Spectroscopic and biochemical characterization of extracellular vesicles derived from ovarian cancer cells - effect of simvastatin, estradiol and progesterone on CD44 expression in TNBC cells
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Date
2024-06-12
Authors
Πανταζή, Λευκοθέα
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Abstract
Breast cancer (BRCA) is the most common type of cancer among women and it is a disease with diverse biological and clinical features. Molecularly, it is divided into five subtypes, with triple negative (TNBC) accounting for approximately 15% of all BRCA cases. The incidence rate and the aggressiveness that characterizes TNBC, combined with the fact that there is no targeted therapy against it, has led to a plethora of research worldwide, aimed at both elucidating the events leading to the development and progression of the disease and finding a cure. Studies focus on the Extracellular Matrix (ECM), which plays a major role in the signaling and, therefore, in the proliferation of cancer cells. One of the major transmembrane receptors of ECM molecules is CD44, a glycoprotein found in many isoforms in both normal and cancer cells, which is the main hyaluronic acid receptor.
Statins have been one of the most effective drugs against hypercholesterolemia, inhibiting the mevalonic acid synthesis pathway. Since this pathway involves a plethora of molecules involved in many other signaling pathways, it is possible that statins may affect tumor growth and progression. Studies have demonstrated the effect of statins on CD44 expression in BRCA cells.
Estradiol (E2) and progesterone (P4) are very essential steroid hormones, responsible for a variety of processes, including proper breast development, through a number of signaling pathways. These pathways are deregulated during BRCA, contributing to tumor progression.
The aim of this project is to investigate the expression of CD44 isoforms in the TNBC cell lines Hs578T and MDA-MB-231 in the presence of simvastatin and hormonal stimulation with E2 or P4, using Real-time PCR. Moreover, the effect of these treatments on cell morphology and circularity was studied. Using bioinformatics, the impact of CD44 expression on overall survival of BRCA patients, the expression of CD44 gene in healthy women and patients, and the potential function of CD44 as a biomarker for TNBC prognosis were investigated.
The results obtained by the databases showed that the high CD44 expression usually tends to favor the survival of TNBC patients. Moreover, the expression of the CD44 gene in patients is lower than in healthy women. Different concentrations of simvastatin, combined with either E2 or P4, altered the morphology and the circularity of the cells, while fluctuations in CD44 isoforms expression were observed among increasing concentrations of simvastatin.
Ovarian cancer (OVCA) is the most lethal among gynecologic malignancies and due to the lack of early symptoms and screening tools, most patients are diagnosed at advanced
stages. Several molecules of the Extracellular Matrix (ECM), such as Matrix Metalloproteinase 14 (MMP14) and integrins, can promote cancer invasion and metastasis. Extracellular Vesicles (EVs) are lipid-layered vesicles that enclose and deliver macromolecules from cell to cell. Therefore, tumor-derived EVs can enhance tumor development and metastasis by carrying a variety of macromolecules that can remodel or degrade the ECM.
The aim of this project is to characterize two OVCA cell lines, ES2 and SKOV3, as well as the EVs they secrete, using biochemical and vibrational spectroscopy approaches. The size and concentration of EVs were measured using the Nanotracking Analysis (NTA) technique. Tetraspanins, well-known EV biomarkers, as well as ECM related surface receptors were detected on the surface of EVs using flow cytometry. Moreover, the expression of MMP14 and several integrin subunits in cell extracts, in parallel to their distribution in EVs, were analyzed using Western blots. The molecular profiles of the cell lines and their EVs were analyzed and compared using FTIR and Raman spectroscopy.
The flow cytometry results showed differences in the protein composition of the ES2- and SKOV3-derived EVs, with well-known tumor markers being expressed more in ES2–derived EVs. The Western blot analysis showed an enrichment of the protein targets in the EVs, especially the ES2–derived ones, compared to the cell extracts. These results align with the role of EVs in tumor progression and demonstrate that the ES2 cell line is more aggressive than the SKOV3 one. The spectroscopic analysis demonstrated differences between the SKOV3 and ES2 cell lines, as well as their corresponding EVs, more specifically in the spectral range corresponding to carbohydrate vibrations.
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Keywords
Breast cancer, Triple negative breast cancer, Extracellular matrix, CD44 isoforms, Simvastatin, Estradiol, Progesterone, Real-time PCR, Ovarian cancer, Extracellular vesicles, MMP14, Integrins, Nanotracking analysis, Flow cytometry, Western blot, FTIR spectroscopy, Raman spectroscopy