Assessment of a Large-Scale Unbiased Malignant Pleural Effusion Proteomics Study of a Real-Life Cohort

Background: Pleural effusion (PE) is common in advanced-stage lung cancer patients and is related to poor prognosis. Identification of cancer cells is the standard method for the diagnosis of a malignant PE (MPE). However, it only has moderate sensitivity. Thus, more sensitive diagnostic tools are urgently needed. Methods: The present study aimed to discover potential protein targets to distinguish malignant pleural effusion (MPE) from other non-malignant pathologies. We have collected PE from 97 patients to explore PE proteomes by applying state-of-the-art liquid chromatography-mass spectrometry (LC-MS) to identify potential biomarkers that correlate with immunohistochemistry assessment of tumor biopsy or with survival data. Functional analyses were performed to elucidate functional differences in PE proteins in malignant and benign samples. Results were integrated into a clinical risk prediction model to identify likely malignant cases. Sensitivity, specificity, and negative predictive value were calculated. Results: In total, 1689 individual proteins were identified by MS-based proteomics analysis of the 97 PE samples, of which 35 were diagnosed as malignant. A comparison between MPE and benign PE (BPE) identified 58 differential regulated proteins after correction of the p-values for multiple testing. Furthermore, functional analysis revealed an up-regulation of matrix intermediate filaments and cellular movement-related proteins. Additionally, gene ontology analysis identified the involvement of metabolic pathways such as glycolysis/gluconeogenesis, pyruvate metabolism and cysteine and methionine metabolism. Conclusion: This study demonstrated a partial least squares regression model with an area under the curve of 98 and an accuracy of 0.92 when evaluated on the holdout test data set. Furthermore, highly significant survival markers were identified (e.g., PSME1 with a log-rank of 1.68 × 10−6 ).info:eu-repo/semantics/publishedVersio

Assessment of a Large-Scale Unbiased Malignant Pleural Effusion Proteomics Study of a Real-Life Cohort

Funding Information: R.M. is supported by Fundação para a Ciência e a Tecnologia (CEEC position, 2019–2025 investigator). This article is a result of the projects (iNOVA4Health—UIDB/04462/2020), supported by Lisboa Portugal Regional Operational Programme (Lisboa2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). This work is also funded by FEDER funds through the COMPETE 2020 Programme and National Funds through FCT—Portuguese Foundation for Science and Technology under the projects number PTDC/BTM-TEC/30087/2017 and PTDC/BTM-TEC/30088/2017. Publisher Copyright: © 2022 by the authors.Background: Pleural effusion (PE) is common in advanced-stage lung cancer patients and is related to poor prognosis. Identification of cancer cells is the standard method for the diagnosis of a malignant PE (MPE). However, it only has moderate sensitivity. Thus, more sensitive diagnostic tools are urgently needed. Methods: The present study aimed to discover potential protein targets to distinguish malignant pleural effusion (MPE) from other non-malignant pathologies. We have collected PE from 97 patients to explore PE proteomes by applying state-of-the-art liquid chromatography-mass spectrometry (LC-MS) to identify potential biomarkers that correlate with immunohistochemistry assessment of tumor biopsy or with survival data. Functional analyses were performed to elucidate functional differences in PE proteins in malignant and benign samples. Results were integrated into a clinical risk prediction model to identify likely malignant cases. Sensitivity, specificity, and negative predictive value were calculated. Results: In total, 1689 individual proteins were identified by MS-based proteomics analysis of the 97 PE samples, of which 35 were diagnosed as malignant. A comparison between MPE and benign PE (BPE) identified 58 differential regulated proteins after correction of the p-values for multiple testing. Furthermore, functional analysis revealed an up-regulation of matrix intermediate filaments and cellular movement-related proteins. Additionally, gene ontology analysis identified the involvement of metabolic pathways such as glycolysis/gluconeogenesis, pyruvate metabolism and cysteine and methionine metabolism. Conclusion: This study demonstrated a partial least squares regression model with an area under the curve of 98 and an accuracy of 0.92 when evaluated on the holdout test data set. Furthermore, highly significant survival markers were identified (e.g., PSME1 with a log-rank of 1.68 × 10−6).publishersversionpublishe

Investigation of VOCs associated with different characteristics of breast cancer cells

The efficacy of breath volatile organic compounds (VOCs) analysis for the screening of patients bearing breast cancer lesions has been demonstrated by using gas chromatography and artificial olfactory systems. On the other hand, in-vitro studies suggest that VOCs detection could also give important indications regarding molecular and tumorigenic characteristics of tumor cells. Aim of this study was to analyze VOCs in the headspace of breast cancer cell lines in order to ascertain the potentiality of VOCs signatures in giving information about these cells and set-up a new sensor system able to detect breast tumor-associated VOCs. We identified by Gas Chromatography-Mass Spectrometry analysis a VOCs signature that discriminates breast cancer cells for: i) transformed condition; ii) cell doubling time (CDT); iii) Estrogen and Progesterone Receptors (ER, PgR) expression, and HER2 overexpression. Moreover, the signals obtained from a temperature modulated metal oxide semiconductor gas sensor can be classified in order to recognize VOCs signatures associated with breast cancer cells, CDT and ER expression. Our results demonstrate that VOCs analysis could give clinically relevant information about proliferative and molecular features of breast cancer cells and pose the basis for the optimization of a low-cost diagnostic device to be used for tumors characterization

