Targeting different domains of gap junction protein to control malignant glioma

Targeting different domains of gap junction protein to control malignant glioma Abstract A rational treatment strategy for glioma, the most common primary central nervous system tumor, should focus on early invasive growth and resistance to current therapeutics. Connexin 43 (Cx43), a gap junction protein, plays important roles not only in the development of the central nervous system and but also in the progression of glioma. The different structural domains of Cx43, including extracellular loops, transmembrane domains, and an intracellular carboxyl terminal, have distinct functions in the invasion and proliferation of gliomas. Targeting these domains of Cx43, which is expressed in distinct patterns in the heterogeneous glioma cell population, can inhibit tumor cell invasion and new tumor formation. Thus, this review summarizes the structural characteristics of Cx43, the effects of regulating different Cx43 domains on the biological characteristics of glioma cells, intervention strategies targeting different domains of Cx43, and future research directions. carboxyl terminal, channel structure, Cx43, glioma, therapeutics Introduction Glioma is the most common primary central nervous system tumor.1 A comprehensive glioma treatment strategy is based on surgical resection combined with chemotherapy and radiotherapy. However, progress to improve the clinical efficacy of glioma treatment remains slow. A more rational treatment strategy for glioma should consist of the following: ① the inhibition of glioma cell migration and invasion to reduce the infiltration of glioma into normal brain tissue and facilitate complete surgical resection; and ② the inhibition of chemoradiotherapy-resistant glioma stem cells (GSCs) that can repopulate the tumor to prevent glioma recurrence.2 Interestingly, recent studies have shown that targeting the different domains of connexin 43 (Cx43) can achieve these goals.3–6 Structural Features of Cx43 The family of connexins contains 21 members in humans, and this family is the basis of direct intercellular communication in many physiological processes.7–9 All connexins share the same topological structure with 4 transmembrane domains connected by the first and second extracellular loops and one cytoplasmic loop; the amino and carboxyl terminal domains are intracellular. Six connexins oligomerize into a connexon to form functional hemichannels at the plasma membrane. These hemichannels are normally closed, but they can open under pathological conditions and thus participate in the development of many diseases.10–12 Signaling molecules and metabolic products (ATP, NAD+, glutamate, glutathione, prostaglandin-E2, and glucose13) can pass through these hemichannels to influence the signal transduction and metabolic status of cells in an autocrine or paracrine manner. Between 2 adjacent cells, connexons connect end-to-end to establish gap junctional intercellular communication (GJIC) and mediate a more extensive exchange of molecules in various organs, including the central nervous system, heart, lens, liver, lung, retina, ear, kidney, testis, ovary, breast, bone, and skin.9,14 The Cx43 protein is encoded by the gap junction protein alpha 1 (GJA1) gene and has a molecular weight of approximately 43 kDa. Cx43 contains 9 structural domains, including 4 transmembrane segments, 2 extracellular loops, 1 amino terminus, 1 cytoplasmic loop, and 1 carboxyl terminus (CT). The hemichannels on the cell membrane and the GJIC between 2 adjacent cells are formed by transmembrane structures of Cx43 to allow the passage of small molecules (ions, second messengers, and metabolites), antigens, and microRNAs.9,15–23 In addition, the 2 extracellular loops of Cx43 play an important role in the formation of gap junctions (Fig. 1A).24 Fig. 1 View largeDownload slide Structural features and function of the Cx43 transmembrane structure and Cx43 carboxyl terminal. (A) At the plasma membrane, functional hemichannels are composed of 6 connexin subunits to allow mass exchange between the cell and the extracellular environment. Between 2 adjacent cells, connexons connect end-to-end to form gap junction structures that mediate a more extensive exchange of molecules (soluble second messengers, amino acids, nucleotides, Ca2+, glucose and its metabolites, nucleotides, amino acids, and peptides), which is termed gap junctional intercellular communication (GJIC). Abbreviations: ATP, adenosine triphosphate; IL-1β, interleukin-1 beta; IP3, inositol triphosphate; NAD, nicotinamide adenine dinucleotide; TNF-α, tumor necrosis factor alpha; cAMP, cyclic adenosine monophosphate. (B) The long carboxyl terminal chain of Cx43 contains a large number of protein-binding sites that interact with a variety of proteins and thereby regulate Cx43 or other proteins; thus, Cx43 exerts a broad range of biological functions. Abbreviations: CIP150, connexin 43-interacting protein 150; TCPTP, T-cell protein tyrosine phosphatase; Cdc2, cell division cycle 2; RPTPμ, receptor protein tyrosine phosphatase μ; MAPK, mitogen-activated protein kinase; NEDD4, neural precursor cell expressed developmentally downregulated protein 4; Hsc70, heat shock protein 70; CK1, casein kinase 1; CNN3/NOV, yr61/connective tissue growth factor/nephroblastoma-overexpressed 3; PKA, protein kinase A; ZO-1, zonula occludens-1. Fig. 1 View largeDownload slide Structural features and function of the Cx43 transmembrane structure and Cx43 carboxyl terminal. (A) At the plasma membrane, functional hemichannels are composed of 6 connexin subunits to allow mass exchange between the cell and the extracellular environment. Between 2 adjacent cells, connexons connect end-to-end to form gap junction structures that mediate a more extensive exchange of molecules (soluble second messengers, amino acids, nucleotides, Ca2+, glucose and its metabolites, nucleotides, amino acids, and peptides), which is termed gap junctional intercellular communication (GJIC). Abbreviations: ATP, adenosine triphosphate; IL-1β, interleukin-1 beta; IP3, inositol triphosphate; NAD, nicotinamide adenine dinucleotide; TNF-α, tumor necrosis factor alpha; cAMP, cyclic adenosine monophosphate. (B) The long carboxyl terminal chain of Cx43 contains a large number of protein-binding sites that interact with a variety of proteins and thereby regulate Cx43 or other proteins; thus, Cx43 exerts a broad range of biological functions. Abbreviations: CIP150, connexin 43-interacting protein 150; TCPTP, T-cell protein tyrosine phosphatase; Cdc2, cell division cycle 2; RPTPμ, receptor protein tyrosine phosphatase μ; MAPK, mitogen-activated protein kinase; NEDD4, neural precursor cell expressed developmentally downregulated protein 4; Hsc70, heat shock protein 70; CK1, casein kinase 1; CNN3/NOV, yr61/connective tissue growth factor/nephroblastoma-overexpressed 3; PKA, protein kinase A; ZO-1, zonula occludens-1. Another important structure of Cx43 is its long-chain CT. The Cx43 CT has a 156 amino acid elongated coil-like structure with a large number of protein-binding sites. Via these sites, Cx43 can interact with different proteins. These interactions affect the phosphorylation status of Cx43 and consequently regulate its degradation, subcellular localization, and assembly processes.25,26 In addition, the CT of Cx43 can regulate the functional status of other protein molecules, affecting downstream signal transduction and regulating the biological function of cells.9,10 It is worth noting that many of the cytoplasmic binding partners (eg, protein kinase C [PKC], c-Src, Hsc70) are key oncogenic pathway members that play important roles in the origin and development of tumors.9,27–29 Furthermore, the same region of the CT of Cx43 can selectively interact with different binding proteins, thereby competitively influencing the subcellular localization of these proteins.30,31 More interestingly, different proteins that interact with the CT of Cx43 can also influence each other. This interaction allows the CT of Cx43 to act as a