Internally Translated Cx43 Isoform GJA1-20k Affects Epithelial to Mesenchymal Transition and Metastatic Cancer Cell Behavior

Epithelial-mesenchymal transition (EMT) is a trans-differentiation program essential for development and wound healing that is pathologically activated during cancer progression. During this process, cells undergo complex changes at the transcriptional and translational levels leading to dissolution of cell-cell junctions, loss of apical-basal polarity, and cytoskeleton reorganization. Transforming Growth Factor-β (TGF-β) is well-established in driving cancer progression through EMT induction. Remodeling of cellular junctions, including gap junctions, is critical to acquiring migratory and invasive characteristics during EMT. The gene GJA1 encodes for Connexin43 (Cx43), the most ubiquitously expressed gap junction protein where altered regulation of Cx43 is associated with cancer progression. Intriguingly, Cx43 mRNA undergoes alternative ‘internal’ translation initiation, generating N-terminally truncated isoforms, including GJA1-20k, which regulates Cx43 gap junction formation. We have previously demonstrated GJA1-20k expression is inhibited during TGF-β-induced EMT, limiting gap junction formation; however, the relationship between GJA1-20k modulation of gap junction localization and cellular invasion and migration remains unknown. Given the role GJA1-20k has in regulating gap junctions, we hypothesize that suppression of GJA1-20k expression promotes metastatic trait acquisition through limiting gap junction formation. Utilizing lentivirally transduced stable mouse mammary gland epithelial (NMuMG) and triple-negative human breast epithelial (MDA-MB-231) cells expressing GJA1-20k, or Lac Z as control, we tested effects on TGF-β-induced EMT induction and metastatic trait induction. Boyden chambers, would/scratch assays were employed to analyze cell invasion and migration respectively. We found GJA1-20k overexpression during EMT results in decreased cell invasion and migration to LacZ controls. Future directions include evaluation of GJA1-20k restoration in a metastatic breast cancer model in vivo. Investigating the underlying role of GJA1-20k in EMT-induced cell junction remodeling could be promising as a potential pharmacological target process independent of transcriptional or post-translational pathways. Ultimately, by adding novel information in the expanding and compelling field of translational control, this work could aid in developing the future of precision medicine as new therapeutic solutions to treat cancer will require limiting cancer cell’s ability to metastasize.

Name thesis:degree_name

Doctor of Philosophy

Level thesis:degree_level

doctoral

Discipline thesis:degree_discipline

Translational Biology, Medicine, and Health

Grantor dc:publisher

Virginia Tech

Year dc:date.issued

2024

Author dc:creator

• Young, Kenneth Lee, II

Chairs dc:contributor.committeechair

• Lamouille, Samy Y.
• Smyth, James W.

Committee members dc:contributor.committeemember

• Farris, Shannon
• Fox, Michael A.
• LaConte, Leslie E. W.

• Connexin43
• GJA1-20k
• epithelial-mesenchymal transition
• breast cancer
• metastasis
• Optical Super Resolution (SoRa) Confocal Microscopy
• Stochastic Optical Reconstruction Microscopy (STORM)
• Trafficking

Statement dc:rights

• CC0 1.0 Universal

Licence dc:rights.uri

• http://creativecommons.org/publicdomain/zero/1.0/

Language dc:language.iso

en

Handle dc:identifier.uri

https://hdl.handle.net/10919/121357

OAI identifier oai:identifier

oai:vtechworks.lib.vt.edu:10919/121357