EGFR and related therapeutic targets in malignant pleural mesothelioma

IntroductionMalignant pleural mesothelioma (MPM) is a rare but aggressive disease and the current first line treatment is associated with a survival rate of 40%. There is currently no second line therapy. This study aimed to explore the expression of proteins in the arachidonic acid pathway, VEGFR-2 pathway, EGFR/HER2 pathway, c-MET pathway and the PI3K/AKT/MTOR pathway in MPM archival tissue samples and cell lines. We also investigated the cytotoxic effect of inhibitors for each individual pathway as single agents and in some instances as combinations.Materials and MethodsImmunohistochemical analysis was performed in 93 archival MPM tissue samples to determine the expression of the HER2, 5-LOX, 12-LOX, VEGFR-2 and c-MET proteins. Mesothelioma cell lines NCI-H2452, NCI-H2052, MSTO-211H and a non-small cell lung cancer cell line A549 were used for western blot analysis, MTS assay and in vitro scratch assay. Western blot analysis was used to evaluate 5-LOX, 12-LOX, VEGFR-2, EGFR, p-ERK, ERK, c-MET, PTEN and p70S6K protein expression in the cell lines. The antiproliferative effect of Baicalein (12-LOX), Zileuton (5-LOX), MK886 (FLAP inhibitor), Celecoxib (COX-2), Cediranib (VEGFR-2), MGCD265 (c-MET/VEGFR inhibitor), Afatinib (EGFR/HER2), Gefitinib (EGFR), Selumetinib (MEK), Tivantinib (c-MET), Crizotinib (c-MET/ALK), SU11274 (c-MET), Onartuzumab (MET monoclonal antibody), NVPBEZ235 (PI3K/AKT/MTOR), VS5584 (PI3K/AKT/MTOR), Ku0063794 (MTOR1/MTOR2), XL388 (MTOR1/MTOR2) was assessed as single agents and in combinations and analysed using the MTS proliferation assay.ResultsPositive 5-LOX and 12-LOX protein expression was seen in 73% (56/77) and 83% (69/83) of archival MPM tissue samples respectively. NCI-H2452, NCI-H2052, MSTO-211H and A549 cells also expressed 5-LOX and 12-LOX proteins. Baicalein was effective in all cell lines. Combination of celecoxib (3 μM) and baicalein (10 μM) was synergistic in the MSTO-211H cell line. Positive VEGFR-2 protein expression was seen in 93.8% (75/80) of archival tissue samples. Cediranib demonstrated cytotoxic effect at doses higher than the clinical relevant dose. MGCD265 also reduced cell proliferation in all cell lines. Positive HER2 expression was seen in 86.2% (69/80) of archival tissue samples. All cell lines expressed EGFR, p-ERK and ERK protein. Gefitinib and Afatinib demonstrated cytotoxic effects at doses significantly higher than their therapeutically relevant doses. Positive c-MET expression was seen in 82% (58/71) of archival tissue samples. NCI-H2452, NCI-H2052, MSTO-211H and A549 cells also expressed c-MET protein. Crizotinib inhibited cell growth by 50% in MSTO-211H cells within its clinically relevant dose. SU11274 also reduced cell growth by 50%. Tivantinib reduced cell growth by 50% in all cell lines at doses significantly lower than its clinically achievable dose of 4 μM. A549 and MSTO-211H cells positively expressed the p70S6K protein and loss of PTEN was also observed in the MSTO-211H cells. PI3K/AKT/MTOR inhibitors, NVPBEZ235 and VS-5584 significantly reduced cell growth by 50% at low nanomolar IC50 values. VS-5584 was combined in turn with Tivantinib and Afatinib. The combination of VS-5584 with Tivantinib demonstrated enhanced growth inhibition in all cell lines compared to either inhibitors alone. Combination of Tivantinib and Afatinib also enhanced the inhibition of cell growth in all cell lines compared to either inhibitors alone. The addition of cisplatin to the tyrosine kinase combinations produced a synergistic effect.ConclusionsOur findings suggest that multiple signalling pathways are active in a significant proportion of MPM samples. Co-targeting the c-MET and PI3K/MTOR pathway might be a potential therapeutic strategy for mesothelioma patients. Further work is required to explore the combination of an EGFR inhibitor and a PI3K/MTOR inhibitor when the EGFR inhibitor is fixed at a therapeutically relevant dose. In addition, understanding the molecular mechanism of Tivantinib, Afatinib and VS-5584, through the use of comparative proteomic platforms, could potentially identify predictive biomarkers of response to these anti-cancer agents in mesothelioma patients