platform or even a hub to integrate multiple signaling pathways, resulting in a broader regulatory effect (Fig. 1B).32–36 Through the above important domains, Cx43 is involved in the regulation of cell proliferation, migration, and other functions; thus, it plays an important role in physiological and pathological processes.9 Function of Cx43 in Gliomas In the early 1960s, tumor cells were shown to influence the cell membrane permeability of normal cells and reduce their intercellular communication. Furthermore, GJIC between tumor cells was nearly absent.37,38 The upregulation of some connexins can restore GJIC and inhibit tumor growth.39,40 Additional studies provided substantial evidence that connexins play important roles in tumor proliferation, apoptosis, invasion, and chemoresistance.41–44 More than 10 isoforms of connexins can be detected in the central nervous system.45,46 Among them, Cx43 is the most abundant member, and its expression level is correlated with the malignancy of glioma. Different grades of glioma show distinct levels of Cx43 protein.47,48 In addition, the role of Cx43 in the pathogenesis and development of glioma is complex49,50 and may differ by glioma cell subsets.3,47,51 Cx43 Inhibits the Expansion of the Glioma Cell Population and Tumor Initiation Many studies have shown that Cx43 negatively regulates the expansion of the glioma population and tumor initiation.29,49,50,52 As noted above, the different domains of Cx43 perform different functions. Thus, this protein regulates glioma cell proliferation and tumor initiation in a variety of ways. The structure of Cx43 consists of 2 extracellular loops and 4 transmembrane domains that allow the formation of gap junctions between neighboring cells, and the resulting gap junction–dependent effects play important roles in the regulation of cell proliferation through the transmission of growth-inhibitory53–56 and apoptotic factors.57–59 These signals derived from the microenvironment or adjacent cells can be delivered through the channel structure, thereby inhibiting the proliferation of glioma cells.54,59 For example, in C6 glioma cells overexpressing Cx43, transfection with cytochrome c (Cytc) can induce apoptosis, and cells in the apoptotic zone can propagate the apoptotic signal through the gap junction channels into adjacent cells that were not transfected with Cytc to expand the apoptosis cascade signal and inhibit glioma cell proliferation in a larger area.59 Cytc can also enter Cx43-expressing C6 glioma cells from the microenvironment in a hemichannel-dependent manner.59 Moreover, some pro-oncogenic factors can downregulate Cx43 and destroy gap junction structures to reduce inhibitory signal transmission; therefore, these factors play a role in promoting tumor cell proliferation and inhibiting tumor cell apoptosis. For example, the downregulation of basic fibroblast growth factor, an important pro-oncogenic growth factor in glioma cells, significantly increases Cx43 expression, restores GJIC, and inhibits glioma cell proliferation, suggesting that basic fibroblast growth factor can regulate the growth of glioma cells via Cx43.60 MicroRNA (miR)-221 and miR-222 are significantly upregulated in clinical glioma samples,61 and they can directly downregulate the level of Cx43 mRNA, thus preventing GJIC and promoting glioma cell proliferation.52 In addition, some exogenous carcinogenic viruses, such as human cytomegalovirus, can promote the degradation of Cx43 via the proteasome-dependent pathway after entering cells and thus inhibit GJIC to promote the proliferation of glioma cells and the expansion of glioma cell populations.62 Interestingly, after the transfection of nonphosphorylated Cx43-216 into glioma cells, although dye transfer capacity did not change significantly, cell expansion was inhibited.63 This finding suggested that another mechanism exists for Cx43 independent of its channel structure to regulate glioma proliferation63,64 and apoptosis.65 Furthermore, the administration of exogenous Cx43 CT was sufficient to inhibit the proliferation of glioma, thereby reversing its malignant phenotype.64 The combined evidence suggests that the CT of Cx43 may play an important role in inhibiting the expansion of the glioma population. As mentioned above, the CT of Cx43 includes many protein-binding sites through which it can affect the stability, activity, and phosphorylation of itself and other proteins via protein-protein interactions, thereby inhibiting glioma cell proliferation.25,28,29,66–68 Previously, all glioma cells in a tumor were considered equal, with the same or similar tumor-initiating potential. However, a growing body of evidence has shown that glioma consists of a heterogeneous stratified cell population. The subpopulation of GSCs with stem cell characteristics and resistance to chemotherapy and radiotherapy is thought to be the root cause of glioma initiation, expansion, and recurrence. Therefore, investigating the relationships between Cx43 and various subgroups of heterogeneous glioma has elevated importance. Interestingly, compared with differentiated glioma cells, a low expression level of Cx43 caused by promoter methylation51 and histone modification47 can be observed in the GSC subpopulation. When these GSCs are placed in serum-containing differentiation medium, the downregulation of the stem cell transcription factor sex determining region Y-box 2 (Sox2) and the upregulation of the differentiation marker glial fibrillary acidic protein are followed by a significant increase in Cx43 expression. In addition, when the expression of Cx43 in GSCs is restored, the activity of c is inhibited, and inhibitor of DNA binding 1 protein (Id-1) and Sox2 are downregulated; these changes inhibit the self-renewal and tumorigenic capacity of GSCs. Moreover, restoring Cx43 expression in GSCs causes a switch in expression from N-cadherin to E-cadherin.3,51 Cx43 can then form complexes with E-cadherin to inhibit the activation of the Wnt/β-catenin pathway and the transcription of its downstream target genes (such as Wnt3a, Atoh1, and POSIN) and stemness-related genes (such as Sox2, Nanog, and Oct4), thereby reducing GSC self-renewal and proliferation as well as tumor invasion and initiation.3,51 These results suggest that low Cx43 expression may be a stem cell characteristic of glioma cells. Because the GSC subpopulation is able to initiate a tumor in an immunodeficiency animal and is thought to drive the recurrence and the therapeutic resistance of glioma, low Cx43 expression likely plays a critical role in glioma development, cell population expansion, and recurrence. It should be noted that the results from Winkler are the opposite: Cx43 is highly expressed in GSCs.49 The reason for this difference may be that the molecular subtypes of the obtained GSCs are different. Using the dataset GSE67089, which contains the expression data of proneural-GSC, mesenchymal-GSC, and non-GSC subpopulations, we have analyzed the expression of Cx43 in these glioma subpopulations. As shown in Fig. 2, the expression of Cx43 (encoded by GJA1) in the mesenchymal-GSC subpopulation is very low or nearly absent, but it is fairly high in proneural-GSCs. Although none of the abovementioned 4 groups performed molecular typing of the GSCs they obtained, it is reasonable to speculate that Lathia,47 Gangoso,3 and Yu51 may have utilized mesenchymal-GSCs, and Winkler49 may have utilized proneural-GSCs. Fig. 2 View largeDownload slide The expression levels of Cx43 and Cx46 in different GBM subpopulations. The expression data of proneural-GSC (n = 18), mesenchymal-GSC (n = 12), and non-GSC (n = 10) are derived from the dataset GSE67089 (https://www.ncbi.nlm.nih.gov/geo) and are shown as mean ± SD. A 2-sided Student’s t-test was used to generate P-values, ****P < 0.0001, *P < 0.05. Abbreviations: PN-GSC, proneural-GSC; MES-GSC, mesenchymal-GSC; non-GSC, differentiated GBM cell. Fig. 2 View largeDownload slide The expression levels of Cx43 and Cx46 in different GBM subpopulations. The expression data of proneural-GSC (n = 18), mesenchymal-GSC (n = 12), and non-GSC (n = 10) are derived from the dataset GSE67089 (https://www.ncbi.nlm.nih.gov/geo) and are shown as mean ± SD. A 2-sided Student’s t-test was used to generate P-values, ****P < 0.0001, *P < 0.05. Abbreviations: PN-GSC, proneural-GSC; MES-GSC, mesenchymal-GSC; non-GSC, differentiated GBM cell. Cx43 Regulates Glioma Invasive Capacity Cx43 plays an important role in normal neuronal migration.69–72 By performing uteroelectroporation with Cx43 short hairpin (sh)RNA plasmids or crossing nestin-Cre transgenic mice with Cx43-floxed mice, the downregulation of Cx43 was shown to significantly delay the migration of neurons during embryonic development.70 When excitatory vertebral neurons migrate radially along radial glial fibers, Cx43 is highly expressed at the contact points between the migrating neurons and radial fibers, providing dynamic adhesion for cell migration. Cx43 also interacts with intracellular cytoskeletal proteins at these points to enable stable cell migration along the radial fibers.69 Cx43 may serve as a regulator to promote the tangential or radial migration of inhibitory interneurons and assist their precise localization during cortical plate entry.71 Meanwhile, Cx43 exerts complex effects on glioma cell migration and invasion via different domains (channel structure, extracellular loop, and CT) (Fig. 3) (reviewed by Sin et al50,72 and Kameritsch et al73). Fig. 3 View largeDownload slide Cx43 structural pattern and targeted therapy strategies. The second extracellular loop (E2) of Cx43 is the connexon component that shares the least homology with other connexins. Thus, it is a suitable target against which to design specific monoclonal antibodies to inhibit the connexon structure, reduce gap junction communication, and consequently reduce glioma cell invasion. The long CT contains several protein-binding sites. Mimic peptides can be designed to target specific protein-binding sites in the CT, such as the 245–283 amino acid region that binds specifically to c-Src, and these peptides can be linked to transmembrane sequences (such as TAT). Under the guidance of TAT, mimic peptides can enter the cell and bind to c-Src to prevent its activation, thereby inhibiting glioma cell proliferation. Abbreviations: N-term, amino terminus of Cx43; MAbE2Cx43, monoclonal antibodies against the E2 extracellular loop of connexin 43; E1/E2, Cx43 first/second extracellular fragment; C-term (COOH), CT of Cx43; TAT, transactivator of transcription cell penetrating sequence. Fig. 3 View largeDownload slide Cx43 structural pattern and targeted therapy strategies. The second extracellular loop (E2) of Cx43 is the connexon component that shares the least homology with other connexins. Thus, it is a suitable target against which to design specific monoclonal antibodies to inhibit the connexon structure, reduce gap junction communication, and consequently reduce glioma cell invasion. The long CT contains several protein-binding sites. Mimic peptides can be designed to target specific protein-binding sites in the CT, such as the 245–283 amino acid region that binds specifically to c-Src, and these peptides can be linked to transmembrane sequences (such as TAT). Under the guidance of TAT, mimic peptides can enter the cell and bind to c-Src to prevent its activation, thereby inhibiting glioma cell proliferation. Abbreviations: N-term, amino terminus of Cx43; MAbE2Cx43, monoclonal antibodies against the E2 extracellular loop of connexin 43; E1/E2, Cx43 first/second extracellular fragment; C-term (COOH), CT of Cx43; TAT, transactivator of transcription cell penetrating sequence. Despite alternate opinions,74 many results have shown that heterocellular GJIC between glioma and astrocytes or endothelial cells promotes the migration and invasion of glioma. Different substrates including microRNAs and Ca++ can be transferred through these heterocellular channel structures formed by Cx43.75–78 For instance, the inhibition of GJIC reduces the invasive capacity of glioma cells in fresh human glioma biopsy slice cultures.77 After being implanted into the brains of mice, Cx43-expressing glioma cells established functional GJIC with host astrocytes and displayed a more invasive phenotype compared with mock and mutant Cx43-transfected cells.75 However, the roles in the migration and invasion of glioma performed by homocellular GJIC between human glioma cells are more complicated. After the knockdown of Cx43 or the treatment with the gap junction inhibitor 18α-GA, GJIC between adjacent U87MG glioma cells was eliminated as assessed by dye coupling. More importantly, the invasive capacity of these cells increased significantly.78 The same results were found by Aftab et al, that the Cx43-mediated homocellular GJIC between adjacent glioma cells causes these glioma cells to migrate in a slower, more clustered manner.79 The destruction of this adjacent homocellular GJIC promotes the glioma cells to migrate in a faster, single-cell manner.79 It is noteworthy that despite the antimigration effect mediated by adjacent homocellular GJIC, the long-distance information transfer through tumor microtubes (TMs),49,80 which is also dependent on the expression of Cx43 at the intersection of TMs, plays a significant role in promoting glioma invasion. In the peritumoral area, many astrocytoma and GBM cells can form long and stable TMs and cross-link with each other to form a multicellular microtube network that promotes glioma cell migration,49 proliferation, and chemoresistance.80 The expression of Cx43 is particularly high at the junctions of this TM network, and calcium waves can propagate via these crossings from one TM to another. The inhibition of Cx43 expression significantly decreases the long-term stabilization of TMs and the tumor volume while significantly prolonging the survival time of mice harboring glioma.49 Interestingly, this TM-like structure also exists between glioma cells and astrocytes.81 In addition to the channel structure–dependent GJIC, the adhesive function mediated by the extracellular loop of Cx43 plays an important role in the regulation of glioma cell invasion (reviewed by Kameritsch et al73). In vitro cultured glioma cells expressing Cx43 aggregated with astrocytes, whereas connexin-free control cells aggregated to a lesser extent.75 Antibodies against the extracellular loops of Cx43 significantly reduced this aggregation between Cx43-expressing glioma cells and astrocytes.75 Intracellular free C-terminal Cx43 protein also affects glioma cell invasion,74 especially in glioma cells expressing low levels of Cx43 primarily in the cytoplasm or around the nucleus, such as GL261 and LN18.82–84 The CT domain of Cx43 contains a large number of phosphorylation sites that can interact with cytoskeletal proteins, such as α-tubulin, β-tubulin, actin, development regulatory brain protein (drebrin), and cortactin.73,85–87 By interacting with these cytoskeletal proteins, the Cx43 CT can regulate the cytoskeleton and alter cellular structures, such as pseudopodia formation, to promote cell migration.83 More interestingly, when the Cx43 channel structure and CT are simultaneously expressed at high levels, cells exhibit migration that is regulated mainly by the Cx43 channel structure.79 The shRNA-mediated downregulation of full-length Cx43 and the simultaneous transduction of the TrCx43 mutant (it can form the channel structure but lacks many phosphorylation and protein-protein interaction sites due to the truncation of the Cx43 tail at amino acid 242) do not significantly affect the migration of U118 cells.79 However, glioma cells transfected with both Cx43 shRNA and the Cx43 T154A mutant (a dominant negative channel mutant that can block gap junction intercellular communication) exhibit a significantly higher migration rate compared with wild-type glioma cells.79 In short, glioma cells expressing high levels of Cx43 in the peritumoral region in vivo utilize the Cx43 channel structure to form membrane TM structures or heterocellular gap junctions with adjacent astrocytes to promote their