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Hyperosmolar Tumor Microenvironment Potentiates Connexin 43-Mediated Signalling to Promote Cervical Cancer Progression and Metastasis
Subhadeep Roy [1], Jayasri Das Sarma [1,2,3]*
1Department of Biological Sciences, Indian Institute of Science Education & Research, Kolkata, Mohanpur, 741246, West Bengal, India
2 Department of Ophthalmology, University of Pennsylvania, Philadelphia, PA 19104, USA
3 Centre for Molecular Neurosciences, Kasturba Medical College, MAHE-Manipal
* Correspondence: dassarmaj@iiserkol.ac.in
The tumor microenvironment (TME) plays a critical role in cervical cancer progression by regulating intercellular communication, epithelial–mesenchymal transition (EMT), and metastatic dissemination. Connexin 43 (Cx43), a major gap junction protein, has emerged as an important regulator of tumor behavior; however, its contribution to cervical cancer progression and its interaction with microenvironmental stressors remain poorly understood. We hypothesized that hyperosmolar conditions within the TME enhance Cx43-dependent tumor-promoting signalling. To investigate this, we compared parental HeLa cells (devoid of Cx43 expression) with Cx43-overexpressing HeLa cells (HeLa-43). HeLa-43 cells formed significantly larger tumors in immunocompromised nude mice, generated larger three-dimensional spheroids, and exhibited enhanced migratory and invasive capacities. These changes were accompanied by EMT-associated molecular alterations, including reduced expression of the epithelial marker occludin and increased expression of the mesenchymal marker vimentin, as well as dysregulation of β-catenin, p53, and heat-shock-associated signalling pathways, supporting a pro-tumorigenic role for Cx43. Given that osmotic stress is an underexplored yet physiologically relevant component of the TME, we next examined its impact on Cx43 signalling. Cervical cancer cells exposed to graded hyperosmolar conditions were analyzed using immunofluorescence microscopy, western blotting, qRT-PCR, migration assays, flow cytometry-based apoptosis analysis, and reactive oxygen species measurements. Hyperosmolar stress induced EMT, characterized by increased vimentin and reduced occludin expression, enhanced migratory potential, and decreased sensitivity to cisplatin-induced apoptosis. Notably, these effects were markedly potentiated in cells expressing elevated levels of Cx43, indicating a functional cooperation between hyperosmolar stress and Cx43-mediated signalling. Collectively, our findings identify Cx43 as a critical mediator of cervical cancer aggressiveness and reveal that a hyperosmolar TME amplifies Cx43-associated EMT, invasion, and therapeutic resistance. These results uncover a previously unrecognized link between osmotic stress and gap junction signalling and suggest that targeting the Cx43–osmotic stress axis could represent a novel therapeutic strategy for cervical cancer.
Keywords: Cervical cancer, Tumor microenvironment (TME), Epithelial-mesenchymal transition (EMT), Connexin 43, Hyperosmolarity. |