Expression analysis of LASP1 in human cancer cell lines, role of Src signaling pathways in endothelial cell permeability and natural compounds in chemoprevention

Part 1 - Expression analysis of LASP1 in human cancer cell lines Cancer takes place by malignant transformation of normal cells; it remains one of the most common causes of death worldwide. LIM and SH3 protein (LASP1), is an actin-binding protein. It is believed that this protein plays a crucial role in cancer dissemination, progression, metastasis, and angiogenesis. I have studied the expression levels of LASP1 by using the Western blot analysis in different breast cancer lines and oral carcinoma line. Two cell lines of breast cancer with different malignancy levels were chosen: MDA-MB-231 (triple-negative for oestrogen receptor, progesterone receptor and human epidermal GF receptor 2) and MCF-7 (poorly aggressive and non-invasive cell line). And the expression levels in oral carcinoma cell lines (HSC-2 (non-invasive, non-metastatic squamous carcinoma) and CAL-27 (poorly differentiated tongue carcinoma)) were analysed: The preliminary hypothesis can be done that the overexpression of LASP1 is correlated with the malignancy of cancer cell line which was confirmed both in breast cancer cell lines and oral carcinoma cell lines. Part 2 - Role of Src signaling pathways in endothelial cell permeability SFKs are the group of nonreceptor tyrosine kinases involved in the regulation of various signaling pathways involved in proliferation, survival, migration, angiogenesis, and metastasis. Src associates with adherens junctions by interacting with VE-cadherin. Upon association in protein complexes, Src can phosphorylate VE-cadherin. Upon VEGF-A binding to its cognate receptors (VEGFR1 and VEGFR2 in vascular ECs), the intrinsic tyrosine kinase activity of these receptors is activated, leading to trans-phosphorylation and direct interaction with many SH-2-containing signalling molecules, including Src. Once bound to VEGFR1 or VEGFR2, Src undergoes a conformational change leading to its activation and the subsequent phosphorylation of VE-cadherin. To understand the role of SFKs member YES in the vascular permeability, the Western blot analyses was performed. The levels of pVEGFR2, total VEGFR2, pSTAT1, STAT1 were studied in HUVECs after VEGF stimulation and in the siYES and siSRC silenced cells. It was noticeable, that siRNA-mediated YES knock-down HUVECs have the lowest expression levels of pSTAT1. Both siYES HUVECs and siSRC HUVECs showed lower pSTAT1 expression in comparison with control. This data suggests that SFKs are involved in the activation of STAT1. Additionally, after Immunostaining analysis of pPAX, it was evident the higher concentratin of pPAX within siYES HUVECs than in control. It was also plausible, that in siYES cells pPAX is more distributed in the focal adhesion sites. The highest level of PAX phosphorylation is observed in siYES cells stimulated for 5 min with VEGFA. The increased adhesion site size phosphorylation of paxillin might provoke increased cell motility. After the studying of cell motility of siYES HUVECs by performing the scratch assay, it was noticeable that the movements of HUVEC siYES were less orchestrated, and the cells were more tended to lose and break adherens junctions. In the contrast, the movements of HUVEC siCTRL were more organised, more structured, and the adherens junctions were more stable in comparison to HUVEC siYES. Better understanding of the mechanisms of SFK influence on cell-cell connection remodelling has an enormous interest in the therapeutic potential of SFK moderation treatment within inflammationrelated illnesses and neoplastic diseases. Part 3 - Natural compounds in chemoprevention Another area of my research focus were natural compounds in chemoprevention/chemotherapy. To structure and highlight all studied information I did literature overview of two natural compounds: piperine and ginger extracts. The chemopreventive molecular mechanisms of these natural compounds include cell cycle arrest, induction of cancer cell death, misbalancing of redox homeostasis, inhibition of cell proliferation, angiogenesis, migration, and dissemination of cancer cells in different cancer types

Proteomic characterization of lung cancer and chronic obstructive pulmonary disease: a bronchoalveolar lavage fluid analysis

Peer Reviewe

Improving the management of lung cancer using mass spectrometry and spectroscopy techniques

Lung cancer is a worldwide health problem associated with poor prognosis. The survival at 5 years remains between 5% and 15% in spite of the development of new drugs. One of the main reasons for this is the disease being diagnosed in late stages when curative treatments might not be available. Therefore, some of the most important factors within improving prognosis are both refining diagnostic techniques for early detection, and better assessing tumour response to treatment. Here lies a need for novel diagnostic tools for lung cancer. Spectroscopic and spectrometric analysis of the molecular underpinnings of the disease may provide biochemical signatures for use in diagnostics. Selected Ion Flow Tube – Mass Spectrometry (SIFT-MS) and Fourier Transform Infrared (FTIR) Spectroscopy may provide the gold standard of diagnostic assessment that is needed. Given both techniques previous contributions and technological advancements, their clinical requirements are being increasingly met. This is leading towards the opportunity for the study of lung cancer to benefit from the rapid, non-destructive and sensitive qualities they have to offer. In this thesis, both techniques have been used with the aim of improving the diagnosis and management of lung cancer