Biology subjects

Lazar, I. M.

Publications and source records attributed to Lazar, I. M..

8 recordsLinked to original sources

bioRxiv2026

XMAn Update - A Database of Homo sapiens Mutated Peptides

Mass spectrometry (MS) is the leading technology for identifying proteins in complex biological samples. It relies on the use of tandem MS alongside a reference database of canonical protein sequences to computationally identify peptides and their parent proteins. The canonical sequences represent the most widely expressed and functionally validated forms of proteins. Consequently, disease-induced or disease-supportive variants, such as those associated with cancer, will evade detection if they are absent from the database. To address this challenge, this study introduces a revised release of the Unkown Mutation Analysis (XMAn) database by incorporating coding missense and nonsense mutations from the latest versions (v103) of the COSMIC Genome Screen Mutants (GSM) and Cancer Gene Census (CGC) datasets in two distinct FASTA-formatted peptide databases comprising 3,848,499 and 312,658 variants, respectively. The mutated peptides were matched to reviewed, non-redundant UniProt Homo sapiens protein entries (18,362 and 746), and characterized in terms of nucleotide- and amino acid mutation frequencies, peptide length distributions, and associations between specific single-nucleotide (SNV) and single amino acid (SAAVs) variants. Applied to the analysis of MDA-MB-231 breast cancer cell-membrane protein fractions, the database enabled the identification of 300+ high-quality variant peptides - several localized to functional protein-binding and catalytic domains - and 23 aberrant protein products mapped to the CGC dataset. The database is hosted and available for download on Zenodo (XMAn/gsm doi: 10.5281/zenodo.21781023; XMAn/cgc doi: 10.5281/zenodo.21781514) or can be accessed through https://sites.google.com/vt.edu/xman-db/home.

bioinformatics↗

bioRxiv2025

Scaffold-free 3D-Cell Co-Culture Model System for the Study of Metastatic Cancer Cell Behavior in the Brain TME

Cancer is a complex disease involving dynamic interactions between cancer, stromal, and infiltrating immune cells, as well as between these cells and the extracellular matrix components of the tumor microenvironment. Brain metastases arise primarily from solid tumors and often result in fatal outcomes. An in-depth understanding of the complex intercellular interactions that evolve in the brain microenvironment is essential to enabling early cancer diagnostics and improving patient outcomes. The protected tumor microenvironment of the brain hinders, however, direct access, impeding the execution of mechanistic studies and limiting the ability to derive meaningful insights. Several in vitro 2D and 3D model systems have been developed to circumvent this problem, none, however, without limitations. The 2D models fail to recapitulate the 3D architecture of the in-vivo environment lacking therefore physiological relevance, while the 3D models present challenges related to the lack of control over cell positioning, lack of vascularization, contamination from non-human scaffolds, batch-to-batch reproducibility, and high production costs. To overcome some of these limitations, we developed an in vitro scaffold-free 3D tumor model system to simulate the in vivo brain metastatic niche. The model was constructed from human brain endothelial cells (HBEC-5i) and two different cancer cell lines derived from breast (MDA-MB-231/triple negative and SK-BR-3/HER2+) and aggressive ovarian (SK-OV-3) cancers. The development of the model relies on a newly identified affinity between the endothelial and cancer cells that enables them to self-assemble in 3D networked constructs, a feature facilitated by the high collagen production by endothelial cells and the secretion of key chemokines by both endothelial and cancer cells. The model mimics the attachment of metastasized cancer cells to the brain microvasculature, enabling the study of temporal changes in endothelial morphology and molecular signaling processes that sustain cancer cell migration, survival, proliferation, and angiogenic processes. Moreover, the model exhibits long-term stability, reproducibility, and effectiveness in evaluating anti-cancer agents. Altogether, the scaffold-free, simple 3D in vitro model systems provides a low cost, physiologically relevant tool for studying the dynamic molecular crosstalk between cancer and brain endothelial cells, and for investigating the fundamental biological processes that unfold in the tumor microenvironment.

cancer biology↗

bioRxiv2024

Proteomic Assessment of SKBR3/HER2+ Breast Cancer Cellular Response to Lapatinib and Investigational Ipatasertib Kinase Inhibitors