own migration, whereas the inhibition of the channel structure can significantly inhibit the invasion of glioma cells. However, for glioma cells expressing low levels of Cx43, which are located at the center of the tumor, the channel structure and the extracellular loop–mediated antimigration effects and Cx43 CT–mediated pro-migration effects were weakened at the same time. Thus, these glioma cells migrated in a faster, single cell manner. As mentioned above, the GSCs of the heterogeneous glioma population express low levels of Cx43, whereas Cx43 expression is relatively high in differentiated glioma cells. Thus, we can speculate that differentiated glioma cells expressing high levels of Cx43 can form heterocellular gap junctions with adjacent astrocytes and long-distance membranous microtube network structures at the edge of the glioma, invade the surrounding area as clusters, and provide support to GSCs (which express low levels of Cx43). Conversely, GSCs expressing low levels of Cx43 may utilize single-cell invasion, which is faster, but they may require the support of TM structures formed by differentiated glioma cells expressing high levels of Cx43 or the guidance of other support structures, such as the ependyma, blood vessels, and white matter fibers. Osswald et al found that the TM networks consisting of glioma cells expressing high levels of Cx43 included small quantities of GSCs that remained stationary for a prolonged time and did not form significant membranous microtube connections with the surrounding glioma cells expressing high levels of Cx43.49 Therefore, the Cx43 channel structure plays a key role in glioma cell invasion, and the destruction or inhibition of this channel structure may be an effective method for the inhibition of glioma cell invasion. Treatment Strategy for Targeting Cx43 In summary, the inhibition of Cx43 expression in invading glioma cell clusters at the periphery of gliomas can reduce the invasive capacity of glioma cells and may be beneficial for surgical resection. The upregulation of Cx43 in GSCs can inhibit GSC self-renewal, which inhibits the proliferation and expansion of the glioma cell population. Thus, combining the 2 strategies may be a promising approach for glioma treatment (Fig. 4). Fig. 4 View largeDownload slide Cx43 plays an important role in glioma cell invasion. Many glioma cells at the periphery of a glioma express high levels of Cx43. These cells connect with each other to form a membranous microtube network structure and promote the migration of glioma cells. A small number of quiescent GSCs expressing low levels of Cx43 are present in these membranous microtube network structures, and these cells may migrate with the help of the membranous microtube structures. The distal region of tumor foci contains small numbers of GSCs expressing low levels of Cx43, and these cells exhibit a scattered distribution along the structure of white matter fibers and constitute the origins of distant glioma recurrence. Fig. 4 View largeDownload slide Cx43 plays an important role in glioma cell invasion. Many glioma cells at the periphery of a glioma express high levels of Cx43. These cells connect with each other to form a membranous microtube network structure and promote the migration of glioma cells. A small number of quiescent GSCs expressing low levels of Cx43 are present in these membranous microtube network structures, and these cells may migrate with the help of the membranous microtube structures. The distal region of tumor foci contains small numbers of GSCs expressing low levels of Cx43, and these cells exhibit a scattered distribution along the structure of white matter fibers and constitute the origins of distant glioma recurrence. Antibody Treatment Targeting the Cx43 Channel Structure in Glioma Cells Based on the features of glioma invasion at the leading edge—① glioma cells form TM structures via Cx43 channels to promote migration and ② glioma cells and adjacent astrocytes or endothelial cells interact via Cx43 channels to promote invasion—inhibiting Cx43 channel structures at the edge of the glioma is a viable treatment strategy.88 The antibody MAbE2Cx43 targeting the second extracellular loop of the Cx43 was generated. This antibody disrupts the functional gap junction between cells by specifically binding to the extracellular loop of Cx43 (Fig. 4).89,90 Isotope-labeled MAbE2Cx43 entered the rat brain 72 hours after being injected into glioma-bearing rats, and their concentration was much higher in the right brain, which harbored a glioma, compared with the left normal brain (0.272% vs 0.005%). This antibody was specifically enriched in brain tissue around the glioma, and a further analysis of the distribution of fluorescence-labeled antibodies showed that MAbE2Cx43 was primarily distributed in peritumoral glioma cells and reactive astrocytes (ie, at the glioma invasion leading edge).88,89 More importantly, MAbE2Cx43 alone or in combination with other drugs can prolong the survival of glioma-bearing animals. Specifically, after treatment with a single dose of MAbE2Cx43 (5 mg/kg), the median survival time of C6 tumor-bearing rats was 39.5 days, which is higher than that of rats treated with a single dose of temozolomide (TMZ) (median survival time, 34 days) or radiotherapy alone (36 Gy, median survival time, 38 days). Overall, MAbE2Cx43 combined with radiotherapy prolonged survival to 60 days.4 In addition, because Cx43 is overexpressed in glioma cells at the leading edge of invasion, Cx43 monoclonal antibodies (mAbs) can be used to treat gliomas in combination with cisplatin nanogel. In this approach, cisplatin is enriched in tissues at the periphery of gliomas, which enhances the efficacy and prolongs survival time. Furthermore, the concentration of cisplatin in normal tissue is decreased, which reduces side effects. The median survival time of the glioma-bearing animals was prolonged by treatment with cisplatin alone (31 days for the free cisplatin-treated group vs 15 days for the control group), but signs of toxicity in the mice treated with cisplatin were significant. Conversely, the MAbE2Cx43 antibody combined with cisplatin nanogel not only increased the median survival time compared with the cisplatin group (42 days for the group treated with cisplatin nanogels with Cx43 mAbs vs 31 days for the free cisplatin-treated group) but also reduced cisplatin toxicity.5,91 Targeted Treatment for GSCs with Low Expression of Cx43 Although GSCs are not numerous and account for only a small proportion of the glioma cell population, they can self-renew, exhibit multipotency, are resistant to TMZ chemotherapy, and can mediate the repopulation process. These cells are considered to be the root cause of glioma chemotherapy failure and malignant recurrence.2 Therefore, strategies that target GSCs are an important direction of glioma treatment to be explored. As mentioned above, Cx43 expression is much lower in GSCs than in differentiated glioma cells and is closely correlated with the malignant phenotype of GSCs. Moreover, the upregulation of Cx43 expression in GSCs inhibits GSC self-renewal and promotes GSC differentiation. In GSCs, the Wnt/β-catenin signaling pathway is overactive and plays an important role in GSC self-renewal and the maintenance of malignant phenotypes.92 The upregulation of Cx43 in GSCs can also promote the complexation of Cx43 with E-cadherin, which reduces the activity of the Wnt/β-catenin signaling pathway and inhibits the self-renewal and other malignant phenotypes of GSCs.51 In addition, the upregulation of Cx43 may also promote GSC differentiation, resulting in the loss of GSC properties and the attenuation of their tumorigenicity.3 The therapeutic significance of Cx43 upregulation in the targeted treatment of cancer stem cells has been shown for other types of tumors; the small molecule sulforaphane can upregulate Cx43 expression via posttranslational regulatory mechanisms, and upregulated Cx43 can suppress tumor stem cell markers (c-Met and CD133), improve GJIC, and ultimately inhibit the malignant