Cell migration and invasiveness significantly contribute to desirable physiological processes, such as wound healing or embryogenesis, as well as to serious pathological processes such as the spread ...of cancer cells to form tumor metastasis. The availability of appropriate methods for studying these processes is essential for understanding the molecular basis of cancer metastasis and for identifying suitable therapeutic targets for anti-metastatic treatment. This review summarizes the current status of these methods: In vitro methods for studying cell migration involve two-dimensional (2D) assays (wound-healing/scratch assay), and methods based on chemotaxis (the Dunn chamber). The analysis of both cell migration and invasiveness in vitro require more complex systems based on the Boyden chamber principle (Transwell migration/invasive test, xCELLigence system), or microfluidic devices with three-dimensional (3D) microscopy visualization. 3D culture techniques are rapidly becoming routine and involve multicellular spheroid invasion assays or array chip-based, spherical approaches, multi-layer/multi-zone culture, or organoid non-spherical models, including multi-organ microfluidic chips. The in vivo methods are mostly based on mice, allowing genetically engineered mice models and transplant models (syngeneic mice, cell line-derived xenografts and patient-derived xenografts including humanized mice models). These methods currently represent a solid basis for the state-of-the art research that is focused on understanding metastatic fundamentals as well as the development of targeted anti-metastatic therapies, and stratified treatment in oncology.
Accurate classification of breast tumors is vital for patient management decisions and enables more precise cancer treatment. Here, we present a quantitative proteotyping approach based on sequential ...windowed acquisition of all theoretical fragment ion spectra (SWATH) mass spectrometry and establish key proteins for breast tumor classification. The study is based on 96 tissue samples representing five conventional breast cancer subtypes. SWATH proteotype patterns largely recapitulate these subtypes; however, they also reveal varying heterogeneity within the conventional subtypes, with triple negative tumors being the most heterogeneous. Proteins that contribute most strongly to the proteotype-based classification include INPP4B, CDK1, and ERBB2 and are associated with estrogen receptor (ER) status, tumor grade status, and HER2 status. Although these three key proteins exhibit high levels of correlation with transcript levels (R > 0.67), general correlation did not exceed R = 0.29, indicating the value of protein-level measurements of disease-regulated genes. Overall, this study highlights how cancer tissue proteotyping can lead to more accurate patient stratification.
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•Proteotyping of 96 breast tumors by SWATH mass spectrometry•Three key proteins for breast tumor classification•Varying degrees of heterogeneity within conventional breast cancer subtypes•Generally modest correlation between protein and transcript levels in tumor tissue
Bouchal et al. explore and confirm the suitability of SWATH-MS for proteotyping of human tumor samples at relatively high throughput. Results indicate that proteotype-based classification resolves more variability than is apparent from conventional subtyping and potentially improves current classification.
Catechol-O-methyl transferase (COMT) is involved in detoxification of catechol estrogens, playing cancer-protective role in cells producing or utilizing estrogen. Moreover, COMT suppressed migration ...potential of breast cancer (BC) cells. To delineate COMT role in metastasis of estrogen receptor (ER) dependent BC, we investigated the effect of COMT overexpression on invasion, transcriptome, proteome and interactome of MCF7 cells, a luminal A BC model, stably transduced with lentiviral vector carrying COMT gene (MCF7-COMT). 2D and 3D assays revealed that COMT overexpression associates with decreased cell invasion (p < 0.0001 for Transwell assay, p < 0.05 for spheroid formation). RNA-Seq and LC-DIA-MS/MS proteomics identified genes associated with invasion (FTO, PIR, TACSTD2, ANXA3, KRT80, S100P, PREX1, CLEC3A, LCP1) being downregulated in MCF7-COMT cells, while genes associated with less aggressive phenotype (RBPMS, ROBO2, SELENBP, EPB41L2) were upregulated both at transcript (|log2FC|> 1, adj. p < 0.05) and protein (|log2FC|> 0.58, q < 0.05) levels. Importantly, proteins driving MET signaling were less abundant in COMT overexpressing cells, and pull-down confirmed interaction between COMT and Kunitz-type protease inhibitor 2 (SPINT2), a negative regulator of MET (log2FC = 5.10, q = 1.04
). In conclusion, COMT may act as tumor suppressor in ER dependent BC not only by detoxification of catechol estrogens but also by suppressing cell invasion and interplay with MET pathway.
