The Impact of Connexin 43 Modulation on the Metastatic Potential of Triple Negative MDA-MB-231 Breast Cancer Cells to the Bone

Abstract

Background: Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer, and a leading cause of cancer-related mortality among women. As TNBC progresses, the cancerous cells detach from the breast and colonize to a secondary tumor site, often the bone, in a process called metastasis. Connexin 43 (Cx43), a gap junction protein, is crucial in tumor progression and has been linked to TNBC's metastasis to the bone.

Aim: The study aims to investigate the role of Connexin 43 (Cx43) modulation in regulating key genes and epithelial-mesenchymal transition (EMT) proteins involved in TNBC progression and metastasis.

Methods: Light and fluorescence microscopy were used to assess cell morphology and confirm successful transfection in modified MDA-MB-231 cells respectively. qPCR and Western blot assays were applied to validate the modification of Cx43 expression. The key gene NPM1, and EMT markers, E-cadherin, N-cadherin, Twist, and Snai1 were evaluated by qPCR to assess the metastatic potential of the modified cells. Immunofluorescence was used to visualize and localize the target proteins, Cx43 and NPM1.

Results: Microscopic evaluation, qPCR and Western Blot assays confirmed the up- regulation of Cx43 in MDA-MB-231 cells (Cx43D), which exhibited an epithelial-like phenotype, as well as the down-regulation of Cx43 in MDA-MB-231 cells (shCx43), which exhibited a mesenchymal-like phenotype. Cx43D manifested an increase in E- cadherin and decreased NPM1, N-cadherin, Twist, and Snail expression on the transcriptional level, indicating a possible shift towards a less metastatic phenotype. Conclusion: The modulation of Connexin 43 affects the metastatic characteristics of MDA-MB-231 TNBC cells.

Keywords: triple negative breast cancer, connexin43, metastasis, epithelial-to- mesenchymal transition, EMT, Cx43D, shCx43.