In-depth proteomic analysis reveals unique subtype-specific signatures in human small-cell lung cancer.

BACKGROUND: Small-cell lung cancer (SCLC) molecular subtypes have been primarily characterized based on the expression pattern of the following key transcription regulators: ASCL1 (SCLC-A), NEUROD1 (SCLC-N), POU2F3 (SCLC-P) and YAP1 (SCLC-Y). Here, we investigated the proteomic landscape of these molecular subsets with the aim to identify novel subtype-specific proteins of diagnostic and therapeutic relevance. METHODS: Pellets and cell media of 26 human SCLC cell lines were subjected to label-free shotgun proteomics for large-scale protein identification and quantitation, followed by in-depth bioinformatic analyses. Proteomic data were correlated with the cell lines' phenotypic characteristics and with public transcriptomic data of SCLC cell lines and tissues. RESULTS: Our quantitative proteomic data highlighted that four molecular subtypes are clearly distinguishable at the protein level. The cell lines exhibited diverse neuroendocrine and epithelial-mesenchymal characteristics that varied by subtype. A total of 367 proteins were identified in the cell pellet and 34 in the culture media that showed significant up- or downregulation in one subtype, including known druggable proteins and potential blood-based markers. Pathway enrichment analysis and parallel investigation of transcriptomics from SCLC cell lines outlined unique signatures for each subtype, such as upregulated oxidative phosphorylation in SCLC-A, DNA replication in SCLC-N, neurotrophin signalling in SCLC-P and epithelial-mesenchymal transition in SCLC-Y. Importantly, we identified the YAP1-driven subtype as the most distinct SCLC subgroup. Using sparse partial least squares discriminant analysis, we identified proteins that clearly distinguish four SCLC subtypes based on their expression pattern, including potential diagnostic markers for SCLC-Y (e.g. GPX8, PKD2 and UFO). CONCLUSIONS: We report for the first time, the protein expression differences among SCLC subtypes. By shedding light on potential subtype-specific therapeutic vulnerabilities and diagnostic biomarkers, our results may contribute to a better understanding of SCLC biology and the development of novel therapies.

Polymorphisms at CYP enzymes, NR1I2 and NR1I3 in association with virologic response to antiretroviral therapy in Brazilian HIV-positive individuals.

Virologic failure of antiretroviral therapy (ART) may be explained by single nucleotide polymorphisms (SNPs) in drug absorption and metabolism genes. Here, we characterized the associations between polymorphisms in cytochrome P450 enzymes' genes CYP2B6 and CYP3A4/A5, nuclear receptor genes NR1I2/3, and initial ART efficacy among 203 HIV-positive individuals from Rio de Janeiro. Association between SNPs and virologic control was evaluated after 6 and 12 months of follow-up using Cox regression models. The SNP rs2307424 (NR1I3) was associated with increased virologic response after 12 months of treatment, while rs1523127 (NR1I2), rs3003596, and rs2502815 (NR1I3) were associated with decreased response. Increased virologic response after 12 months (adjHR = 1.54; p = 0.02) was also observed among carriers of the NR1I3 haplotype rs2502815G-rs3003596A-rs2307424A versus the reference haplotype G-A-G. Our results suggest that NR1I2 and NR1I3 variants are associated with virologic responses to ART among Brazilians.

Mapping the Melanoma Plasma Proteome (MPP) Using Single-Shot Proteomics Interfaced with the WiMT Database.

