Connexins in cancer: bridging the gap to the clinic

Abstract

Gap junctions comprise arrays of intercellular channels formed by connexin proteins and provide for the direct communication between adjacent cells. This type of intercellular communication permits the coordination of cellular activities and plays key roles in the control of cell growth and differentiation and in the maintenance of tissue homoeostasis. After more than 50 years, deciphering the links among connexins, gap junctions and cancer, researchers are now beginning to translate this knowledge to the clinic. The emergence of new strategies for connexin targeting, combined with an improved understanding of the molecular bases underlying the dysregulation of connexins during cancer development, offers novel opportunities for clinical applications. However, different connexin isoforms have diverse channel-dependent and -independent functions that are tissue and stage specific. This can elicit both pro- and anti-tumorigenic effects that engender significant challenges in the path towards personalised medicine. Here, we review the current understanding of the role of connexins and gap junctions in cancer, with particular focus on the recent progress made in determining their prognostic and therapeutic potential.

Fig. 1

Connexins, connexons and gap junctions. a Connexins are tetraspanning integral membrane proteins with cytosolic C and N termini. Six connexins oligomerize to form a connexon. At the plasma membrane, the connexon can dock head-to-head with a connexon in an adjacent cell to form a gap junction intercellular channel. b Biosynthesis, intracellular trafficking and degradation of connexins. (1) Connexins are cotranslationally inserted into the endoplasmic reticulum. (2) A subpool of newly synthesised connexins undergo endoplasmic reticulum-associated degradation. (3) During their trafficking to the plasma membrane, connexins oligomerise into connexons. (4) After their arrival at the plasma membrane, the connexons can dock with connexons from adjacent cells to form gap junction channels. (5) Connexons may also form functional channels at the non-junctional areas of the plasma membrane (also known as hemichannels). (6) Gap junction endocytosis results in the formation of a connexosome (also known as annular gap junction). (7) The connexosome may be degraded by autophagy. (8) Alternatively, the connexosome may change its morphology to that of a connexin-enriched multivesicular endosome in a process that is associated with the fusion between the connexosome and early endosomes. (9) Connexins are sorted from early endosomes to lysosomes via late endosomes. (10) Under certain conditions, endocytosed connexons may undergo recycling to the plasma membrane. Hemichannels also undergo endocytosis and recycling, but their endocytic and recycling pathways are poorly characterised (question marks). (11) Multivesicular endosomes can fuse with the plasma membrane to secrete exosomes containing connexons. (12) Microvesicles containing connexons can be formed by the outward budding of the plasma membrane

Fig. 2

Dysregulation of connexins in cancer: therapeutic opportunities. Multiple stages of connexin life cycle are subject to dysregulation during cancer progression, as exemplified by GJA1 (Cx43). (1) Transcription: connexin expression is often reduced (but sometimes increased) in human tumours at the mRNA expression level, of which multiple pathways are therapeutic targets (text highlighted in red for key targets), including transcription factor activity and epigenetic silencing by histone acetylation and promoter methylation (promoter region in green, with C and M illustrating the non-methylated and methylated sites, respectively; blue, some important transcription factors regulating Cx43 expression). Histone acetylation can be modified by targeting histone acetyltransferase enzymes (HATs) or histone deacetylases (HDACs), typically promoting and repressing transcription, respectively. Transcriptional silencing due to promoter hypermethylation by DNA methyltransferase enzymes (DNMTs) may also be amenable to therapeutic intervention leading to the restoration of GJIC. (2) mRNA regulation: mRNA stability and translation is subject to regulation by multiple cancer-associated microRNAs. Moreover, alternative translation initiation, resulting in the synthesis of truncated forms of Cx43, might regulate Cx43 and have important implications for its dysregulation in cancer. This process is regulated by key cancer signalling pathways such as mTOR and Mnk1/2 and is altered during pathological conditions such as hypoxia. Truncated forms of Cx43, notably the 20-kDa form named GJA1–20k, may be important for the efficient targeting of Cx43 to the membrane. Indeed, Smad3/ERK-dependent repression of GJA1–20k was recently shown to reduce Cx43 gap junctions during epithelial-to-mesenchymal transition (EMT). (3) Post-translational regulation: connexins frequently display an aberrant localisation in cancer cells. Phosphorylation and other multiple post-translational events, occurring mainly at their C terminus, regulate connexin trafficking and stability at the plasma membrane. Cx43 is regulated by several kinases that are frequently overactivated or overexpressed during cancer development and susceptible to pharmacological inhibition, such as mitogen-activated protein kinase (MAPK), protein kinase C (PKC), protein kinase A (PKA), cdc2/cyclin B and v-src/c-src. Cx43 is also regulated by acetylation, ubiquitination and SUMOylation

