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. / R01 HL132236 JWS R41 CA250874 SL R01HL132236 Diversity Supplement KLY II 23PRE1025483 AHA Predoctoral Fellowship KLY II / Doctor of Philosophy / Every organ system relies upon cell-to-cell communication to properly function and is the basis of multi-cellular life. Gap junctions are nanoscale conduits allowing the passage of small signaling molecules and ions between adjacent cells, similar to telephone tubes. Gap junctions are formed from proteins called connexins. Interestingly, it is well known that shorter pieces of connexin proteins can regulate the formation of gap junctions and are uniquely created by a process called alternative ‘internal’ translation. Changes in the amounts of short-length and full-length connexin proteins are often found in cancer cells. Cancer is the uncontrolled growth of abnormal cells. Commonly, the morphology of cancer cells, and the way they communicate with neighboring cells, is altered. Cancer progression is aided by changes in cell signaling molecules, including TGF-β which can drive cancer cells to leave primary tumor sites and grow elsewhere in the body. This is important for the cancer cells to continue dividing and eventually metastasizing (invading other organ systems). Treating cancer once it has spread to other regions of the body is difficult and is the main cause of cancer deaths worldwide. Using TGF-β to model metastatic changes in mouse and human cell lines, we studied how short-length connexin protein affects metastatic cancer cell behavior. With this information we will be able to guide the development of druggable alternative ‘internal’ translation targets, by restoring the proper communication between neighboring cells and therefore preventing spread of cancer cells.