Jakub Faktor

Proteomics in investigation of cancer metastasis: functional and clinical consequences and methodological challenges.

Metastases are responsible for most of the cases of death in patients with solid tumors. There is thus an urgent clinical need of better understanding the exact molecular mechanisms and finding novel therapeutics targets and biomarkers of metastatic disease of various tumors. Metastases are formed in a complicated biological process called metastatic cascade. Up to now, proteomics has enabled the identification of number of metastasis‐associated proteins and potential biomarkers in cancer tissues, microdissected cells, model systems, and secretomes. Expression profiles and biological role of key proteins were confirmed in verification and functional experiments. This communication reviews these observations and analyses the methodological aspects of the proteomics approaches used. Moreover, it reviews contribution of current proteomics in the field of functional characterization and interactome analysis of proteins involved in various events in metastatic cascade. It is evident that ongoing technical progress will further increase proteome coverage and sample capacity of proteomics technologies, giving complex answers to clinical and functional questions asked.

Comparison of targeted proteomics approaches for detecting and quantifying proteins derived from human cancer tissues.

Targeted mass spectrometry‐based proteomics approaches enable the simultaneous and reproducible quantification of multiple protein analytes across numerous conditions in biology and clinical studies. These approaches involve e.g. selected reaction monitoring (SRM) typically conducted on a triple quadrupole mass spectrometer, its high‐resolution variant named pseudo‐SRM (p‐SRM), carried out in a quadrupole coupled with an TOF analyzer (qTOF), and “sequential window acquisition of all theoretical spectra” (SWATH). Here we compared these methods in terms of signal‐to‐noise ratio (S/N), coefficient of variance (CV), fold change (FC), limit of detection and quantitation (LOD, LOQ). We have shown the highest S/N for p‐SRM mode, followed by SRM and SWATH, demonstrating a trade‐off between sensitivity and level of multiplexing for SRM, p‐SRM, and SWATH. SRM was more sensitive than p‐SRM based on determining their LOD and LOQ. Although SWATH has the worst S/N, it enables peptide multiplexing with post‐acquisition definition of the targets, leading to better proteome coverage. FC between breast tumors of different clinical‐pathological characteristics were highly correlated (R2>0.97) across three methods and consistent with the previous study on 96 tumor tissues. Our technical note presented here, therefore, confirmed that outputs of all the three methods were biologically relevant and highly applicable to cancer research.

Rearrangement of mitochondrial pyruvate dehydrogenase subunit dihydrolipoamide dehydrogenase protein–protein interactions by the MDM2 ligand nutlin-3

Drugs targeting MDM2s hydrophobic pocket activate p53. However, these agents act allosterically and have agonist effects on MDM2s protein interaction landscape. Dominant p53‐independent MDM2‐drug responsive‐binding proteins have not been stratified. We used as a variable the differential expression of MDM2 protein as a function of cell density to identify Nutlin‐3 responsive MDM2‐binding proteins that are perturbed independent of cell density using SWATH‐MS. Dihydrolipoamide dehydrogenase, the E3 subunit of the mitochondrial pyruvate dehydrogenase complex, was one of two Nutlin‐3 perturbed proteins identified fours hour posttreatment at two cell densities. Immunoblotting confirmed that dihydrolipoamide dehydrogenase was induced by Nutlin‐3. Depletion of MDM2 using siRNA also elevated dihydrolipoamide dehydrogenase in Nutlin‐3 treated cells. Mitotracker confirmed that Nutlin‐3 inhibits mitochondrial activity. Enrichment of mitochondria using TOM22+ immunobeads and TMT labeling defined key changes in the mitochondrial proteome after Nutlin‐3 treatment. Proximity ligation identified rearrangements of cellular protein–protein complexes in situ. In response to Nutlin‐3, a reduction of dihydrolipoamide dehydrogenase/dihydrolipoamide acetyltransferase protein complexes highlighted a disruption of the pyruvate dehydrogenase complex. This coincides with an increase in MDM2/dihydrolipoamide dehydrogenase complexes in the nucleus that was further enhanced by the nuclear export inhibitor Leptomycin B. The data suggest one therapeutic impact of MDM2 drugs might be on the early perturbation of specific protein–protein interactions within the mitochondria. This methodology forms a blueprint for biomarker discovery that can identify rearrangements of MDM2 protein–protein complexes in drug‐treated cells.

Mass spectrometry analysis of the oxidation states of the pro-oncogenic protein anterior gradient-2 reveals covalent dimerization via an intermolecular disulphide bond

Anterior Gradient-2 (AGR2) is a component of a pro-oncogenic signalling pathway that can promote p53 inhibition, metastatic cell migration, limb regeneration, and cancer drug-resistance. AGR2 is in the protein-disulphide isomerase superfamily containing a single cysteine (Cys-81) that forms covalent adducts with its client proteins. We have found that mutation of Cysteine-81 attenuates its biochemical activity in its sequence-specific peptide docking function, reduces binding to Reptin, and reduces its stability in cells. As such, we evaluated how chemical oxidation of its cysteine affects its biochemical properties. Recombinant AGR2 spontaneously forms covalent dimers in the absence of reductant whilst DTT promotes dimer to monomer conversion. Mutation of Cysteine-81 to alanine prevents peroxide catalysed dimerization of AGR2 in vitro, suggesting a reactive cysteine is central to covalent dimer formation. Both biochemical assays and ESI mass spectrometry were used to demonstrate that low levels of a chemical oxidant promote an intermolecular disulphide bond through formation of a labile sulfenic acid intermediate. However, higher levels of oxidant promote sulfinic or sulfonic acid formation thus preventing covalent dimerization of AGR2. These data together identify the single cysteine of AGR2 as an oxidant responsive moiety that regulates its propensity for oxidation and its monomeric-dimeric state. This has implications for redox regulation of the pro-oncogenic functions of AGR2 protein in cancer cells.