phenotype of pancreatic ductal cancer stem cells.93 This behavior suggests that Cx43 may serve as a candidate for targeted GSC therapy.47,51 Because the Cx43 CT plays a central role in suppressing the expansion of the glioma cell population and tumor repopulation, directly introducing the Cx43 CT into GSCs, which express low levels of Cx43 or no Cx43, may exert an important therapeutic effect. To this end, Gangoso et al synthesized a mimetic peptide containing amino acids 245–283 of the Cx43 CT and a transmembrane sequence of HIV.3 This mimetic peptide can enter GSCs and specifically inhibit c-Src Tyr-416 (Y416 c-Src) phosphorylation, thereby reversing the malignant phenotype of GSCs, which endogenously express low levels of Cx43 (Fig. 4).3 Specifically, this peptide inhibits Id-1 and Sox2 expression, causes a switch in expression from N-cadherin to E-cadherin, reduces GSC spheroid formation, and facilitates the differentiation of GSC into oligodendrocyte cells (O4 upregulation).3 Discussion and Perspectives Based on the important role of Cx43 in gliomas, a treatment strategy targeting the different domains of Cx43 warrants further exploration. Glioma cells at the peritumoral invasion front express high levels of Cx43, which can form heterocellular channels and a special TM network that facilitates the migration of glioma cells and may even provide structural support for the invasion of GSCs. Monoclonal antibodies designed against the extracellular domain of Cx43 can effectively block the intercellular channel–mediated communication among glioma cells and between glioma cells and normal brain tissue cells, thus inhibiting glioma invasion. In addition, Cx43 utilizes a large number of protein-binding sites on its CT to regulate its stability, activity, and phosphorylation status and to regulate other proteins via protein-protein interactions; these activities negatively regulate glioma cell population expansion and proliferation. However, Cx43 expression is low or absent in GSCs, the key subgroup regulating tumor population expansion and reconstruction. Therefore, introducing a Cx43-CT mimetic peptide into the GSC cell subset with low or absent Cx43 expression may inhibit these cells and prevent the expansion of the glioma cell population. However, the strategies described here targeting different domains of Cx43 use either antibodies or mimetic peptides to achieve therapeutic purposes, and the application of these therapeutics in the clinic is inconvenient and limited. Future research should focus on integrating these 2 strategies. For example, we can construct a therapeutic fusion peptide consisting of a single-chain soluble antibody against the Cx43 extracellular domain + linker sequence (which can be cleaved by matrix metalloproteinases) + Cx43-CT mimetic peptide + transmembrane sequence (Fig. 5). When a therapeutic fusion peptide reaches the glioma, it can be cleaved into 2 segments by local high concentrations of matrix metalloproteinases. For Cx43-expressing glioma cells, the single-domain soluble antibody moiety binds to the extracellular domain of Cx43, destroying the heterocellular gap junction channel and inhibiting TM network–mediated invasion. For the GSC subgroup expressing low levels of Cx43 or in which Cx43 expression is absent, the Cx43-CT mimetic peptide can enter cells under the guidance of the transmembrane sequence and bind to c-Src, Hsc70, and PKC, thus inhibiting the proliferation and self-renewal and other malignant phenotypes of GSCs. Our lab is performing these experiments. Moreover, we are also using a similar strategy to create chimeric antigen receptor (CAR)–modified T cells (Fig. 6) in an effort to apply this strategy for the treatment of glioma. However, before solving the problem of fusion peptides or new CAR T cells passing through the blood–brain barrier to reach brain lesions, intraoperative or intrathecal administration may be a method worth trying. In addition, the disruption of glioma connexins by small molecules, as summarized by Osswald et al, is also a promising strategy.94 The orally bioavailable modulators of gap junctions meclofenamate and tonabersat disrupt the astrocyte gap junctional network and attenuate established brain metastasis.95 Fig. 5 View largeDownload slide Schematics of the therapeutic fusion peptide for the treatment of glioma. This fusion peptide consists of a single-domain soluble antibody recognizing the Cx43 extracellular domain linked to a mimetic peptide of the Cx43 CT, which is followed by a transmembrane sequence in which the linker comprises a specific amino acid sequence cleavable by matrix metalloproteinases. When this fusion peptide enters the body, it can be cleaved into 2 parts by endogenous matrix metalloproteinases generated locally in tumors. The first part is the single-domain soluble antibody recognizing the Cx43 extracellular domain, which can bind specifically to the Cx43 extracellular domain in glioma cells expressing high levels of Cx43 to destroy the gap junction channel and thereby inhibit glioma invasion mediated by the membrane microtube structure. The second fragment is the Cx43-CT mimetic peptide with a transmembrane sequence, which can enter glioma cell subsets (such as GSCs) expressing low levels of Cx43 under the guidance of the transmembrane sequence and mimic the function of the endogenous Cx43 CT to inhibit the proliferation and self-renewal of cells. Abbreviations: scFv, single chain antibody fragment; TAT, transactivator of transcription cell penetrating sequence. Fig. 5 View largeDownload slide Schematics of the therapeutic fusion peptide for the treatment of glioma. This fusion peptide consists of a single-domain soluble antibody recognizing the Cx43 extracellular domain linked to a mimetic peptide of the Cx43 CT, which is followed by a transmembrane sequence in which the linker comprises a specific amino acid sequence cleavable by matrix metalloproteinases. When this fusion peptide enters the body, it can be cleaved into 2 parts by endogenous matrix metalloproteinases generated locally in tumors. The first part is the single-domain soluble antibody recognizing the Cx43 extracellular domain, which can bind specifically to the Cx43 extracellular domain in glioma cells expressing high levels of Cx43 to destroy the gap junction channel and thereby inhibit glioma invasion mediated by the membrane microtube structure. The second fragment is the Cx43-CT mimetic peptide with a transmembrane sequence, which can enter glioma cell subsets (such as GSCs) expressing low levels of Cx43 under the guidance of the transmembrane sequence and mimic the function of the endogenous Cx43 CT to inhibit the proliferation and self-renewal of cells. Abbreviations: scFv, single chain antibody fragment; TAT, transactivator of transcription cell penetrating sequence. Fig. 6 View largeDownload slide Schematic representation of a Cx43 fusion peptide CAR-modified T cell killing a glioma cell. This CAR consists of the Cx43 carboxyl-terminal mimetic peptide connected to a transmembrane peptide, which is linked to a single-domain soluble antibody recognizing the second extracellular loop (E2) of Cx43 via a linker (containing a matrix metalloproteinase-specific cleavage site) plus the T-cell transmembrane structure TM (CD28) and intracellular cascade signal 4-1BB-CD3zeta. After T cells modified by this receptor enter the body, the receptor can be digested into 2 parts by the local high concentrations of endogenous matrix metalloproteinases in the tumor. After the Cx43 carboxyl-terminal mimetic peptide containing the transmembrane sequence (TAT) is released from the T-cell surface, it enters glioma cell subpopulations expressing low levels of Cx43, such as GSCs, under the guidance of the transmembrane sequence. The peptide mimics the function of the endogenous Cx43 CT to inhibit GSC proliferation and self-renewal; the Cx43-E2 single-domain CAR remains on the T-cell surface, directing T cells to specifically recognize glioma cells expressing high levels of Cx43 and transducing the signal into T cells via the transmembrane (TM) structure. This signal activates the intracellular signaling cascade 4-1BB-CD3zeta, which triggers the targeted killing of glioma cells expressing high levels of Cx43 by T cells. Abbreviations: Cx43 CT, CT of Cx43; Cx43-E2 single chain antibody, single chain antibody against the E2 extracellular loop of connexin 43; VH, heavy chain; VL, light chain; TM, transmembrane region; TAT, transactivator of transcription cell penetrating sequence. Fig. 6 View largeDownload slide Schematic representation of a Cx43 fusion peptide CAR-modified T cell killing a glioma cell. This CAR consists of the Cx43 carboxyl-terminal mimetic peptide connected to a transmembrane peptide, which is linked to a single-domain soluble antibody recognizing the second extracellular loop (E2) of Cx43 via a linker (containing a matrix metalloproteinase-specific cleavage site) plus the T-cell transmembrane structure TM (CD28) and intracellular cascade signal 4-1BB-CD3zeta. After T cells modified by this receptor enter the body, the receptor can be digested into 2 parts by the local high concentrations of endogenous matrix metalloproteinases in the tumor. After the Cx43 carboxyl-terminal mimetic peptide containing the transmembrane sequence (TAT) is released from the T-cell surface, it enters glioma cell subpopulations expressing low levels of Cx43, such as GSCs, under the guidance of the transmembrane sequence. The peptide mimics the function of the endogenous Cx43 CT to inhibit GSC proliferation and self-renewal; the Cx43-E2 single-domain CAR remains on the T-cell surface, directing T cells to specifically recognize glioma cells expressing high levels of Cx43 and transducing the signal into T cells via the transmembrane (TM) structure. This signal activates the intracellular signaling cascade 4-1BB-CD3zeta, which triggers the targeted killing of glioma cells expressing high levels of Cx43 by T cells. Abbreviations: Cx43 CT, CT of Cx43; Cx43-E2 single chain antibody, single chain antibody against the E2 extracellular loop of connexin 43; VH, heavy chain; VL, light chain; TM, transmembrane region; TAT, transactivator of transcription cell penetrating sequence. The channel function of Cx43 is primarily mediated by the 4 transmembrane domains and 2 extracellular loops. In addition to targeting the second extracellular loop, studies need to be performed to determine the effects on Cx43 channel function of targeting the 4 transmembrane structures and the first extracellular loop. In addition, the Cx43 CT, which interacts with c-Src, E-cadherin, zonula occludens 1, cytoskeleton proteins, Hsc70, and PKC, contains many protein-binding sites. Our previous study showed that some sequences of the Cx43 CT could specifically bind to protein kinase B (Akt) and extracellular signal-regulated kinase (ERK) 1 and 2, thus inhibiting Akt and ERK1/2 phosphorylation in GSCs. This finding suggests that the CT of Cx43 can act as a platform for the accumulation of proteins in various signaling pathways, resulting in more complex interactions and a wider regulation of cells.9,32–34,96 Future studies need to explore strategies to more rationally inhibit GSCs by utilizing the large number of protein-binding sites on the Cx43 CT. Of course, such studies require a more in-depth understanding of the mechanisms mediating interactions with the Cx43 CT. Assessing the expression and functional status of Cx43 in the GSC subpopulation relies mainly on surface makers—for example, CD133. However, it should be noted that CD133 is not only expressed in GSCs but can also be expressed in other cells, such as normal neural stem cells. A series of relatively specific markers of GSCs,97,98 such as PBK, CENPA, KIF15, DEPDC1, CDC6, DLG7, KIF18A, EZH2, HMMR, and cadherin-19, which have been identified recently, should be used to investigate the expression and functional status of gap junction proteins in heterogeneous glioma cell populations. In addition, compared with the data generated from primary culture models (primary glioblastoma cells kept in serum-free medium, neurosphere-forming conditions), data from glioma cell line models (U251, U87, C6, etc, kept in serum-containing medium, monolayer conditions) should be presented very differently. These culture conditions might actually explain much of the contradictory findings regarding the relationship between Cx43 and glioma invasiveness. In future studies, the simultaneous detection of the expression and the functional status of gap junction proteins in glioma cell lines, primary glioma cells, human specimens, and mouse spontaneous tumors, as a recent series of studies have performed, may be a better strategy.49,95 In addition to Cx43, other connexins, especially Cx46, play important roles in the development and progression of glioma.47,99,100 The expression of Cx46 was negatively correlated with Cx43 expression in gliomas. The expression of Cx46 is high in GSCs, whereas Cx43 is low in this subpopulation; in contrast, in the non-GSC subgroup with high Cx43 expression, Cx46 expression was low or absent. More importantly, the expression of Cx46 and the GJIC mediated by Cx46 were closely related to the self-renewal and tumorigenicity of GSCs, and the survival time of patients with high Cx46 expression was significantly shortened.47 Therefore, the combined targeting of different connexins is worth exploring in the future. However, as shown in Fig. 2, the expression levels of Cx43 and Cx46 might be different in distinct GSCs; thus, the molecular subtypes of GSCs need to be evaluated prior to the application of this strategy. In addition, the gap junction channels formed by different connexins have obvious selectivity for the passage of substances. For example, adenosine passage is approximately 12-fold greater through channels formed by Cx32 compared with channels formed by Cx43. In contrast, ATP passage is 300-fold greater through channels formed by Cx43.101 Thus, the therapeutic implications of this selectivity are worth considering in the development of connexin-targeting strategies for glioma therapy. In summary, Cx43 is a multidomain transmembrane protein involved in the multifaceted regulation of many biological characteristics of gliomas via the channel structures formed by Cx43 and the long CT of Cx43. We believe that these findings and progress from related research will result in strategies targeting Cx43 that have promising applications in the clinical treatment of glioma. Funding This study was supported by grants from the National Key Research and Development Program of China (2016YFA0202104 to S-C.Y.), the National Natural Science Foundation of China (81572880 and 81172071 to S-C.Y., 81302193 to Q-K.Y.), the Outstanding Youth Science Foundation of Chongqing (CSTC2013JCYJJQ10003 to S-C.Y.), the Postgraduate Education Foundation of Chongqing (YJG153062 to S-C.Y.), and the Key Clinical Research Program of Southwest Hospital (SWH2016ZDCX1005 to S-C.Y.). Acknowledgments We thank Miss Ying Ji for assistance with preparing schematic diagrams. Conflict of interest statement The authors declare that they have no conflicts of interest. References 1. Siegel R , Naishadham D , Jemal A . Cancer statistics, 2013 . CA Cancer J Clin . 2013 ; 63 ( 1 ): 11 – 30 . 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Google Scholar CrossRef Search ADS PubMed © The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Neuro-Oncology Oxford University Press

Targeting different domains of gap junction protein to control malignant glioma

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Abstract

