Browsing by Author "Karcini, Arba"

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    The Cell Membrane Proteome of the SKBR3/HER2+ Cells and Implications for Cancer Targeted Therapies
    Karcini, Arba (Virginia Tech, 2023-06-02)
    Breast cancer is the second most common type of cancer among women in the US and the second leading cause of cancer death. HER2+ breast cancers represent ~20% of all cancer types, are highly invasive, and can be treated by using targeted therapies against the HER2 receptor. However, these therapies are challenged by the development of drug resistance, often induced by the presence of mutations in the cell-membrane proteins and receptors and/or by alternative signaling pathways that cross-talk with- or transactivate HER2+ triggered signaling. This study was aimed at investigating the cell membrane proteome of SKBR3 cells, representative of HER2+ breast cancers, and the signaling landscape and cellular responses elicited by the cell membrane receptors when the cells are stimulated with either growth factors or therapeutic drugs. It was hypothesized that the identification of a broad range of cell membrane proteins with roles in cancer progression and signaling crosstalk will lead to a more comprehensive understanding of the biological processes that sustain the proliferation of cancer cells, and will guide the selection of more efficient drug targets. The project was conceptualized in three stages: (1) profiling the cell membrane proteins of SKBR3 cells, (2) determining the functional role of the detected cell membrane proteins in the context of cancer hallmarks and exploring their mutational profile, and (3) analyzing the cellular events that occur in response to treatment with a single therapeutic agent or a combination of drugs. Mass spectrometry technologies were used for performing proteomic and phosphoproteomic profiling of SKBR3 cells, detecting changes in the abundance of the detected proteins, and identifying the presence of mutations in the cell membrane proteins. Orthogonal enrichment methods were developed for profiling the low-abundance cell membrane proteins, for generating a rich landscape of cell membrane receptors with various functional roles and relevance to the cancer hallmarks, and for enabling the detection of potentially new drivers of aberrant proliferation. The analysis of serum-starved, stimulated (with growth factors), or inhibited (with kinase inhibitors) cells revealed alternative protein players and crosstalk activities that determine the fate of cells, and that may fuel the development of resistance to treatment with drugs. The proteome profiles that were generated in this project expand the opportunities for targeting cancer-relevant processes beyond proliferation, which is commonly attempted, broadening the landscape to also include apoptosis, invasion, and metastasis. Altogether, the findings that emerged from this work will lay the ground for future studies that aim at developing more complex and effective targeted cancer treatment approaches.
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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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    Preferential phosphatidylinositol 5-phosphate binding contributes to a destabilization of the VHS domain structure of Tom1
    Xiong, Wen; Tang, Tuo-Xian; Littleton, Evan S.; Karcini, Arba; Lazar, Iuliana M.; Capelluto, Daniel G. S. (Springer Nature, 2019-07-26)
    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.
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    Proteogenomic Analysis of Protein Sequence Alterations in Breast Cancer Cells
    Lazar, Iuliana M.; Karcini, Arba; Ahuja, Shreya; Estrada-Palma, Carly (Nature Research, 2019-07-18)
    Cancer evolves as a result of an accumulation of mutations and chromosomal aberrations. Developments in sequencing technologies have enabled the discovery and cataloguing of millions of such mutations. The identification of protein-level alterations, typically by using reversed-phase protein arrays or mass spectrometry, has lagged, however, behind gene and transcript-level observations. In this study, we report the use of mass spectrometry for detecting the presence of mutations-missense, indels and frame shifts-in MCF7 and SKBR3 breast cancer, and non-tumorigenic MCF10A cells. The mutations were identified by expanding the database search process of raw mass spectrometry files by including an in-house built database of mutated peptides (XMAn-v1) that complemented a minimally redundant, canonical database of Homo sapiens proteins. The work resulted in the identification of nearly 300 mutated peptide sequences, of which ~50 were characterized by quality tandem mass spectra. We describe the criteria that were used to select the mutated peptide sequences, evaluate the parameters that characterized these peptides, and assess the artifacts that could have led to false peptide identifications. Further, we discuss the functional domains and biological processes that may be impacted by the observed peptide alterations, and how protein-level detection can support the efforts of identifying cancer driving mutations and genes. Mass spectrometry data are available via ProteomeXchange with identifier PXD014458.
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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.