Cx43 and Associated Cell Signaling Pathways Regulate Tunneling Nanotubes in Breast Cancer Cells

Simple Summary The gap junction protein connexin 43 (Cx43) facilitates direct intercellular communication and displays a complex dichotomous role in cancer. Here, we used breast cancer cell lines and CRISPR/Cas9 technology to dissect some of the poorly understood multimodal functions of Cx43 in this setting. Our data show that Cx43 directly modulates the formation of pro-tumorigenic tunneling nanotubes (TNTs), which are cellular protrusions that connect non-adjacent cells to facilitate cellular communication, including the direct cell–cell transfer of organelles. We identify several important cancer signaling pathways that affect TNT formation and show that this effect is strongly influenced by the presence or absence of Cx43. Finally, we demonstrate that Cx43 can affect TNT formation by modulating the cellular secretome. This work provides important insight into the pro-tumorigenic role of Cx43 and its interconnections with TNTs. Abstract Connexin 43 (Cx43) forms gap junctions that mediate the direct intercellular diffusion of ions and small molecules between adjacent cells. Cx43 displays both pro- and anti-tumorigenic properties, but the mechanisms underlying these characteristics are not fully understood. Tunneling nanotubes (TNTs) are long and thin membrane projections that connect cells, facilitating the exchange of not only small molecules, but also larger proteins, organelles, bacteria, and viruses. Typically, TNTs exhibit increased formation under conditions of cellular stress and are more prominent in cancer cells, where they are generally thought to be pro-metastatic and to provide growth and survival advantages. Cx43 has been described in TNTs, where it is thought to regulate small molecule diffusion through gap junctions. Here, we developed a high-fidelity CRISPR/Cas9 system to knockout (KO) Cx43. We found that the loss of Cx43 expression was associated with significantly reduced TNT length and number in breast cancer cell lines. Notably, secreted factors present in conditioned medium stimulated TNTs more potently when derived from Cx43-expressing cells than from KO cells. Moreover, TNT formation was significantly induced by the inhibition of several key cancer signaling pathways that both regulate Cx43 and are regulated by Cx43, including RhoA kinase (ROCK), protein kinase A (PKA), focal adhesion kinase (FAK), and p38. Intriguingly, the drug-induced stimulation of TNTs was more potent in Cx43 KO cells than in wild-type (WT) cells. In conclusion, this work describes a novel non-canonical role for Cx43 in regulating TNTs, identifies key cancer signaling pathways that regulate TNTs in this setting, and provides mechanistic insight into a pro-tumorigenic role of Cx43 in cancer.