Professor Virginia Commonwealth University, United States
Introduction: Epithelial-Mesenchymal Transition (EMT) is a crucial biological process where epithelial cells transition to a mesenchymal phenotype, enhancing their migratory and invasive capabilities. The cytokine transforming growth factor-beta1 (TGF-β1) is a key promoter of EMT, driving the assembly and production of fibronectin (FN), a critical component of the extracellular matrix (ECM). Cells secrete TGF-β1 in a latent form, comprising the latency-associated peptide (LAP) and latent TGF-β1 binding protein 1 (LTBP-1). Integrins are integral to the TGF-β1-FN crosstalk. Integrin αvβ6 binds to ECM proteins such as FN and the LAP-TGF-β1 complex, facilitating the activation of TGF-β1. Additionally, integrin α5β1 predominantly binds to FN, initiating ECM assembly and activating several intracellular signaling pathways.
This study aims to investigate how integrin inhibitors, including ATN-161 (targeting α5β1), STX-100 (targeting αvβ6), and 28-G11 (targeting LAP+TGF-β1), disrupt integrin-mediated signaling during EMT. We hypothesize that inhibiting integrin signaling will interrupt the downstream effects of TGF-β1 and FN assembly. By modulating these critical pathways, integrin inhibitors have the potential to prevent EMT-associated pathologies, such as cancer metastasis.
Materials and
Methods: In these experiments, an immortalized non-tumorigenic epithelial cell line (MCF10A), isolated from the mammary gland of a female with fibrocystic breasts, was used. To ensure that the integrin inhibitor concentrations did not adversely impact cell health, a cell viability experiment was conducted. MCF10A cells were cultured to approximately 70% confluency, seeded at about 250,000 cells per well, and treated with the following conditions: 10 μg/mL ATN-161 ± 4 ng/mL TGF-β1, 1 μg/mL STX-100 ± 4 ng/mL TGF-β1, or 1 μg/mL 28-G11 ± 4 ng/mL TGF-β1, along with a media control condition. After two days in culture, the cells were analyzed.
To identify nuclei, actin, E-cadherin, and fibronectin, the MCF10A cells were quantified using immunofluorescence imaging. Additionally, the cells were probed for EMT transcription factors (SNAI1, ZEB1) and EMT markers (CDH1, CDH2, FN1). Cell morphology was assessed using immunofluorescence imaging followed by image processing and analysis with a custom MATLAB code. Changes in the gene expression of EMT markers and transcription factors were measured using RT-qPCR.
Statistical analysis was performed using GraphPad Prism 10, with each experiment conducted in triplicate (N=3). A two-way ANOVA with multiple comparisons test was used to compare cell means within their respective rows and columns, with a significance level of α < 0.05.
Results, Conclusions, and Discussions: Integrin and LAP inhibitors, with or without TGF-β1, impact MCF10A cell morphology. Various metrics, such as average cell area, nuclear area, FN mean fibril size and total fibril area, percentage area of cell coverage, and E-cadherin junctional area, were analyzed using MATLAB. It was observed that both average cell area and nuclear area increased in response to co-culture with inhibitors and TGF-β1. FN fibril size exhibited a statistically significant increase in response to TGF-β1, and the inhibitors appeared to prevent EMT relative to positive TGF-β1 controls. As expected, FN fibril area increased in response to TGF-β1 and the E-cadherin junctional area decreased with inhibitor and TGF-β1 treatments.
Overall, the integrin and LAP inhibitors did not significantly reduce EMT progression as initially hypothesized. Future experiments will alter integrin inhibitor concentrations, vary cell densities, and extend treatment durations. The RT-qPCR experiments revealed that inhibition of integrins had varied effects on EMT marker gene expression. Relative fold changes were determined by RT-qPCR analysis of EMT markers, normalized to 18S rRNA expression. The results for EMT transcription factors (SNAI1, ZEB1) and EMT markers (CDH1, CDH2, FN1) indicated that integrin inhibitors and TGF-β1 conditions had an inconclusive impact on TGF-β1-FN crosstalk.
To refine these findings, future studies will repeat RT-qPCR experiments with optimized integrin inhibitor concentrations, cell densities, and longer treatment durations. Collectively, these data will enhance our understanding of TGF-β1-FN crosstalk and help identify potential therapeutic targets to prevent EMT-related disease progression, including cancer metastasis.