Fig. 3

Interactions between connexins and proteins that affect tumour growth and migration. Examples of proteins that interact with specific regions of connexins and may act as therapy targets. a The interaction between Cx43 and tubulin is involved in the regulation of cell migration. Similar mechanisms have been proposed for other proteins associated with the cytoskeleton, such as cadherins, catenins, vinculin, ZO-1 and drebrin. In addition, Cx43 may compete with the tubulin–Smad2/3 interaction causing Smad2/3 release. Cx43 binds to c-Src and its endogenous inhibitors CSK and PTEN, promoting c-Src inhibition. Cx43, by interacting with β-catenin, prevents the transcriptional activity of β-catenin in the nucleus, where it regulates the expression of genes involved in promoting cell malignancy. A similar sequestration mechanism may occur with drebrin, ezrin or ZO-1. These proteins, and many others such as Nedd4, also have important roles in regulating Cx43 gap junction plaques, which influence GJIC and therefore may have therapeutic potential. b Cx26 has been proposed to maintain a cancer stem cell phenotype specifically in triple-negative breast cancer cells through its interaction with NANOG and focal adhesion kinase (FAK). c Cx32 binds to the scaffold protein discs large homologue 1 (Dlgh1) and may control cell proliferation in liver cells through its interaction with and maintenance of Dlgh1 at the plasma membrane. The interaction of Dlgh with Cx43 has also been associated with cancer progression through a mechanism involving the oncoprotein E6 (see section “Connexins and tumour viruses”). d Cx50 interacts with and promotes auto-ubiquitination and the subsequent degradation of Skp2, a key negative regulator of the cyclin-dependent kinase (CDK) inhibitor p27. a–d To complicate this scenario, the phosphorylation of connexins modifies their binding affinities to various protein partners. For instance, c-Src phosphorylation affects the binding of several Cx43 partners. GJIC, gap junction intercellular communication

Fig. 4

Connexins and the tumour stroma. GJIC can occur between cancer cells or in a heterocellular manner between cancer cells and nearby cells such as noncancerous epithelial tissue cells and stromal cells, including cancer-associated fibroblasts, immune cells and vascular and lymphatic endothelial cells. In addition, there is crosstalk via the hemichannel release of autocrine and paracrine signals. These signals influence tumour growth both positively and negatively in a context-dependent manner and help to regulate apoptosis, proliferation, invasion, intravasation and extravasation. In addition, connexins are thought to be implicated in other communication forms, as a part of tunnelling nanotubes (microtubes) or extracellular vesicle function (e.g. exosomes). Other tumours, or parts of tumours, are devoid of GJIC and may or may not express connexins at high levels in the cytoplasm or nucleus, thus escaping the direct GJIC with surrounding cells. This may be associated with a reduced polarity and cell–cell adhesion. The benefits and drawbacks of maintained GJIC are likely tissue and stage dependent. An understanding of this complex network of signals is essential to move forward with additional therapeutic strategies of targeting connexins in cancer. GJIC, gap junction intercellular communication