Modern cancer treatment approaches aim at achieving cancer remission by using targeted and personalized therapies, as well as harnessing the power of the immune system to recognize and eliminate the cancer cells. To overcome a relatively short-lived response due to the development of resistance to the administered drugs, combination therapies have been pursued, as well. To expand the outlook of combination therapies, the objective of this study was to use high-throughput data generation technologies such as mass spectrometry and proteomics to investigate the response of HER2+ breast cancer cells to a mixture of two kinase inhibitors that has not been adopted yet as a standard treatment regime. The broader landscape of biological processes that are affected by inhibiting two major pathways that sustain the growth and survival of cancer cells, i.e., EGFR and PI3K/AKT, was investigated by treating SKBR3/HER2+ breast cancer cells with Lapatinib or a mixture of Lapatinib/Ipatasertib small molecule drugs. Changes in protein expression and/or activity in response to the drug treatments were assessed by using two complementary quantitative proteomic approaches based on peak area and peptide spectrum match measurements. Over 900 proteins matched by three unique peptide sequences (FDR<0.05) were affected by the exposure of cells to the drugs. The work corroborated the anti-proliferative activity of Lapatinib and Ipatasertib, and, in addition to cell cycle and growth arrest processes enabled the identification of several multi-functional proteins with roles in cancer-supportive hallmark processes. Among these, immune response, adhesion and migration emerged as particularly relevant to the ability to effectively suppress the proliferation and dissemination of cancer cells. The supplementation of Lapatinib with Ipatasertib further affected the expression or activity of additional transcription factors and proteins involved in gene expression, trafficking, DNA repair, and development of multidrug resistance. Furthermore, over fifty of the affected proteins represented approved or investigational targets in the DrugBank database, which through their protein-protein interaction networks can inform the selection of effective therapeutic partners. Altogether, our findings exposed a broad plethora of yet untapped opportunities that can be further explored for enhancing the anti-cancer effects of each drug as well as of many other multi-drug therapies that target the EGFR/ERBB2 and PI3K/AKT pathways. The data are available via ProteomeXchange with identifier PXD051094.

pharmacology and toxicology↗

bioRxiv2023

Proteomic Insights into Metastatic Breast Cancer Response to Brain Cell-Secreted Factors

The most devastating feature of cancer cells is their ability to metastasize to distant sites in the body. HER2+ and TN breast cancers frequently metastasize to the brain and stay potentially dormant for years until favorable conditions support their proliferation. The sheltered and delicate nature of the brain prevents, however, early disease detection and effective delivery of therapeutic drugs. Moreover, the challenges associated with the acquisition of brain biopsies add compounding difficulties to exploring the mechanistic aspects of tumor development, leading to slow progress in understanding the drivers of disease progression and response to therapy. To provide insights into the determinants of cancer cell behavior at the brain metastatic site, this study was aimed at exploring the early response of HER2+ breast cancer cells (SKBR3) to factors present in the brain perivascular niche. The neural microenvironment was simulated by using the secretome of a set of brain cells that come first in contact with the cancer cells upon crossing the blood brain barrier, i.e., endothelial cells (HBEC5i), astrocytes (NHA), and microglia (HMC3). Cytokine microarrays were used to investigate the secretome mediators of intercellular communication, and proteomic technologies for assessing the changes in the behavior of cancer cells upon exposure to the brain cell-secreted factors. The cytokines, growth factors and enzymes detected in the brain secretomes were supportive of inflammatory conditions, indicating a collective functional contribution to cell activation, defense, inflammatory responses, chemotaxis, adhesion, angiogenesis, and ECM organization. The SKBR3 cells, on the other hand, secreted numerous cancer-promoting growth factors that were either absent or present in lower abundance in the brain cell cultures, indicating that upon exposure the SKBR3 cells may have been deprived of favorable conditions for optimal growth. Altogether, the results suggest that the subjection of SKBR3 cells to the brain cell-secreted factors altered their growth potential and drove them toward a state of quiescence, with broader overall outcomes that affected cellular metabolism, adhesion and immune response processes. The findings of this study underscore the key role played by the neural niche in shaping the behavior of metastasized cancer cells, provide insights into the cellular cross-talk that may lead cancer cells into dormancy, and highlight novel opportunities for the development of metastatic breast cancer therapeutic strategies.

