Abstract
Connexins (Cx) are primary components of gap junctions that selectively allow molecules to be exchanged between adjacent cells, regulating multiple cellular functions. Along with their channel forming functions, connexins play a variety of roles in different stages of tumorigenesis and their roles in tumor initiation and progression is isoform- and tissue-specific. While Cx26 and Cx43 were downregulated during breast tumorigenesis, Cx32 was accumulated in the cytoplasm of the cells in lymph node metastasis of breast cancers and Cx32 was further upregulated in metastasis. Cx32’s effect on cell proliferation, gap junctional communication, hemichannel activity, cellular motility and epithelial-to-mesenchymal transition (EMT) were investigated by overexpressing Cx32 in Hs578T and MCF7 breast cancer cells. Additionally, the expression and localization of Cx26 and Cx43 upon Cx32 overexpression were examined by Western blot and immunostaining experiments, respectively. We observed that MCF7 cells had endogenous Cx32 while Hs578T cells did not and when Cx32 was overexpressed in these cells, it caused a significant increase in the percentages of Hs578T cells at the S phase in addition to increasing their proliferation. Further, while Cx32 overexpression did not induce hemichannel activity in either cell, it decreased gap junctional communication between Hs578T cells. Additionally, Cx32 was mainly observed in the cytoplasm in both cells, where it did not form gap junction plaques but Cx32 overexpression reduced Cx43 levels without affecting Cx26. Moreover, migration and invasion potentials of Hs578T and migration in MCF7 were reduced upon Cx32 overexpression. Finally, the protein level of mesenchymal marker N-cadherin decreased while epithelial marker ZO-1 and E-cadherin increased in Hs578T cells. We observed that Cx32 overexpression altered cell proliferation, communication, migration and EMT in Hs578T, suggesting a tumor suppressor role in these cells while it had minor effects on MCF7 cells.
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Abbreviations
- Cx:
-
Connexin
- GJIC:
-
Gap junctional intercellular communication
- GFP:
-
Green fluorescent protein
- CBX:
-
Carbenoxolone
- EMT:
-
Epithelial-to-mesenchymal transition
- MET:
-
Mesenchymal-to-epithelial transition
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Acknowledgements
We thank Dr. Steven Scherer from University of Pennsylvania, PA, USA for kindly providing the pIRES2-EGFP2-Cx32 vector. The Biotechnology and Bioengineering Research and Application Center, Izmir Institute of Technology staff and Ms Yagmur Ceren Unal is gratefully acknowledged for their expert technical help.
Funding
This work was supported by The Scientific and Technological Research Council of Turkey (114Z874 to GM) and Izmir Institute of Technology Research Fund (2019IYTE0235 to GM). The Young Investigator Award by the Turkish Academy of Sciences (GM) is also highly appreciated.
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GM conceived the project; DU performed the majority of experiments with contribution from TBG (Cx26 and Cx43 immunostaining experiments), SY (Golgi-Cx32 co-immunostaining experiments), OYO and GM; GM, DU, OYO and EO designed the experiments; DU, EO and GM analyzed the data; GM and DU wrote the manuscript; all authors edited the manuscript.
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Ugur, D., Gungul, T.B., Yucel, S. et al. Connexin 32 overexpression increases proliferation, reduces gap junctional intercellular communication, motility and epithelial-to-mesenchymal transition in Hs578T breast cancer cells. J. Cell Commun. Signal. 16, 361–376 (2022). https://doi.org/10.1007/s12079-021-00665-9
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DOI: https://doi.org/10.1007/s12079-021-00665-9