lymph node positive. In vitro experiments showed that the interferon-induced increase in Stat1 abundance correlates with increased migration and invasion in cultured cells. When CD74 was overexpressed, it increased cell adhesion on matrigel. This effect was accompanied with a marked increase in the membrane expression of beta-catenin, MUC18, plexins, integrins, and other proteins involved in cell adhesion and cancer metastasis. Taken together, our results show that Stat1/CD74 positive triple-negative tumors are more aggressive and suggest an approach for development of better diagnostics and more targeted therapies for triple negative breast cancer. This article is part of a Special Issue entitled: Proteomics: The clinical link.

Plasticity in the proteome of Emiliania huxleyi CCMP 1516 to extremes of light is highly targeted

Optimality principles are often applied in theoretical studies of microalgal ecophysiology to predict changes in allocation of resources to different metabolic pathways, and optimal acclimation is likely to involve changes in the proteome, which typically accounts for > 50% of cellular nitrogen (N). We tested the hypothesis that acclimation of the microalga Emiliania huxleyi CCMP 1516 to suboptimal vs supraoptimal light involves large changes in the proteome as cells rebalance the capacities to absorb light, fix CO2 , perform biosynthesis and resist photooxidative stress. Emiliania huxleyi was grown in nutrient-replete continuous culture at 30 (LL) and 1000 μmol photons m(-2) s(-1) (HL), and changes in the proteome were assessed by LC-MS/MS shotgun proteomics. Changes were most evident in proteins involved in the light reactions of photosynthesis; the relative abundance of photosystem I (PSI) and PSII proteins was 70% greater in LL, light-harvesting fucoxanthin-chlorophyll proteins (Lhcfs) were up to 500% greater in LL and photoprotective LI818 proteins were 300% greater in HL. The marked changes in the abundances of Lhcfs and LI818s, together with the limited plasticity in the bulk of the E. huxleyi proteome, probably reflect evolutionary pressures to provide energy to maintain metabolic capabilities in stochastic light environments encountered by this species in nature.

Profiling signatures of ovarian cancer tumour suppression using 2D-DIGE and 2D-LC-MS/MS with tandem mass tagging

Epithelial ovarian cancer (EOC) is the most common form of gynaecological malignancy in the developed world and has a poor prognosis due to its late detection. Identifying molecular markers of the disease may provide novel approaches to screening and could enable targeted treatment and the design of novel therapies. Although blood is recognized as a highly important source of disease-related biomarkers, the complexity and dynamic range of protein abundance in body fluids has hampered proteomic biomarker discovery and alternative approaches using cell models may be more successful. Herein, we have utilized two cellular models of EOC, where transfer of normal chromosome 18 material into the EOC cell lines TOV-112D and TOV-21G induced in vitro and in vivo suppression of their tumourigenic phenotype. A combination of quantitative two-dimensional difference gel electrophoresis (2D-DIGE) and two-dimensional-liquid chromatography tandem mass spectrometry (2D-LC-MS/MS) with tandem mass tagging (TMT) was employed to examine the whole cell, secreted and crude membrane proteomes of the parental and hybrid cell models to identify differentially expressed proteins as potential markers of tumour suppression. Protein changes of interest were confirmed by immunoblotting in additional hybrid and revertant cell lines where incorporated chromosome 18 material was lost. One candidate marker was also tested in sera from a set of ovarian cancer cases and controls. We have identified a list of promising candidate biomarkers for further testing and functional characterization.

Genomic modelling of the ESR1 Y537S mutation for evaluating function and new therapeutic approaches for metastatic breast cancer

Drugs that inhibit estrogen receptor-α (ER) activity have been highly successful in treating and reducing breast cancer progression in ER-positive disease. However, resistance to these therapies presents a major clinical problem. Recent genetic studies have shown that mutations in the ER gene are found in >20% of tumours that progress on endocrine therapies. Remarkably, the great majority of these mutations localize to just a few amino acids within or near the critical helix 12 region of the ER hormone binding domain, where they are likely to be single allele mutations. Understanding how these mutations impact on ER function is a prerequisite for identifying methods to treat breast cancer patients featuring such mutations. Towards this end, we used CRISPR-Cas9 genome editing to make a single allele knock-in of the most commonly mutated amino acid residue, tyrosine 537, in the estrogen-responsive MCF7 breast cancer cell line. Genomic analyses using RNA-seq and ER ChIP-seq demonstrated that the Y537S mutation promotes constitutive ER activity globally, resulting in estrogen-independent growth. MCF7-Y537S cells were resistant to the anti-estrogen tamoxifen and fulvestrant. Further, we show that the basal transcription factor TFIIH is constitutively recruited by ER-Y537S, resulting in ligand-independent phosphorylation of Serine 118 (Ser118) by the TFIIH kinase, cyclin-dependent kinase (CDK)7. The CDK7 inhibitor, THZ1 prevented Ser118 phosphorylation and inhibited growth of MCF7-Y537S cells. These studies confirm the functional importance of ER mutations in endocrine resistance, demonstrate the utility of knock-in mutational models for investigating alternative therapeutic approaches and highlight CDK7 inhibition as a potential therapy for endocrine-resistant breast cancer mediated by ER mutations.

Pseudopodial and β-arrestin-interacting proteomes from migrating breast cancer cells upon PAR2 activation.