Hydrogen can serve as an electron donor for chemolithotrophic acidophiles, especially in the deep terrestrial subsurface and geothermal ecosystems. Nevertheless, the current knowledge of hydrogen ...utilization by mesophilic acidophiles is minimal. A multi-omics analysis was applied on
growing on hydrogen, and a respiratory model was proposed. In the model, NiFe hydrogenases oxidize hydrogen to two protons and two electrons. The electrons are used to reduce membrane-soluble ubiquinone to ubiquinol. Genetically associated iron-sulfur proteins mediate electron relay from the hydrogenases to the ubiquinone pool. Under aerobic conditions, reduced ubiquinol transfers electrons to either cytochrome
oxidase via cytochrome
complex and cytochrome
or the alternate directly to cytochrome
oxidase, resulting in proton efflux and reduction of oxygen. Under anaerobic conditions, reduced ubiquinol transfers electrons to outer membrane cytochrome
(ferrireductase) via cytochrome
complex and a cascade of electron transporters (cytochrome
, cytochrome
, rusticyanin, and high potential iron-sulfur protein), resulting in proton efflux and reduction of ferric iron. The proton gradient generated by hydrogen oxidation maintains the membrane potential and allows the generation of ATP and NADH. These results further clarify the role of extremophiles in biogeochemical processes and their impact on the composition of the deep terrestrial subsurface.
Abstract
Background
Desmocollin-1 (DSC1) is a desmosomal transmembrane glycoprotein that maintains cell-to-cell adhesion. DSC1 was previously associated with lymph node metastasis of luminal A breast ...tumors and was found to increase migration and invasion of MCF7 cells in vitro. Therefore, we focused on DSC1 role in cellular and molecular mechanisms in luminal A breast cancer and its possible therapeutic modulation.
Methods
Western blotting was used to select potential inhibitor decreasing DSC1 protein level in MCF7 cell line. Using atomic force microscopy we evaluated effect of DSC1 overexpression and modulation on cell morphology. The LC–MS/MS analysis of total proteome on Orbitrap Lumos and RNA-Seq analysis of total transcriptome on Illumina NextSeq 500 were performed to study the molecular mechanisms associated with DSC1. Pull-down analysis with LC–MS/MS detection was carried out to uncover DSC1 protein interactome in MCF7 cells.
Results
Analysis of DSC1 protein levels in response to selected inhibitors displays significant DSC1 downregulation (
p
-value ≤ 0.01) in MCF7 cells treated with NF-κB inhibitor parthenolide. Analysis of mechanic cell properties in response to DSC1 overexpression and parthenolide treatment using atomic force microscopy reveals that DSC1 overexpression reduces height of MCF7 cells and conversely, parthenolide decreases cell stiffness of MCF7 cells overexpressing DSC1. The LC–MS/MS total proteome analysis in data-independent acquisition mode shows a strong connection between DSC1 overexpression and increased levels of proteins LACRT and IGFBP5, increased expression of IGFBP5 is confirmed by RNA-Seq. Pathway analysis of proteomics data uncovers enrichment of proliferative MCM_BIOCARTA pathway including CDK2 and MCM2-7 after DSC1 overexpression. Parthenolide decreases expression of LACRT, IGFBP5 and MCM_BIOCARTA pathway specifically in DSC1 overexpressing cells. Pull-down assay identifies DSC1 interactions with cadherin family proteins including DSG2, CDH1, CDH3 and tyrosine kinase receptors HER2 and HER3; parthenolide modulates DSC1-HER3 interaction.
Conclusions
Our systems biology data indicate that DSC1 is connected to mechanisms of cell cycle regulation in luminal A breast cancer cells, and can be effectively modulated by parthenolide.
Graphical Abstract
Lymph node status is one of the best prognostic factors in breast cancer, however, its association with distant metastasis is not straightforward. Here we compare molecular mechanisms of nodal and ...distant metastasis in molecular subtypes of breast cancer, with major focus on luminal A patients. We analyze a new cohort of 706 patients (MMCI_706) as well as an independent cohort of 836 primary tumors with full gene expression information (SUPERTAM_HGU133A). We evaluate the risk of distant metastasis, analyze targetable molecular mechanisms in Gene Set Enrichment Analysis and identify relevant inhibitors. Lymph node positivity is generally associated with NF-κB and Src pathways and is related to high risk (OR: 5.062 and 2.401 in MMCI_706 and SUPERTAM_HGU133A, respectively, p < 0.05) of distant metastasis in luminal A patients. However, a part (≤15%) of lymph node negative tumors at the diagnosis develop the distant metastasis which is related to cell proliferation control and thrombolysis. Distant metastasis of lymph node positive patients is mostly associated with immune response. These pro-metastatic mechanisms further vary in other molecular subtypes. Our data indicate that the management of breast cancer and prevention of distant metastasis requires stratified approach based on targeted strategies.
