Tishchenko Alexander, Azorín Daniel D, Vidal-Brime Laia, Muñoz María José, Arenas Pol Jiménez, Pearce Christopher, Girao Henrique, Ramón Y Cajal Santiago, Aasen Trond
Patologia Molecular Translacional, Vall d'Hebron Institut de Recerca (VHIR), Vall d'Hebron Hospital Universitari, Vall d'Hebron Barcelona Hospital Campus, Passeig Vall d'Hebron 119-129, 08035 Barcelona, Spain.
Biomedical Research (iCBR), Faculty of Medicine, University of Coimbra, Azinhaga de Santa Comba, Celas, 3000-548 Coimbra, Portugal.
Cancers (Basel). 2020 Sep 29;12(10):2798. doi: 10.3390/cancers12102798.
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.
连接蛋白43（Cx43）形成间隙连接，介导离子和小分子在相邻细胞间的直接细胞间扩散。Cx43具有促肿瘤和抗肿瘤特性，但其潜在机制尚未完全明确。隧道纳米管（TNTs）是连接细胞的细长膜性突起，不仅促进小分子交换，还能促进更大的蛋白质、细胞器、细菌和病毒的交换。通常，TNTs在细胞应激条件下形成增加，在癌细胞中更突出，一般认为它们具有促转移作用，并提供生长和生存优势。Cx43已在TNTs中被描述，被认为可通过间隙连接调节小分子扩散。在此，我们开发了一种高保真CRISPR/Cas9系统来敲除（KO）Cx43。我们发现，Cx43表达缺失与乳腺癌细胞系中TNT长度和数量显著减少有关。值得注意的是，与敲除细胞相比，条件培养基中存在的分泌因子在源自表达Cx43的细胞时对TNTs的刺激更强。此外，抑制几种关键的癌症信号通路可显著诱导TNT形成，这些信号通路既调节Cx43，又受Cx43调节，包括RhoA激酶（ROCK）、蛋白激酶A（PKA）、粘着斑激酶（FAK）和p38。有趣的是，药物诱导的TNTs刺激在Cx43敲除细胞中比在野生型（WT）细胞中更有效。总之，这项工作描述了Cx43在调节TNTs中的一种新的非经典作用，确定了在这种情况下调节TNTs的关键癌症信号通路，并提供了对Cx43在癌症中的促肿瘤作用的机制性见解。