Metastatic cancer cells form pseudopodia (PD) to facilitate their migration. The proteinase-activated receptor-2 (PAR-2) transduces migratory signals from proteases, and it forms protein complexes with β-arrestin and other signalling molecules that are enriched in pseudopodia. More generally, however, pseudopodial regulation is poorly understood. Here, we purified the pseudopodial proteomes of breast cancer cells after activation of the endogenous PAR-2 and we combined gel-based approaches with label-free high-resolution mass spectrometry to identify proteins that accumulate at the pseudopodia upon PAR-2-mediated migration. We identified >410 proteins in the cell body and >380 in the pseudopodia upon PAR2 activation, of which 93 were enriched in the pseudopodia. One of the pathways strongly enriched in the PD was the clathrin-mediated endocytosis signalling pathway, highlighting the importance of the scaffolding function of β-arrestin in PAR-2 signalling via its endocytosis. We therefore immunoprecipitated β-arrestins, and with mass spectrometry we identified 418 novel putative interactors. These data revealed novel β-arrestin functions that specifically control PAR-2-regulated signalling in migrating breast cancer cells but also showed that some β-arrestin functions are universal between GPCRs and cell types. In conclusion, this study reveals novel proteins and signalling pathways potentially important for migration of breast cancer cells.

Testing breast cancer serum biomarkers for early detection and prognosis in pre-diagnosis samples

Background:Breast cancer is a leading cause of morbidity and mortality worldwide. Although mammography screening is available, there is an ongoing interest in improved early detection and prognosis. Herein, we have analysed a combination of serological biomarkers in a case–control cohort of sera taken before diagnosis.Methods:This nested case–control study within the UK Collaborative Trial of Ovarian Cancer Screening (UKCTOCS) used serum samples from 239 women who subsequently developed breast cancer and 239 matched cancer-free controls. Sera were screened by ELISA for 9 candidate markers. Univariate and multivariate analyses were performed to examine associations with clinico-pathological features and between case controls in different time groups before diagnosis.Results:Significant associations with clinico-pathological features related to prognosis were found for several candidates (CA15-3, HSP90A and PAI-1). However, there were no consistent differences between cases and controls for any candidate in the lead up to diagnosis. Whilst combination models outperformed single markers, there was no increase in performance towards diagnosis.Conclusions:This study using unique pre-diagnosis samples shows that CA15-3, HSP90A and PAI-1 have potential as early prognostic markers and warrant further investigation. However, none of the candidates or combinations would be useful for screening.

miR‐204 is Dysregulated in Metastatic Prostate Cancer In Vitro

During cancer progression, the genome instability incurred rearrangement could possibly turn some of the tumor suppressor micro‐RNAs into pro‐oncogenic ones. We aimed to investigate miR‐204 in the context of prostate cancer progression using a cell line model of different levels of genome instability (LNCaP, PC3, VCaP and NCI H660), as demonstrated by the availability of ERG fusion. We studied the effect of miR‐204 modulation on master transcription factors important for lineage development, cell differentiation and prostate cancer bone marrow metastasis. We followed c‐MYB, ETS1 and RUNX2 transcript and protein expression and the miR‐204 affected global proteome. We further investigated if these transcription factors exert an effect on miR‐204 expression (qPCR, luciferase reporter assay) by silencing them using esiRNA. We found dualistic miR‐204 effects, either acting as a tumor suppressor on c‐MYB, or as an oncomiR on ETS1. RUNX2 and ETS1 regulation by miR‐204 was ERG fusion dependent, demonstrating regulatory circuitry disruption in advanced metastatic models. miR‐204 also differentially affected mRNA splicing and protein stability. miR‐204 levels were found dependent on cancer hypermethylation and supported by positive feedback induced by all three transcription factors. In this regulatory circuitry among miR‐204, c‐MYB, RUNX2 and ETS1, the c‐MYB was found to induce all three other members, but its expression was differentially affected by the methylation status in lymph node vs. bone metastasis. We demonstrate that not only tumor suppressor micro‐RNA loss, but also significant genome rearrangement‐driven regulatory loop perturbations play a role in the advanced cancer progression, conferring better pro‐survival and metastatic potential.

Quantitative proteome profiling of lymph node-positive vs. -negative colorectal carcinomas pinpoints MX1 as a marker for lymph node metastasis

We used high‐resolution mass spectrometry to measure the abundance of more than 9,000 proteins in 19 individually dissected colorectal tumors representing lymph node metastatic (n = 10) and nonmetastatic (n = 9) phenotypes. Statistical analysis identified MX1 and several other proteins as overexpressed in lymph node‐positive tumors. MX1, IGF1‐R and IRF2BP1 showed significantly different expression in immunohistochemical validation (Wilcoxon test p = 0.007 for IGF1‐R, p = 0.04 for IRF2BP1 and p = 0.02 for MX1 at the invasion front) in the validation cohort. Knockout of MX1 by siRNA in cell cultures and wound healing assays provided additional evidence for the involvement of this protein in tumor invasion. The collection of identified and quantified proteins to our knowledge is the largest tumor proteome dataset available at the present. The identified proteins can give insights into the mechanisms of lymphatic metastasis in colorectal carcinoma and may act as prognostic markers and therapeutic targets after further prospective validation.

A peptide-centric approach to breast cancer biomarker discovery utilizing label-free multiple reaction monitoring mass spectrometry

We report an approach for multiplex analysis of cancer biomarkers based on the measurement of diagnostic peptides in whole tissue protein digests. Label‐free quantitation with MS3 multiple reaction monitoring (MRM) was developed to afford accurate analysis of prospective marker peptides in a panel of breast tumors. This approach provides an economical and robust alternative to stable isotope‐based methods. It is equally applicable to the analysis of samples derived from tissue biopsy, aspirate, or plasma and can be easily translated to clinic.