Plasma analysis by mass spectrometry-based proteomics remains a challenge due to its large dynamic range of 10 orders in magnitude. We created a methodology for protein identification known as Wise MS Transfer (WiMT). Melanoma plasma samples from biobank archives were directly analyzed using simple sample preparation. WiMT is based on MS1 features between several MS runs together with custom protein databases for ID generation. This entails a multi-level dynamic protein database with different immunodepletion strategies by applying single-shot proteomics. The highest number of melanoma plasma proteins from undepleted and unfractionated plasma was reported, mapping >1200 proteins from >10,000 protein sequences with confirmed significance scoring. Of these, more than 660 proteins were annotated by WiMT from the resulting ~5800 protein sequences. We could verify 4000 proteins by MS1t analysis from HeLA extracts. The WiMT platform provided an output in which 12 previously well-known candidate markers were identified. We also identified low-abundant proteins with functions related to (i) cell signaling, (ii) immune system regulators, and (iii) proteins regulating folding, sorting, and degradation, as well as (iv) vesicular transport proteins. WiMT holds the potential for use in large-scale screening studies with simple sample preparation, and can lead to the discovery of novel proteins with key melanoma disease functions.

The human melanoma proteome atlas-Defining the molecular pathology.

The MM500 study is an initiative to map the protein levels in malignant melanoma tumor samples, focused on in-depth histopathology coupled to proteome characterization. The protein levels and localization were determined for a broad spectrum of diverse, surgically isolated melanoma tumors originating from multiple body locations. More than 15,500 proteoforms were identified by mass spectrometry, from which chromosomal and subcellular localization was annotated within both primary and metastatic melanoma. The data generated by global proteomic experiments covered 72% of the proteins identified in the recently reported high stringency blueprint of the human proteome. This study contributes to the NIH Cancer Moonshot initiative combining detailed histopathological presentation with the molecular characterization for 505 melanoma tumor samples, localized in 26 organs from 232 patients.

Investigation of a new oxazolidine derivative in human resistance acute leukemia cells: deciphering its mechanism of action by label-free proteomic.

The present study aimed to evaluate the mechanism of action of the antineoplastic activity of an oxazolidine derivative, LPSF/NB-3 (5-(4-cloro-benzilideno)-3-etil-2-tioxo-oxazolidin-4-ona). Cytotoxicity assays were performed in peripheral blood mononuclear cells (PBMCs) and resistant acute leukemia cell line (HL-60/MX1) by the MTT method. LPSF/NB-3 exhibited cytotoxicity in HL-60/MX1, but it was not toxic to healthy cells in the highest dose tested (100 µM). The protein extract of HL-60/MX1 cells treated with LPSF/NB-3 was subjected to proteomic analysis using two-dimensional chromatography coupled to mass spectrometry. We could identify a total of 2652 proteins, in which 633 were statistically modulated. Within the group of protein considered for the quantitative analysis with the established criteria, 262 were differentially expressed, 146 with increased expression and 116 with decreased expression in the sample treated with LPSF/NB-3 compared to the control. The following differentially expressed pathways were found: involving regulation of the cytoskeleton, DNA damage, and transduce cellular signals. Networks that were highlighted are related to the immune system. The ELISA technique was used to assess the immunomodulatory potential of LPSF/NB-3 in PBMCs. We observed significant decrease of IFNγ (p < 0.01) and dose-response pattern of the cytokines IL-6, IL-17A, IL-22, and IL-10. Therefore, results suggest that LPSF/NB-3 appears to modulate important pathways, including cell cycle and immune system regulatory pathways.

Proteomic Workflows for High-Quality Quantitative Proteome and Post-Translational Modification Analysis of Clinically Relevant Samples from Formalin-Fixed Paraffin-Embedded Archives.

Well-characterized archival formalin-fixed paraffin-embedded (FFPE) tissues are of much value for prospective biomarker discovery studies, and protocols that offer high throughput and good reproducibility are essential in proteomics. Therefore, we implemented efficient paraffin removal and protein extraction from FFPE tissues followed by an optimized two-enzyme digestion using suspension trapping (S-Trap). The protocol was then combined with TMTpro 16plex labeling and applied to lung adenocarcinoma patient samples. In total, 9585 proteins were identified, and proteins related to the clinical outcome were detected. Because acetylation is known to play a major role in cancer development, a fast on-trap acetylation protocol was developed for studying endogenous lysine acetylation, which allows identification and localization of the lysine acetylation together with quantitative comparison between samples. We demonstrated that FFPE tissues are equivalent to frozen tissues to study the degree of acetylation between patients. In summary, we present a reproducible sample preparation workflow optimized for FFPE tissues that resolves known proteomic-related challenges. We demonstrate compatibility of the S-Trap with isobaric labeling and for the first time, we prove that it is feasible to study endogenous lysine acetylation stoichiometry in FFPE tissues, contributing to better utility of the existing global tissue archives. The MS proteomic data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifiers PXD020157, PXD021986, and PXD021964.