Abstract A rational treatment strategy for glioma, the most common primary central nervous system tumor, should focus on early invasive growth and resistance to current therapeutics. Connexin 43 (Cx43), a gap junction protein, plays important roles not only in the development of the central nervous system and but also in the progression of glioma. The different structural domains of Cx43, including extracellular loops, transmembrane domains, and an intracellular carboxyl terminal, have distinct functions in the invasion and proliferation of gliomas. Targeting these domains of Cx43, which is expressed in distinct patterns in the heterogeneous glioma cell population, can inhibit tumor cell invasion and new tumor formation. Thus, this review summarizes the structural characteristics of Cx43, the effects of regulating different Cx43 domains on the biological characteristics of glioma cells, intervention strategies targeting different domains of Cx43, and future research directions. carboxyl terminal, channel structure, Cx43, glioma, therapeutics Introduction Glioma is the most common primary central nervous system tumor.1 A comprehensive glioma treatment strategy is based on surgical resection combined with chemotherapy and radiotherapy. However, progress to improve the clinical efficacy of glioma treatment remains slow. A more rational treatment strategy for glioma should consist of the following: ① the inhibition of glioma cell migration and invasion to reduce the infiltration of glioma into normal brain tissue and facilitate complete surgical resection; and ② the inhibition of chemoradiotherapy-resistant glioma stem cells (GSCs) that can repopulate the tumor to prevent glioma recurrence.2 Interestingly, recent studies have shown that targeting the different domains of connexin 43 (Cx43) can achieve these goals.3–6 Structural Features of Cx43 The family of connexins contains 21 members in humans, and this family is the basis of direct intercellular communication in many physiological processes.7–9 All connexins share the same topological structure with 4 transmembrane domains connected by the first and second extracellular loops and one cytoplasmic loop; the amino and carboxyl terminal domains are intracellular. Six connexins oligomerize into a connexon to form functional hemichannels at the plasma membrane. These hemichannels are normally closed, but they can open under pathological conditions and thus participate in the development of many diseases.10–12 Signaling molecules and metabolic products (ATP, NAD+, glutamate, glutathione, prostaglandin-E2, and glucose13) can pass through these hemichannels to influence the signal transduction and metabolic status of cells in an autocrine or paracrine manner. Between 2 adjacent cells, connexons connect end-to-end to establish gap junctional intercellular communication (GJIC) and mediate a more extensive exchange of molecules in various organs, including the central nervous system, heart, lens, liver, lung, retina, ear, kidney, testis, ovary, breast, bone, and skin.9,14 The Cx43 protein is encoded by the gap junction protein alpha 1 (GJA1) gene and has a molecular weight of approximately 43 kDa. Cx43 contains 9 structural domains, including 4 transmembrane segments, 2 extracellular loops, 1 amino terminus, 1 cytoplasmic loop, and 1 carboxyl terminus (CT). The hemichannels on the cell membrane and the GJIC between 2 adjacent cells are formed by transmembrane structures of Cx43 to allow the passage of small molecules (ions, second messengers, and metabolites), antigens, and microRNAs.9,15–23 In addition, the 2 extracellular loops of Cx43 play an important role in the formation of gap junctions (Fig. 1A).24 Fig. 1 View largeDownload slide Structural features and function of the Cx43 transmembrane structure and Cx43 carboxyl terminal. (A) At the plasma membrane, functional hemichannels are composed of 6 connexin subunits to allow mass exchange between the cell and the extracellular environment. Between 2 adjacent cells, connexons connect end-to-end to form gap junction structures that mediate a more extensive exchange of molecules (soluble second messengers, amino acids, nucleotides, Ca2+, glucose and its metabolites, nucleotides, amino acids, and peptides), which is termed gap junctional intercellular communication (GJIC). Abbreviations: ATP, adenosine triphosphate; IL-1β, interleukin-1 beta; IP3, inositol triphosphate; NAD, nicotinamide adenine dinucleotide; TNF-α, tumor necrosis factor alpha; cAMP, cyclic adenosine monophosphate. (B) The long carboxyl terminal chain of Cx43 contains a large number of protein-binding sites that interact with a variety of proteins and thereby regulate Cx43 or other proteins; thus, Cx43 exerts a broad range of biological functions. Abbreviations: CIP150, connexin 43-interacting protein 150; TCPTP, T-cell protein tyrosine phosphatase; Cdc2, cell division cycle 2; RPTPμ, receptor protein tyrosine phosphatase μ; MAPK, mitogen-activated protein kinase; NEDD4, neural precursor cell expressed developmentally downregulated protein 4; Hsc70, heat shock protein 70; CK1, casein kinase 1; CNN3/NOV, yr61/connective tissue growth factor/nephroblastoma-overexpressed 3; PKA, protein kinase A; ZO-1, zonula occludens-1. Fig. 1 View largeDownload slide Structural features and function of the Cx43 transmembrane structure and Cx43 carboxyl terminal. (A) At the plasma membrane, functional hemichannels are composed of 6 connexin subunits to allow mass exchange between the cell and the extracellular environment. Between 2 adjacent cells, connexons connect end-to-end to form gap junction structures that mediate a more extensive exchange of molecules (soluble second messengers, amino acids, nucleotides, Ca2+, glucose and its metabolites, nucleotides, amino acids, and peptides), which is termed gap junctional intercellular communication (GJIC). Abbreviations: ATP, adenosine triphosphate; IL-1β, interleukin-1 beta; IP3, inositol triphosphate; NAD, nicotinamide adenine dinucleotide; TNF-α, tumor necrosis factor alpha; cAMP, cyclic adenosine monophosphate. (B) The long carboxyl terminal chain of Cx43 contains a large number of protein-binding sites that interact with a variety of proteins and thereby regulate Cx43 or other proteins; thus, Cx43 exerts a broad range of biological functions. Abbreviations: CIP150, connexin 43-interacting protein 150; TCPTP, T-cell protein tyrosine phosphatase; Cdc2, cell division cycle 2; RPTPμ, receptor protein tyrosine phosphatase μ; MAPK, mitogen-activated protein kinase; NEDD4, neural precursor cell expressed developmentally downregulated protein 4; Hsc70, heat shock protein 70; CK1, casein kinase 1; CNN3/NOV, yr61/connective tissue growth factor/nephroblastoma-overexpressed 3; PKA, protein kinase A; ZO-1, zonula occludens-1. Another important structure of Cx43 is its long-chain CT. The Cx43 CT has a 156 amino acid elongated coil-like structure with a large number of protein-binding sites. Via these sites, Cx43 can interact with different proteins. These interactions affect the phosphorylation status of Cx43 and consequently regulate its degradation, subcellular localization, and assembly processes.25,26 In addition, the CT of Cx43 can regulate the functional status of other protein molecules, affecting downstream signal transduction and regulating the biological function of cells.9,10 It is worth noting that many of the cytoplasmic binding partners (eg, protein kinase C [PKC], c-Src, Hsc70) are key oncogenic pathway members that play important roles in the origin and development of tumors.9,27–29 Furthermore, the same region of the CT of Cx43 can selectively interact with different binding proteins, thereby competitively influencing the subcellular localization of these proteins.30,31 More interestingly, different proteins that interact with the CT of Cx43 can also influence each other. This interaction allows the CT of Cx43 to act as a platform or even a hub to integrate multiple signaling pathways, resulting in a broader regulatory effect (Fig. 1B).32–36 Through the above important domains, Cx43 is involved in the regulation of cell proliferation, migration, and other functions; thus, it plays an important role in physiological and pathological processes.9 Function of Cx43 in Gliomas In the early 1960s, tumor cells were shown to influence the cell membrane permeability of normal cells and reduce their intercellular communication. Furthermore, GJIC between tumor cells was nearly absent.37,38 The upregulation of some connexins can restore GJIC and inhibit tumor growth.39,40 Additional studies provided substantial