Browsing by Author "Lazar, Iulia M."

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    Anti-inflammatory cytokine stimulation of HMC3 cells: Proteome dataset
    Ahuja, Shreya; Lazar, Iulia M. (Elsevier, 2023-07-20)
    The immunoprotective functions of microglia in the brain are mediated by the inflammatory M1 phenotype. This phenotype is challenged by anti-inflammatory cytokines which polarize the microglia cells to an immunosuppressive M2 phenotype, a trait that is often exploited by cancer cells to evade immune recognition and promote tumor growth. Investigating the molecular determinants of this behavior is crucial for advancing the understanding of the mechanisms that cancer cells use to escape immune attack. In this article, we describe liquid chromatography (LC)-mass spectrometry (MS)/proteomic data acquired with an EASY-nanoLC 1200-Q ExactiveTM OrbitrapTM mass spectrometer that reflect the response of human microglia cells (HMC3) to stimulation with potential cancer-released anti-inflammatory cytokines known to be key players in promoting tumorigenesis in the brain (IL-4, IL-13, IL-10, TGFB and MCP-1). The MS files were processed with the Proteome Discoverer v.2.4 software package. The cell culture conditions, the sample preparation protocols, the MS acquisition parameters, and the data processing approach are described in detail. The RAW and processed MS files associated with this work were deposited in the PRIDE partner repository of the ProteomeXchange Consortium with the dataset identifiers PXD023163 and PXD023166, and the analyzed data in the Mendeley Data cloud-based repository with DOI 10.17632/fvhw2zwt5d.1. The biological interpretation of the data can be accessed in the research article “Systems-Level Proteomics Evaluation of Microglia Response to Tumor-Supportive Anti-inflammatory Cytokines” (Shreya Ahuja and Iulia M. Lazar, Frontiers in Immunology 2021 [1]). The proteome data described in this article will benefit researchers who are either interested in re-processing the data with alternative search engines and filtering criteria, and/or exploring the data in more depth to advance the understanding of cancer progression and the discovery of novel biomarkers or drug targets.
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    Mapping the cell-membrane proteome of the SKBR3/HER2+ cell line to the cancer hallmarks
    Lazar, Iulia M.; Karcini, Arba; Haueis, Joshua R. S. (PLOS, 2022-08-01)
    The hallmarks of biological processes that underlie the development of cancer have been long recognized, yet, existing therapeutic treatments cannot prevent cancer from continuing to be one of the leading causes of death worldwide. This work was aimed at exploring the extent to which the cell-membrane proteins are implicated in triggering cancer hallmark processes, and assessing the ability to pinpoint tumor-specific therapeutic targets through a combined membrane proteome/cancer hallmark perspective. By using GO annotations, a database of human proteins associated broadly with ten cancer hallmarks was created. Cell-membrane cellular subfractions of SKBR3/HER2+ breast cancer cells, used as a model system, were analyzed by high resolution mass spectrometry, and high-quality proteins (FDR<3%) identified by at least two unique peptides were mapped to the cancer hallmark database. Over 1,400 experimentally detected cell-membrane or cell-membrane associated proteins, representing ~18% of the human cell-membrane proteome, could be matched to the hallmark database. Representative membrane constituents such as receptors, CDs, adhesion and transport proteins were distributed over the entire genome and present in every hallmark category. Sustained proliferative signaling/cell cycle, adhesion/tissue invasion, and evasion of immune destruction emerged as prevalent hallmarks represented by the membrane proteins. Construction of protein-protein interaction networks uncovered a high level of connectivity between the hallmark members, with some receptor (EGFR, ERBB2, FGFR, MTOR, CSF1R), antigen (CD44), and adhesion (MUC1) proteins being implicated in most hallmark categories. An illustrative subset of 138 hallmark proteins that included 42 oncogenes, 24 tumor suppressors, 9 oncogene/tumor suppressor, and 45 approved drug targets was subjected to a more in-depth analysis. The existing drug targets were implicated mainly in signaling processes. Network centrality analysis revealed that nodes with high degree, rather than betweenness, represent a good resource for informing the selection of putative novel drug targets. Through heavy involvement in supporting cancer hallmark processes, we show that the functionally diverse and networked landscape of cancer cell-membrane proteins fosters unique opportunities for guiding the development of novel therapeutic interventions, including multi-agent, immuno-oncology and precision medicine applications.
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    A proteomics study to investigate the role of the neural niche in the development of metastatic HER2+ breast cancer
    Ahuja, Shreya (Virginia Tech, 2022-06-13)
    Advanced stage tumors can acquire the ability to divide uncontrollably, invade the surrounding matrix, and circulate through the bloodstream or lymphatic system to distant organs in a process known as metastasis. The brain, which is shielded from the environment by the blood brain barrier, offers an immunocompetent lodging spot for the circulating cancer cells. Therefore, it is a "popular" destination for metastasized cancers which even surpass the incidents of primary brain tumors. It is hypothesized that the disseminated cancer cells engage with the host cells of the perivascular neural niche in a poorly understood crosstalk of molecular factors, that in turn augment the metastatic colonization of cancer cells. A better understanding of this crosstalk is indispensable to apprehending the complexity of the metastasis process, and to facilitating the discovery of biomarkers that predict metastatic potential and improve patient prognosis. The larger goal of this study was to adopt a mass spectrometry-based systems biology approach to investigate the molecular mechanisms and regulatory networks that underlie the complex phenomenon of breast cancer propagation at the brain metastatic site. To achieve this, the study was divided in three sub-projects designed around the following objectives, i.e., (a) to comprehensively characterize the protein landscape of the neural niche or the brain microenvironment comprised of astrocytes, microglia and endothelial cells, (b) to explore the immunological protein networks activated in microglia cells upon stimulation with anti-inflammatory cytokines released by tumor cells in the brain, and (c) to investigate the protein-level changes elicited in HER2+ breast cancer cells when grown under conditions that simulate the brain microenvironment in-vitro. Detailed characterization of the neural niche enabled us to propose molecular mechanisms that allow for the seeding and outgrowth of metastasized cancer cells in the brain. The study further provided novel insights into the signaling networks that regulate the immune functions of the microglia and their role during cancer development. Lastly, an in-depth investigation of breast cancer cells cultured in the presence of neural niche factors revealed potential novel mechanisms of cancer cell dormancy during metastasis. Altogether, large-scale proteomics data generated in this work will help clarify the mechanisms of metastatic cancer development, and will lay the groundwork for future studies that aim at the discovery of novel biomarkers and druggable targets for the treatment of brain metastatic cancers.