Toxicoproteomics Disclose Pesticides as Downregulators of TNF-α, IL-1ß and Estrogen Receptor Pathways in Breast Cancer Women Chronically Exposed.

Deleterious effects have been widely associated with chronic pesticide exposure, including cancer development. In spite of several known consequences that pesticides can trigger in the human body, few is known regarding its impact on breast cancer women that are chronically exposed to such substances during agricultural work lifelong. In this context, the present study performed a high-throughput toxicoproteomic study in association with a bioinformatics-based design to explore new putative processes and pathways deregulated by chronic pesticide exposure in breast cancer patients. To reach this goal, we analyzed comparatively non-depleted plasma samples from exposed (n = 130) and non-occupationally exposed (n = 112) women diagnosed with breast cancer by using a label-free proteomic tool. The list of proteins differentially expressed was explored by bioinformatics and the main pathways and processes further investigated. The toxicoproteomic study revealed that women exposed to pesticides exhibited mainly downregulated events, linked to immune response, coagulation and estrogen-mediated events in relation to the unexposed ones. Further investigation shown that the identified deregulated processes and pathways correlated with significant distinct levels tumor necrosis factor alpha and interleukin 1 beta in the blood, and specific clinicopathological characteristics pointed out by bioinformatics analysis as adipose-trophic levels, menopause and intratumoral clots formation. Altogether, these findings reinforce pesticides as downregulators of several biological process and highlight that these compounds can be linked to poor prognosis in breast cancer.

Gene doping and genomic science in sports: where are we?

The misuse of sport-related gene transfer methods in elite athletes is a real and growing concern. The success of gene therapy in the treatment of hereditary diseases has been most evident since targets in gene therapy products can be used in healthy individuals to improve sports performance. Performing these practices threatens the sporting character of competitions and may pose potential health hazards. Since the World Anti-Doping Agency pronouncement on the prohibition of such practices in 2003, several researchers have been trying to address the challenge of developing an effective method for the detection of genetic doping. This review presents an overview of the published methods developed for this purpose, the advantages and limitations of technologies and the putative target genes. At last, we present the perspective related to the application of the detection methods in the doping control field.

Label-Free Proteomics Revealed Oxidative Stress and Inflammation as Factors That Enhance Chemoresistance in Luminal Breast Cancer.

Breast cancer is the leading cause of cancer-associated death among women worldwide. Its high mortality rate is related to resistance towards chemotherapies, which is one of the major challenges of breast cancer research. In this study, we used label-free mass spectrometry- (MS-) based proteomics to investigate the differences between circulating proteins in the plasma of patients with chemoresponsive and chemoresistant luminal A breast cancer. MS analysis revealed 205 differentially expressed proteins. Furthermore, we used in silico tools to build protein-protein interaction networks. Most of the upregulated proteins in the chemoresistant group were closely related and tightly linked. The predominant networks were related to oxidative stress, the inflammatory response, and the complement cascade. Through this analysis, we identified inflammation and oxidative stress as central processes of breast cancer chemoresistance. Furthermore, we confirmed our hypothesis by evaluating oxidative stress and performing cytokine profiling in our cohort. The connections among oxidative stress, inflammation, and the complement system described in our study seem to indicate a pivotal axis in breast cancer chemoresistance. Hence, these findings will have significant clinical implications for improving therapies to bypass breast cancer chemoresistance in the future.

Doping control analysis at the Rio 2016 Olympic and Paralympic Games.

