The role of cytoskeletal components in the epithelial-mesenchymal transition of tumor cells
2025-07-23 12:18
The role of cytoskeletal components in the epithelial-mesenchymal transition of tumor cells
Project goal: Study of changes in cytoskeleton and motility of tumor cells during induced epithelial-mesenchymal transition (EMT). It is hypothesized that EMT is accompanied by a change in expression profile of cytoskeletal proteins, resulting in accelerated microtubule growth and activated cortical actin dynamics, which increases invasiveness of cancer cells.
Project description: Epithelial-mesenchymal transition (EMT) is an important process that occurs during normal development, but it also contributes to some pathological processes (e.g., cancer progression and metastasis). During EMT, epithelial cells lose intercellular contacts and become more motile and invasive. The gold standard for confirming EMT involves description of changes in cell morphology and transcriptional profile of a cell.
There are numerous changes in the transcriptional profile of a cell during EMT, including decreased expression of E-cadherin at cell-cell junctions and increased expression of vimentin and fibronectin, which serve to strengthen cellular interactions with the extracellular matrix. Although the transcriptional aspects of EMT have been well studied, and transcription factors such as Zeb, Snail, and Twist have been identified as key inducers of this process, morphological changes during EMT have been described to a much lesser degree.
Morphological changes in epithelial cells during EMT are usually described as the loss of a cobblestone-like shape with apicobasal polarity and the acquisition of an extended spindle shape typical for mesenchymal cells. Changes in cell shape and morphology occur due to the synergistic contribution of two dynamic components of the cytoskeleton: actin filaments and microtubule networks.
The role of the actin cytoskeleton in maintaining cell shape is relatively well studied. F-actin rearrangements affect cellular morphology, ability to migrate and invasiveness. During TGF-β-induced EMT remodeling of the actin cytoskeleton is triggered by activation of the small GTPase RhoA, which stimulates the assembly of stress fibrils and also induces the destruction of E-cadherin in cell contacts, contributing to changes in the morphology of transforming cells. Reorganization of the actin cytoskeleton is a clearly identifiable driving force that allows cells to separate from each other and to migrate, but the mechanisms that control F-actin dynamics during EMT remain unclear.
Microtubules are another highly dynamic component of the cytoskeleton. Evidence has been obtained that even minor changes in microtubule dynamics can noticeably influence cell migration due to numerous signaling cascades associated with microtubules. It is also known that inhibitors of microtubule dynamics can inhibit EMT and stop the dissemination of tumor cells, slowing down the formatting of metastases in some types of cancer, since inhibition of microtubule dynamics by paclitaxel or nocodazole sharply reduces the motility of mesenchymal cells. However, the contribution of microtubule dynamics to EMT still remains largely unknown.
We plan to study changes in the expression profile of cytoskeletal proteins and the morphological and dynamic parameters of the microtubule network and actin cytoskeleton in EMT, as well as analyze their role in increasing the migratory and invasive potential of tumor cells.
Our central hypothesis is that EMT is regulated by changes in the expression profile of cytoskeletal proteins, resulting in accelerated microtubule growth and activation of cortical actin dynamics, which plays a decisive role in stimulating the invasive potential of tumor cells.
Project facilitators: Ivan Vorobyev - PI Alina Saidova - Senior researcher Madina Tlegenova - Junior researcher Marina Janibekova - Junior researcher Dana Yerpasheva - Research assistant
Realisation period: 2024-2026
Expected results: The following outcomes are expected to be achieved upon completion of the project:
1. Obtain stable tumor cell lines MCF-7, CaCo-2 and 22Rv1 with inducible expression of EMT inducer proteins, obtain control cell lines with constant expression of β-3 tubulin.
2. In model cell lines, confirm the overexpression of EMT inducing proteins using real-time PCR (at the mRNA level) and Western blot (at the protein level)
3. Assess changes in cell morphology during EMT and describe the expression profile of EMT markers in model cell lines
4. Work out a protocol for transfection of model cell lines with a plasmid encoding the microtubule plus terminal protein EB3
5.Analyze the expression profile of actin and tubulin isoforms in model cell lines before and after EMT induction
6.Obtain data on the dynamics of microtubules in model cell lines before and after EMT
7.Describe rearrangement of the actin filament network in model cell lines before and after EMT
8.Assess changes in the migratory potential of model cell lines before and after EMT on an experimental wound model .
9.Assess changes in the invasive potential of model cell lines using a modified Boyden chamber model (transwell assay)
Methodology: In our study, we employ a comprehensive approach that includes advanced methods in cellular and molecular biology, such as high-throughput fluorescence microscopy and live-cell imaging, super-resolution confocal microscopy, the generation of stable cell lines with inducible expression of EMT-related proteins, real-time PCR, immunoblotting, flow cytometry and imaging cytometry with visualization, as well as cell sorting.