Targeted proteomics driven verification of biomarker candidates associated with breast cancer aggressiveness.

Breast cancer is the most common and molecularly relatively well characterized malignant disease in women, however, its progression to metastatic cancer remains lethal for 78% of patients 5years after diagnosis. Novel markers could identify the high risk patients and their verification using quantitative methods is essential to overcome genetic, inter-tumor and intra-tumor variability and translate novel findings into cancer diagnosis and treatment. We recently identified 13 proteins associated with estrogen receptor, tumor grade and lymph node status, the key factors of breast cancer aggressiveness, using untargeted proteomics. Here we verified these findings in the same set of 96 tumors using targeted proteomics based on selected reaction monitoring with mTRAQ labeling (mTRAQ-SRM), transcriptomics and immunohistochemistry and validated in 5 independent sets of 715 patients using transcriptomics. We confirmed: (i) positive association of anterior gradient protein 2 homolog (AGR2) and periostin (POSTN) and negative association of annexin A1 (ANXA1) with estrogen receptor status; (ii) positive association of stathmin (STMN1), cofilin-1 (COF1), plasminogen activator inhibitor 1 RNA-binding protein (PAIRBP1) and negative associations of thrombospondin-2 (TSP2) and POSTN levels with tumor grade; and (iii) positive association of POSTN, alpha-actinin-4 (ACTN4) and STMN1 with lymph node status. This study highlights a panel of gene products that can contribute to breast cancer aggressiveness and metastasis, the understanding of which is important for development of more precise breast cancer treatment.

Combined Proteomics and Transcriptomics Identifies Carboxypeptidase B1 and Nuclear Factor κB (NF-κB) Associated Proteins as Putative Biomarkers of Metastasis in Low Grade Breast Cancer.

Current prognostic factors are insufficient for precise risk-discrimination in breast cancer patients with low grade breast tumors, which, in disagreement with theoretical prognosis, occasionally form early lymph node metastasis. To identify markers for this group of patients, we employed iTRAQ-2DLC-MS/MS proteomics to 24 lymph node positive and 24 lymph node negative grade 1 luminal A primary breast tumors. Another group of 48 high-grade tumors (luminal B, triple negative, Her-2 subtypes) was also analyzed to investigate marker specificity for grade 1 luminal A tumors. From the total of 4405 proteins identified (FDR < 5%), the top 65 differentially expressed together with 30 previously identified and control markers were analyzed also at transcript level. Increased levels of carboxypeptidase B1 (CPB1), PDZ and LIM domain protein 2 (PDLIM2), and ring finger protein 25 (RNF25) were associated specifically with lymph node positive grade 1 tumors, whereas stathmin 1 (STMN1) and thymosin beta 10 (TMSB10) associated with aggressive tumor phenotype also in high grade tumors at both protein and transcript level. For CPB1, these differences were also observed by immunohistochemical analysis on tissue microarrays. Up-regulation of putative biomarkers in lymph node positive (versus negative) luminal A tumors was validated by gene expression analysis of an independent published data set (n = 343) for CPB1 (p = 0.00155), PDLIM2 (p = 0.02027) and RELA (p = 0.00015). Moreover, statistically significant connections with patient survival were identified in another public data set (n = 1678). Our findings indicate unique pro-metastatic mechanisms in grade 1 tumors that can include up-regulation of CPB1, activation of NF-κB pathway and changes in cell survival and cytoskeleton. These putative biomarkers have potential to identify the specific minor subpopulation of breast cancer patients with low grade tumors who are at higher than expected risk of recurrence and who would benefit from more intensive follow-up and may require more personalized therapy.

Targeted Proteomics Driven Verification of Biomarker Candidates Associated with Breast Cancer Aggressiveness.

Breast cancer is the most common and molecularly well-characterized malignant cancer in women; however, its progression to metastatic cancer remains lethal for 78% of patients within 5 years of diagnosis. Identifying novel markers in high risk patients using quantitative methods is essential to overcome genetic, inter-tumor, and intra-tumor variability, and to translate novel findings into cancer diagnosis and treatment. Using untargeted proteomics, we recently identified 13 proteins associated with some key factors of breast cancer aggressiveness: estrogen receptors, tumor grade, and lymph node status. Here we verified these findings in a set of 96 tumors using targeted proteomics based on selected reaction monitoring with mTRAQ labeling (mTRAQ-SRM). This study highlights a panel of gene products that could contribute to breast cancer aggressiveness and metastasis, and can help develop more precise breast cancer treatments.