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    The SKBR3 cell-membrane proteome reveals telltales of aberrant cancer cell proliferation and targets for precision medicine applications
    Karcini, Arba; Lazar, Iulia M. (Nature Portfolio, 2022-06-27)
    The plasma membrane proteome resides at the interface between the extra- and intra-cellular environment and through its various roles in signal transduction, immune recognition, nutrient transport, and cell-cell/cell-matrix interactions plays an absolutely critical role in determining the fate of a cell. Our work was aimed at exploring the cell-membrane proteome of a HER2+ breast-cancer cell line (SKBR3) to identify triggers responsible for uncontrolled cell proliferation and intrinsic resources that enable detection and therapeutic interventions. To mimic environmental conditions that enable cancer cells to evolve adaptation/survival traits, cell culture was performed under serum-rich and serum-deprived conditions. Proteomic analysis enabled the identification of similar to 2000 cell-membrane proteins. Classification into proteins with receptor/enzymatic activity, CD antigens, transporters, and cell adhesion/junction proteins uncovered overlapping roles in processes that drive cell growth, apoptosis, differentiation, immune response, adhesion and migration, as well as alternate pathways for proliferation. The large number of tumor markers (> 50) and putative drug targets (> 100) exposed a vast potential for yet unexplored detection and targeting opportunities, whereas the presence of 15 antigen immunological markers enabled an assessment of epithelial, mesenchymal or stemness characteristics. Serum-starved cells displayed altered processes related to mitochondrial OXPHOS/ATP synthesis, protein folding and localization, while serum-treated cells exhibited attributes that support tissue invasion and metastasis. Altogether, our findings advance the understanding of the biological triggers that sustain aberrant cancer cell proliferation, survival and development of resistance to therapeutic drugs, and reveal vast innate opportunities for guiding immunological profiling and precision medicine applications aimed at target selection or drug discovery.
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    Systems-Level Proteomics Evaluation of Microglia Response to Tumor-Supportive Anti-Inflammatory Cytokines
    Ahuja, Shreya; Lazar, Iulia M. (2021-09-09)
    Background Microglia safeguard the CNS against injuries and pathogens, and in the presence of certain harmful stimuli are capable of inducing a disease-dependent inflammatory response. When exposed to anti-inflammatory cytokines, however, these cells possess the ability to switch from an inflammatory to an immunosuppressive phenotype. Cancer cells exploit this property to evade the immune system, and elicit an anti-inflammatory microenvironment that facilitates tumor attachment and growth. Objective The tumor-supportive biological processes that are activated in microglia cells in response to anti-inflammatory cytokines released from cancer cells were explored with mass spectrometry and proteomic technologies. Methods Serum-depleted and non-depleted human microglia cells (HMC3) were treated with a cocktail of IL-4, IL-13, IL-10, TGF beta, and CCL2. The cellular protein extracts were analyzed by LC-MS/MS. Using functional annotation clustering tools, statistically significant proteins that displayed a change in abundance between cytokine-treated and non-treated cells were mapped to their biological networks and pathways. Results The proteomic analysis of HMC3 cells enabled the identification of similar to 10,000 proteins. Stimulation with anti-inflammatory cytokines resulted in the activation of distinct, yet integrated clusters of proteins that trigger downstream a number of tumor-promoting biological processes. The observed changes could be classified into four major categories, i.e., mitochondrial gene expression, ECM remodeling, immune response, and impaired cell cycle progression. Intracellular immune activation was mediated mainly by the transducers of MAPK, STAT, TGF beta, NFKB, and integrin signaling pathways. Abundant collagen formation along with the expression of additional receptors, matrix components, growth factors, proteases and protease inhibitors, was indicative of ECM remodeling processes supportive of cell-cell and cell-matrix adhesion. Overexpression of integrins and their modulators was reflective of signaling processes that link ECM reorganization with cytoskeletal re-arrangements supportive of cell migration. Antigen processing/presentation was represented by HLA class I histocompatibility antigens, and correlated with upregulated proteasomal subunits, vesicular/viral transport, and secretory processes. Immunosuppressive and proangiogenic chemokines, as well as anti-angiogenic factors, were detectable in low abundance. Pronounced pro-inflammatory, chemotactic or phagocytic trends were not observed, however, the expression of certain receptors, signaling and ECM proteins indicated the presence of such capabilities. Conclusions Comprehensive proteomic profiling of HMC3 cells stimulated with anti-inflammatory cytokines revealed a spectrum of microglia phenotypes supportive of cancer development in the brain via microenvironment-dependent biological mechanisms.