This paper summarises the results obtained from the doping control analyses performed during the Summer XXXI Olympic Games (August 3-21, 2016) and the XV Paralympic Games (September 7-18, 2016). The analyses of all doping control samples were performed at the Brazilian Doping Control Laboratory (LBCD), a World Anti-Doping Agency (WADA)-accredited laboratory located in Rio de Janeiro, Brazil. A new facility at Rio de Janeiro Federal University (UFRJ) was built and fully operated by over 700 professionals, including Brazilian and international scientists, administrative staff, and volunteers. For the Olympic Games, 4913 samples were analysed. In 29 specimens, the presence of a prohibited substance was confirmed, resulting in adverse analytical findings (AAFs). For the Paralympic Games, 1687 samples were analysed, 12 of which were reported as AAFs. For both events, 82.8% of the samples were urine, and 17.2% were blood samples. In total, more than 31 000 analytical procedures were conducted. New WADA technical documents were fully implemented; consequently, state-of-the-art analytical toxicology instrumentation and strategies were applied during the Games, including different types of mass spectrometry (MS) analysers, peptide, and protein detection strategies, endogenous steroid profile measurements, and blood analysis. This enormous investment yielded one of the largest Olympic legacies in Brazil and South America. Copyright © 2017 John Wiley & Sons, Ltd.

Identifying potential markers in Breast Cancer subtypes using plasma label-free proteomics.

Breast Cancer (BC) is the most common neoplasia among women and has a high mortality rate worldwide. Over the past several decades, increasing molecular knowledge of BC has resulted in its stratification into 4 major molecular subtypes according to hormonal receptor expression. Unfortunately, although the data accumulated thus far has improved BC prognosis and treatment, there have been few achievements in its diagnosis. In this study, we applied a Label-free Nano-LC/MSMS approach to reveal systemic molecular features and possible plasma markers for BC patients. Compared to healthy control plasma donors, we identified 191, 166, 182, and 186 differentially expressed proteins in the Luminal, Lumina-HER2, HER2, and TN subtypes. In silico analysis demonstrated an overall downregulation of cellular basal machinery and, more importantly, brought new focus to the known pathways and signaling molecules in BC that are related to immune system alterations. Moreover, using western blot analysis, we verified high levels of BCAS3, IRX1, IRX4 and IRX5 in BC plasma samples, thus highlighting the potential use of plasma proteomics in investigations into cancer biomarkers. SIGNIFICANCE: The results of this study provide new insight into Breast Cancer (BC). We determined the plasma proteomic profile of BC subtypes. Furthermore, we report that the signaling pathways correlating with late processes in BC already exhibit plasma alterations in less aggressive subtypes. Additionally, we validated the high levels of particular proteins in patient samples, which suggests the use of these proteins as potential disease markers.

Clinical proteomics in cancer: Where we are.

Proteomics has emerged as a promising field in the post-genomic era. Notwithstanding the great advances provided by gene expression analysis in cancer, the lack of a correlation between gene expression and protein levels has highlighted the need for a proteomic focus on cancer. Although the increasing knowledge regarding cancer biology, a reliable marker to improve diagnosis, prognosis and treatment for cancer patients is not a reality at present. In this review, we address the main considerations regarding proteomics-based studies and their clinical applications on cancer research, highlighting some considerations related to strengths and limitations of proteomics-based studies and its application to clinical practice.

Early downregulation of acute phase proteins after doxorubicin exposition in patients with breast cancer.

Chemotherapy remains the first-choice option for adjuvant therapy in breast cancer. Here, we investigated the impact of the first chemotherapic cycle of doxorubicin on the plasmatic-proteomic profiling of women diagnosed with breast cancer (n = 87). Blood samples were obtained from the same patient before and after doxorubicin infusion (1 h, 60 mg/m(2)) and processed for label-free LC-MS proteomic screening. A total of 80 proteins were downregulated after chemotherapy. In silico analysis revealed that the main biological process enrolled was inflammation and canonical pathways involving acute phase proteins. TNF-α, IL-1ß, IL-12, TGF-ß1, clusterin, and gelsolin were chosen as relevant for further validation. All selected targets presented reduced plasmatic levels after treatment. Our results indicate that doxorubicin downregulated acute phase proteins immediately after its infusion. Since such proteins are cancer promoting, its downregulation could support the effectiveness of doxorubicin along treatment.