cancer biology↗

bioRxiv2022

Mapping the Cell-Membrane Proteome to the Cancer Hallmarks

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 novel 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 1100 experimentally detected cell-membrane or cell-membrane associated proteins, representing ~8 % of the human cell-membrane proteome, were mapped 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 116 hallmark proteins that included 44 oncogenes, 28 tumor suppressors, and 38 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.

cancer biology↗

bioRxiv2021

The SKBR3 Cell-Membrane Proteome: Role in Aberrant Cancer Cell Proliferation and Resource for Precision Medicine Applications

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 and cell-matrix interactions plays an absolutely critical role in determining the fate of a cell. Our work was aimed at exploring the landscape of the cancer cell-membrane proteome responsible for sustaining uncontrolled cell proliferation, and its intrinsic resources for enabling detection and therapeutic interventions. SKBR3/HER2+ breast cancer cells were used as a model system and mass spectrometry for characterizing the proteome. The number of identified cell-membrane proteins exceeded 2,000, with ~1,300 being matched by two or more unique peptides. Classification in four major categories, i.e., proteins with receptor or enzymatic activity, CD antigens, transporters, and cell adhesion proteins, uncovered overlapping roles in biological processes that drive cell growth, apoptosis, differentiation, immune response, adhesion and migration, as well as capabilities for signaling crosstalk and 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 the vast innate opportunities for guiding immunological profiling and precision medicine applications aimed at target selection or drug discovery.

cancer biology↗

bioRxiv2021

Systems-Level Proteomics Evaluation of Microglia Response to Tumor-Supportive Anti-inflammatory Cytokines

BackgroundMicroglia safeguard the CNS against injuries and pathogens by inducing an inflammatory response. When exposed to anti-inflammatory cytokines, 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. ObjectiveThe 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. MethodsSerum-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. ResultsThe proteomic analysis of HMC3 cells enabled the identification of [~]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, enabled ECM remodeling processes supportive of cell-cell and cell-matrix adhesion. Overexpression of integrins and their modulators was reflective of signaling processes that correlated 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, and vesicular/viral transport and secretory processes. Immunosuppressive and proangiogenic chemokines were detectable in low abundance. Pronounced pro-inflammatory, chemotactic or phagocytic trends were not observed, however, the expression of certain receptors and ECM proteins indicated the presence of such capabilities. ConclusionsComprehensive proteomic profiling of HMC3 cells stimulated with anti-inflammatory cytokines revealed a microglia phenotype that provides novel insights into the tumor microenvironment-driven mechanisms that fuel cancer development in the brain.

immunology↗

bioRxiv2019

Preferential phosphatidylinositol 5-phosphate binding contributes to a destabilization of the VHS domain structure of Tom1

Tom1 transports endosomal ubiquitinated proteins that are targeted for degradation in the lysosomal pathway. Infection of eukaryotic cells by Shigella flexneri boosts oxygen consumption and promotes the synthesis of phosphatidylinositol-5-phosphate [PtdIns5P], which triggers Tom1 translocation to signaling endosomes. Removing Tom1 from its cargo trafficking function hinders protein degradation in the host and, simultaneously, enables bacterial survival. Tom1 preferentially binds PtdIns5P via its VHS domain, but the effects of a reducing environment as well as PtdIns5P on the domain structure and function are unknown. Thermal denaturation studies demonstrate that, under reducing conditions, the monomeric Tom1 VHS domain switches from a three-state to a two-state transition behavior. PtdIns5P reduced thermostability, interhelical contacts, and conformational compaction of Tom1 VHS, suggesting that the phosphoinositide destabilizes the protein domain. Destabilization of Tom1 VHS structure was also observed with other phospholipids. Isothermal calorimetry data analysis indicates that, unlike ubiquitin, Tom1 VHS endothermically binds to PtdIns5P through two noncooperative binding sites, with its acyl chains playing a relevant role in the interaction. Altogether, these findings provide mechanistic insights about the recognition of PtdIns5P by the VHS domain that may explain how Tom1, when in a different VHS domain conformational state, interacts with downstream effectors under S. flexneri infection.

biophysics↗