The present pilot study constitutes a proof-of-principle in the use of a quantitative LC-MS/MS based proteomic method for the comparative analysis of representative low-grade breast primary tumor ...tissues with and without metastases and metastasis in lymph node relative to the nonmetastatic tumor type. The study method incorporated iTRAQ stable isotope labeling, two-dimensional liquid chromatography, nanoelectrospray ionization and high resolution tandem mass spectrometry using the hybrid QqTOF platform (iTRAQ-2DLC-MS/MS). The principal aims of this study were (1) to define the protein spectrum obtainable using this approach, and (2) to highlight potential candidates for verification and validation studies focused on biomarkers involved in metastatic processes in breast cancer. The study resulted in the reproducible identification of 605 nonredundant proteins (p ≤ 0.05). A quantitative comparison revealed 3/3 proteins with significantly increased/decreased level in metastatic primary tumor and 13/6 proteins with increased/decreased level in lymph node metastasis compared to nonmetastatic primary tumor (p < 0.01). Changes in selected differentially expressed proteins were verified with qRT-PCR. Although our pilot scale study does not warrant general biological conclusions, the synergic regulation of some proteins with related function (e.g., heme binding proteins, proteins of energetic metabolism, interferon induced proteins, proteins with adhesive function) determined in our sample set reflects the ability of our method in providing biologically meaningful data. The main conclusion from this pilot study was that our quantitative proteomic method constitutes a novel way of analyzing cancerous breast tissue biopsy samples that can be extended as part of a larger scale biomarker discovery program.
Renal cell carcinoma (RCC) represents about 2–3% of all cancers with over 400,000 new cases per year. Sunitinib, a vascular endothelial growth factor tyrosine kinase receptor inhibitor, has been used ...mainly for first-line treatment of metastatic clear-cell RCC with good or intermediate prognosis. However, about one-third of metastatic RCC patients do not respond to sunitinib, leading to disease progression. Here, we aim to find and characterize proteins associated with poor sunitinib response in a pilot proteomics study. Sixteen RCC tumors from patients responding (8) vs. non-responding (8) to sunitinib 3 months after treatment initiation were analyzed using data-independent acquisition mass spectrometry, together with their adjacent non-cancerous tissues. Proteomics analysis quantified 1996 protein groups (FDR = 0.01) and revealed 27 proteins deregulated between tumors non-responding vs. responding to sunitinib, representing a pattern of deregulated proteins potentially contributing to sunitinib resistance. Gene set enrichment analysis showed an up-regulation of epithelial-to-mesenchymal transition with transgelin as one of the most significantly abundant proteins. Transgelin expression was silenced by CRISPR/Cas9 and RNA interference, and the cells with reduced transgelin level exhibited significantly slower proliferation. Our data indicate that transgelin is an essential protein supporting RCC cell proliferation, which could contribute to intrinsic sunitinib resistance.
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.
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.
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•Transgelin is upregulated in lymph node positive breast cancer samples.•Its expression is not driven by known breast cancer molecular markers.•Transgelin is expressed by the cells of tumor stroma.•Transgelin is downregulated in high grade vs. low grade breast cancer samples.•This reflects lower content of stromal cells in high vs. low grade tumors.
Elemental sulfur oxidation by ferric iron in
Acidithiobacillus ferrooxidans
was investigated. The apparent Michaelis constant for ferric iron was 18.6 mM. An absence of anaerobic ferric iron ...reduction ability was observed in bacteria maintained on elemental sulfur for an extended period of time. Upon transition from ferrous iron to elemental sulfur medium, the cells exhibited similar kinetic characteristics of ferric iron reduction under anaerobic conditions to those of cells that were originally maintained on ferrous iron. Nevertheless, a total loss of anaerobic ferric iron reduction ability after the sixth passage in elemental sulfur medium was demonstrated. The first proteomic screening of total cell lysates of anaerobically incubated bacteria resulted in the detection of 1599 protein spots in the master two-dimensional electrophoresis gel. A set of 59 more abundant and 49 less abundant protein spots that changed their protein abundances in an anaerobiosis-dependent manner was identified and compared to iron- and sulfur-grown cells, respectively. Proteomic analysis detected a significant increase in abundance under anoxic conditions of electron transporters, such as rusticyanin and cytochrome c
552
, involved in the ferrous iron oxidation pathway. Therefore we suggest the incorporation of
rus
-operon encoded proteins in the anaerobic respiration pathway. Two sulfur metabolism proteins were identified, pyridine nucleotide-disulfide oxidoreductase and sulfide-quinone reductase. The important transcription regulator, ferric uptake regulation protein, was anaerobically more abundant. The anaerobic expression of several proteins involved in cell envelope formation indicated a gradual adaptation to elemental sulfur oxidation.
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