Kaurenoic Acid Possesses Leishmanicidal Activity by Triggering a NLRP12/IL-1ß/cNOS/NO Pathway.

Leishmania amazonensis (L. amazonensis) infection can cause severe local and diffuse injuries in humans, a condition clinically known as American cutaneous leishmaniasis (ACL). Currently, the therapeutic approach for ACL is based on Glucantime, which shows high toxicity and poor effectiveness. Therefore, ACL remains a neglected disease with limited options for treatment. Herein, the in vitro antiprotozoal effect and mechanisms of the diterpene kaurenoic acid [ent-kaur-16-en-19-oic acid] (KA) against L. amazonensis were investigated. KA exhibited a direct antileishmanial effect on L. amazonensis promastigotes. Importantly, KA also reduced the intracellular number of amastigote forms and percentage of infected peritoneal macrophages of BALB/c mice. Mechanistically, KA treatment reestablished the production of nitric oxide (NO) in a constitutive NO synthase- (cNOS-) dependent manner, subverting the NO-depleting escape mechanism of L. amazonensis. Furthermore, KA induced increased production of IL-1ß and expression of the inflammasome-activating component NLRP12. These findings demonstrate the leishmanicidal capability of KA against L. amazonensis in macrophage culture by triggering a NLRP12/IL-1ß/cNOS/NO mechanism.

The positive is inside the negative: HER2-negative tumors can express the HER2 intracellular domain and present a HER2-positive phenotype.

Overexpression of human epithelial growth factor receptor 2 (HER2) is a poor prognostic factor in breast cancer. HER2 is a transmembrane receptor comprising an extracellular domain (ECD), a single transmembrane domain, and an intracellular domain (ICD) with tyrosine-kinase activity. Receptor dimerization triggers pivotal effector pathways in cancer, such as phosphatidylinositol 3-kinase (PI3K) signaling. Currently, screening of HER2 in breast tumors for prognostic and therapeutic purposes involves immunohistochemical (IHC) phenotyping for the ECD, in which tumors with IHC scores below 2+ are reported as HER2-negative. We used a label-free liquid chromatography-mass spectrometry (LC-MS) proteomic approach to compare plasma samples from patients with HER2-positive breast tumors and patients with HER2-negative tumors. Patients with HER2-negative tumors expressed higher circulating levels of calpain-10 than patients with HER2-positive tumors. Calpains cleave HER2, releasing its ECD and transforming phenotypically positive tumors into phenotypically negative tumors. Therefore, we investigated the expression of the ICD in HER2-negative samples that overexpressed calpain-10. We found that 16% of HER2-negative tumors were positive for HER2-ICD, which was associated with circulating HER2-ECD. HER2 gene amplification was also observed in some HER2-negative tumors. Positive staining for the PI3K pathway was observed in the HER2-negative, ICD-positive tumors, similar to the HER2-positive cohort. Microarray analysis revealed that HER2-negative, ICD-positive samples clustered between HER2-positive tumors and triple-negative tumors. Survival analysis revealed that outcome in women with HER2-negative, ICD-positive tumors was better than in women bearing HER2-negative, ICD-negative (triple negative) tumors but was quite similar to HER2-positive tumors and worse than women with luminal A tumors. Moreover, in vitro analyses revealed that MDA-MB 231, a triple negative cell line, possesses calpain-10 and HER2-p-ICD up-regulation and blockage of calpain-10 activity promoted an increase in HER2-p-ICD and p-AKT levels, suggesting an increase in these pathways signaling. These data indicate that HER2-negative tumors with HER2-ICD positivity exhibit clinical behavior closer to that of HER2-positive tumors. This indicates a need for HER2-ICD screening when determining the molecular profile of breast tumors. These findings further indicate that lapatinib should be investigated as a target therapy for HER2-ICD-positive breast tumors.