Transgelin is upregulated in stromal cells of lymph node positive breast cancer.

Transgelin and transgelin-2 have been discussed as potential markers of various cancers. Here we identified increased transgelin level in lymph node positive vs. negative, low grade primary breast cancer tissues using 2-DE in the cohort of 12 patients. We further clinically validated 2-DE results in an independent cohort of 48 low grade breast cancer patients through untargeted and targeted proteomics analysis (iTRAQ-2D-LC-MS/MS, mTRAQ-SRM), at transcript level and using immunohistochemistry. Another group of 48 high grade tumors of different breast cancer subtypes was analyzed together with the low grade samples to test transgelin specificity for low grade tumors and to study transgelin relation to known molecular markers and histological features. The results confirmed transgelin connection with the lymph node metastasis. As a marker of a reactive tumor stroma, transgelin can be connected with the higher risk of metastasis development. Moreover, we observed significant down-regulation of transgelin in high vs. low grade tumors caused by decreased content of stromal cells (mainly expressing transgelin) in high grade tumor tissue. We also analyzed expression of transgelin-2 in the second cohort using proteomics and immunohistochemistry. Transgelin-2 was mainly expressed by epithelial cancer cells and its levels were increased in metastatic and poorly differentiated tumors. BIOLOGICAL SIGNIFICANCE: Both transgelin and transgelin-2 have been previously described as potential markers of many types of cancer. We are specifying this connection to metastatic affection of lymph nodes and cell differentiation in breast cancer. In the wider context, the results of our study highlight tumor stroma as a source of cancer biomarkers and point out how measured levels of tissue markers can actually reflect cellular feature of cancer mass.

Proteomic responses to a methyl viologen-induced oxidative stress in the wild type and FerB mutant strains of Paracoccus denitrificans.

FerB is a cytoplasmic flavoprotein from the soil bacterium Paracoccus denitrificans with a putative role in defense against oxidative stress. To further explore this hypothesis, we compared protein variations upon methyl viologen treatment in wild-type and FerB mutant strains by a quantitative proteomic analysis based on iTRAQ-3DLC-MS/MS analysis. The proteins showing the most prominent increase in abundance were assigned to carbon fixation and sulfur assimilatory pathways. By employing these proteins as indirect markers, oxidative stress was found to be 15% less severe in the wild-type than in the FerB-deficient mutant cells. Oxidative stress altered the levels of proteins whose expression is dependent on the transcriptional factor FnrP. The observed down-regulation of the fnrP regulon members, most notably that of nitrous oxide reductase, was tentatively explained by an oxidative degradation of the [4Fe-4S] center of FnrP leading to a protein form which no longer activates transcription. While the level of FerB remained relatively constant, two proteins homologous to FerB accumulated during oxidative stress. When their genes were expressed in Escherichia coli, neither of the protein products contained a bound flavin, whereas they both had a high activity of flavin reductase, one preferentially utilizing NADH and the other NADPH.

Proteome-wide dataset generated by iTRAQ-3DLCMS/MS technique for studying the role of FerB protein in oxidative stress in Paracoccus denitrificans.

3DLC protein- and peptide-fractionation technique combined with iTRAQ-peptide labeling and Orbitrap mass spectrometry was employed to quantitate Paracoccus dentirificans total proteome with maximal coverage. This resulted in identification of 24,948 peptides representing 2627 proteins (FDR<0.01) in P. dentirificans wild type and ferB mutant strains grown in the presence or absence of methyl viologen as an oxidative stressor. The data were generated for assessment of FerB protein role in oxidative stress as published by Pernikárová et al.; proteomic responses to a methyl viologen-induced oxidative stress in the wild type and FerB mutant strains of P. denitrificans, J. Proteomics 2015;125:68-75. Dataset is supplied in the article.