PO-244 Catechol-o-methyltransferase: a dual-role player in different breast cancer subtypes?

Introduction Catechol-O-methyltransferase (COMT) plays an essential role in detoxification of catechols by transferring the methyl group from S-adenosyl-l-methionine to the substrate. In breast cancer, it catalyses methylation of oestrogen metabolites to block their oestrogenicity which prevents their oxidation to carcinogenic quinones. In this study we investigated whether its tumour suppressor role is limited to oestrogen receptor dependent breast cancer, or whether it has a general validity. Material and methods A differential cell surface proteomics analysis with SILAC-LC-MS quantification was performed on MDA-MB-231 breast cancer cell line and its clone selected for higher migration capacity. Analysis of migration and invasiveness of MCF7 cells stably transfected with COMT was performed using Transwell assay. The protein-level expression of COMT in different breast cancer subtypes was determined by next-generation, data independent acquisition based proteomics (96 patients) and immunohistochemistry (621 patients). Results and discussions A quantitative cell surface proteomics identified the membrane-bound isoform of COMT as a cell surface protein overexpressed in a more migrating clone of MDA-MB-231 cells, indicating the association of COMT with cell motility in triple negative breast cancer. On the other hand, overexpression of COMT in MCF-7 cells decreased cell migration and invasiveness, supporting tumour suppressor role in oestrogen receptor dependent breast cancer. Analysis of 96 primary breast cancer tumours using next generation proteomics showed increased COMT protein levels in more aggressive tumours: in grade 3 vs. grade 1, in luminal B vs. luminal A, in triple negative vs. luminal A, and in triple negative lymph node positive vs. triple negative lymph node negative tumours. Conclusion Based on our results, we hypothesise a dual role of COMT in breast cancer: while tumour suppressor role is further supported by our data in ER +breast cancer cells, in tissues COMT seems to be associated with more aggressive phenotype, namely in ER independent, triple negative breast cancer. It is thus evident that the dominance of catecholoestrogen methylation activity may be limited to ER dependent breast cancer. This may alter the perspective how COMT is considered as a potential diagnostic or therapeutic target. We thank Czech Science Foundation, project No. 17–05957S, for financial support.

Pull-down Assay on Streptavidin Beads and Surface Plasmon Resonance Chips for SWATH-MS-based Interactomics

Background/Aim: Pul-down assay is a popular in vitro method for identification of physical interactors of selected proteins. Here, for the first time, we compared three conventional variants of pull-down assay with the streptavidin-modified surface plasmon resonance (SPR) chips for the detection of PDZ and LIM domain protein 2 (PDLIM2) interaction partners. Materials and Methods: PDLIM2 protein–protein interactions were analysed by three variants of pull-down assay on streptavidin beads using LC-MS/MS in “Sequential Window Acquisition of all Theoretical fragment ion spectra (SWATH)” mode and compared with LC-SWATH-MS/MS data from SPR chips. Results: The results showed that (i) the use of SPR chip led to comparable data compared to on-column streptavidin beads, (ii) gravity flow and microflow in wash and elution steps provided better results than centrifugation, and (iii) type and concentration of detergent did not significantly affect the interactome data of cancer-associated PDLIM2. Conclusion: Our study supports further application of SPR-based affinity purification with SWATH mass spectrometry for reproducible and controlled characterization of cancer-associated interactomes.

Tamoxifen-Dependent Induction of AGR2 Is Associated with Increased Aggressiveness of Endometrial Cancer Cells.

ABSTRACT Tamoxifen treatment in breast cancer patients is associated with increased risk of endometrial malignancies. Significantly, higher AGR2 expression was found in endometrial cancers that developed in women previously treated with tamoxifen compared to those who had not been exposed to tamoxifen. An association of elevated AGR2 level with myometrial invasion occurrence and invasion depth was also found. In vitro analyses identified a stimulatory effect of AGR2 on cellular proliferation. Although adverse tamoxifen effects on endometrial cells remain elusive, our work identifies elevated AGR2 as a candidate tamoxifen-dependent mechanism of action responsible for increased incidence of endometrial cancer.

Building Mass Spectrometry Spectral Libraries of Human Cancer Cell Lines

BACKGROUND Cancer research often focuses on protein quantification in model cancer cell lines and cancer tissues. SWATH (sequential windowed acquisition of all theoretical fragment ion spectra), the state of the art method, enables the quantification of all proteins included in spectral library. Spectral library contains fragmentation patterns of each detectable protein in a sample. Thorough spectral library preparation will improve quantitation of low abundant proteins which usually play an important role in cancer. AIM Our research is focused on the optimization of spectral library preparation aimed at maximizing the number of identified proteins in MCF-7 breast cancer cell line. First, we optimized the sample preparation prior entering the mass spectrometer. We examined the effects of lysis buffer composition, peptide dissolution protocol and the material of sample vial on the number of proteins identified in spectral library. Next, we optimized mass spectrometry (MS) method for spectral library data acquisition. CONCLUSION Our thorough optimized protocol for spectral library building enabled the identification of 1,653 proteins (FDR < 1%) in 1 µg of MCF-7 lysate. This work contributed to the enhancement of protein coverage in SWATH digital biobanks which enable quantification of arbitrary protein from physically unavailable samples. In future, high quality spectral libraries could play a key role in preparing of patient proteome digital fingerprints.Key words: biomarker - mass spectrometry - proteomics - digital biobanking - SWATH - protein quantificationThis work was supported by the project MEYS - NPS I - LO1413.The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study.The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.Submitted: 7. 5. 2016Accepted: 9. 6. 2016.