Label-free proteomic analysis of breast cancer molecular subtypes.

To better characterize the cellular pathways involved in breast cancer molecular subtypes, we performed a proteomic study using a label-free LC-MS strategy for determining the proteomic profile of Luminal A, Luminal-HER2, HER2-positive, and triple-negative (TN) breast tumors compared with healthy mammary tissue. This comparison aimed to identify the aberrant processes specific for each subtype and might help to refine our understanding regarding breast cancer biology. Our results address important molecular features (both specific and commonly shared) that explain the biological behavior of each subtype. Changes in proteins related to cytoskeletal organization were found in all tumor subtypes, indicating that breast tumors are under constant structural modifications to invade and metastasize. We also found changes in cell-adhesion processes in all molecular subtypes, corroborating that invasiveness is a common property of breast cancer cells. Luminal-HER2 and HER2 tumors also presented altered cell cycle regulation, as shown by the several DNA repair-related proteins. An altered immune response was also found as a common process in the Luminal A, Luminal-HER2, and TN subtypes, and complement was the most important pathway. Analysis of the TN subtype revealed blood coagulation as the most relevant biological process.

Oxidative stress, redox signaling and cancer chemoresistance: putting together the pieces of the puzzle.

Chemotherapy continues to be the main treatment option for cancer. Although systemic chemotherapy can efficiently eradicate cancer cells, a significant proportion of patients carry tumors that present a chemoresistant phenotype, resulting in disease progression, cancer relapse, and reduced survival. It has also become clear that the effect of most chemotherapeutic drugs is associated with their capacity to generate reactive species (RS) that bind to specific structures within the cancer cell and promote cell death. Due to repeated exposure to chemotherapeutic agents, the redox homeostasis of cancer cells is continuously disturbed, which can result in changes to the cell's ability to cope with excessive RS levels through the production of protective molecules. It is thought that the imbalance resulting from this process-- oxidative stress--is toxic to cancer cells. Paradoxically, the metabolites produced during oxidative stress can favor the survival of some cancer subpopulations, which present specific gene signatures that confer a chemoresistant phenotype on these clones. Despite the huge amount of information generated by currently available technologies, we cannot predict whether this resistance will arise during chemotherapy and we still do not fully understand the mechanism by which it arises. In this review, we discuss the main findings regarding the role of oxidative stress signaling in cancer chemotherapy and the key redox molecules and pathways that lead to the development of chemoresistance.

Mechanisms of kidney repair by human mesenchymal stromal cells after ischemia: a comprehensive view using label-free MS(E).

Acute kidney injury (AKI) is one of the more frequent and lethal pathological conditions seen in intensive care units. Currently available treatments are not totally effective but stem cell-based therapies are emerging as promising alternatives, especially the use of mesenchymal stromal cells (MSC), although the signaling pathways involved in their beneficial actions are not fully understood. The objective of this study was to identify signaling networks and key proteins involved in the repair of ischemia by MSC. Using an in vitro model of AKI to investigate paracrine interactions and label-free high definition 2D-NanoESI-MS(E) , differentially expressed proteins were identified in a human renal proximal tubule cell lineage (HK-2) exposed to human MSC (hMSC) after an ischemic insult. In silico analysis showed that hMSC stimulated antiapoptotic activity, normal ROS handling, energy production, cytoskeleton organization, protein synthesis, and cell proliferation. The proteomic data were validated by parallel experiments demonstrating reduced apoptosis in HK-2 cells and recovery of intracellular ATP levels. qRT-PCR for proteins implicated in the above processes revealed that hMSC exerted their effects by stimulating translation, not transcription. Western blotting of proteins associated with ROS and energy metabolism confirmed their higher abundance in HK-2 cells exposed to hMSC.