evidence that connexins play important roles in tumor proliferation, apoptosis, invasion, and chemoresistance.41–44 More than 10 isoforms of connexins can be detected in the central nervous system.45,46 Among them, Cx43 is the most abundant member, and its expression level is correlated with the malignancy of glioma. Different grades of glioma show distinct levels of Cx43 protein.47,48 In addition, the role of Cx43 in the pathogenesis and development of glioma is complex49,50 and may differ by glioma cell subsets.3,47,51 Cx43 Inhibits the Expansion of the Glioma Cell Population and Tumor Initiation Many studies have shown that Cx43 negatively regulates the expansion of the glioma population and tumor initiation.29,49,50,52 As noted above, the different domains of Cx43 perform different functions. Thus, this protein regulates glioma cell proliferation and tumor initiation in a variety of ways. The structure of Cx43 consists of 2 extracellular loops and 4 transmembrane domains that allow the formation of gap junctions between neighboring cells, and the resulting gap junction–dependent effects play important roles in the regulation of cell proliferation through the transmission of growth-inhibitory53–56 and apoptotic factors.57–59 These signals derived from the microenvironment or adjacent cells can be delivered through the channel structure, thereby inhibiting the proliferation of glioma cells.54,59 For example, in C6 glioma cells overexpressing Cx43, transfection with cytochrome c (Cytc) can induce apoptosis, and cells in the apoptotic zone can propagate the apoptotic signal through the gap junction channels into adjacent cells that were not transfected with Cytc to expand the apoptosis cascade signal and inhibit glioma cell proliferation in a larger area.59 Cytc can also enter Cx43-expressing C6 glioma cells from the microenvironment in a hemichannel-dependent manner.59 Moreover, some pro-oncogenic factors can downregulate Cx43 and destroy gap junction structures to reduce inhibitory signal transmission; therefore, these factors play a role in promoting tumor cell proliferation and inhibiting tumor cell apoptosis. For example, the downregulation of basic fibroblast growth factor, an important pro-oncogenic growth factor in glioma cells, significantly increases Cx43 expression, restores GJIC, and inhibits glioma cell proliferation, suggesting that basic fibroblast growth factor can regulate the growth of glioma cells via Cx43.60 MicroRNA (miR)-221 and miR-222 are significantly upregulated in clinical glioma samples,61 and they can directly downregulate the level of Cx43 mRNA, thus preventing GJIC and promoting glioma cell proliferation.52 In addition, some exogenous carcinogenic viruses, such as human cytomegalovirus, can promote the degradation of Cx43 via the proteasome-dependent pathway after entering cells and thus inhibit GJIC to promote the proliferation of glioma cells and the expansion of glioma cell populations.62 Interestingly, after the transfection of nonphosphorylated Cx43-216 into glioma cells, although dye transfer capacity did not change significantly, cell expansion was inhibited.63 This finding suggested that another mechanism exists for Cx43 independent of its channel structure to regulate glioma proliferation63,64 and apoptosis.65 Furthermore, the administration of exogenous Cx43 CT was sufficient to inhibit the proliferation of glioma, thereby reversing its malignant phenotype.64 The combined evidence suggests that the CT of Cx43 may play an important role in inhibiting the expansion of the glioma population. As mentioned above, the CT of Cx43 includes many protein-binding sites through which it can affect the stability, activity, and phosphorylation of itself and other proteins via protein-protein interactions, thereby inhibiting glioma cell proliferation.25,28,29,66–68 Previously, all glioma cells in a tumor were considered equal, with the same or similar tumor-initiating potential. However, a growing body of evidence has shown that glioma consists of a heterogeneous stratified cell population. The subpopulation of GSCs with stem cell characteristics and resistance to chemotherapy and radiotherapy is thought to be the root cause of glioma initiation, expansion, and recurrence. Therefore, investigating the relationships between Cx43 and various subgroups of heterogeneous glioma has elevated importance. Interestingly, compared with differentiated glioma cells, a low expression level of Cx43 caused by promoter methylation51 and histone modification47 can be observed in the GSC subpopulation. When these GSCs are placed in serum-containing differentiation medium, the downregulation of the stem cell transcription factor sex determining region Y-box 2 (Sox2) and the upregulation of the differentiation marker glial fibrillary acidic protein are followed by a significant increase in Cx43 expression. In addition, when the expression of Cx43 in GSCs is restored, the activity of c is inhibited, and inhibitor of DNA binding 1 protein (Id-1) and Sox2 are downregulated; these changes inhibit the self-renewal and tumorigenic capacity of GSCs. Moreover, restoring Cx43 expression in GSCs causes a switch in expression from N-cadherin to E-cadherin.3,51 Cx43 can then form complexes with E-cadherin to inhibit the activation of the Wnt/β-catenin pathway and the transcription of its downstream target genes (such as Wnt3a, Atoh1, and POSIN) and stemness-related genes (such as Sox2, Nanog, and Oct4), thereby reducing GSC self-renewal and proliferation as well as tumor invasion and initiation.3,51 These results suggest that low Cx43 expression may be a stem cell characteristic of glioma cells. Because the GSC subpopulation is able to initiate a tumor in an immunodeficiency animal and is thought to drive the recurrence and the therapeutic resistance of glioma, low Cx43 expression likely plays a critical role in glioma development, cell population expansion, and recurrence. It should be noted that the results from Winkler are the opposite: Cx43 is highly expressed in GSCs.49 The reason for this difference may be that the molecular subtypes of the obtained GSCs are different. Using the dataset GSE67089, which contains the expression data of proneural-GSC, mesenchymal-GSC, and non-GSC subpopulations, we have analyzed the expression of Cx43 in these glioma subpopulations. As shown in Fig. 2, the expression of Cx43 (encoded by GJA1) in the mesenchymal-GSC subpopulation is very low or nearly absent, but it is fairly high in proneural-GSCs. Although none of the abovementioned 4 groups performed molecular typing of the GSCs they obtained, it is reasonable to speculate that Lathia,47 Gangoso,3 and Yu51 may have utilized mesenchymal-GSCs, and Winkler49 may have utilized proneural-GSCs. Fig. 2 View largeDownload slide The expression levels of Cx43 and Cx46 in different GBM subpopulations. The expression data of proneural-GSC (n = 18), mesenchymal-GSC (n = 12), and non-GSC (n = 10) are derived from the dataset GSE67089 (https://www.ncbi.nlm.nih.gov/geo) and are shown as mean ± SD. A 2-sided Student’s t-test was used to generate P-values, ****P < 0.0001, *P < 0.05. Abbreviations: PN-GSC, proneural-GSC; MES-GSC, mesenchymal-GSC; non-GSC, differentiated GBM cell. Fig. 2 View largeDownload slide The expression levels of Cx43 and Cx46 in different GBM subpopulations. The expression data of proneural-GSC (n = 18), mesenchymal-GSC (n = 12), and non-GSC (n = 10) are derived from the dataset GSE67089 (https://www.ncbi.nlm.nih.gov/geo) and are shown as mean ± SD. A 2-sided Student’s t-test was used to generate P-values, ****P < 0.0001, *P < 0.05. Abbreviations: PN-GSC, proneural-GSC; MES-GSC, mesenchymal-GSC; non-GSC, differentiated GBM cell. Cx43 Regulates Glioma Invasive Capacity Cx43 plays an important role in normal neuronal migration.69–72 By performing uteroelectroporation with Cx43 short hairpin (sh)RNA plasmids or crossing nestin-Cre transgenic mice with Cx43-floxed mice, the downregulation of Cx43 was shown to significantly delay the migration of neurons during embryonic development.70 When excitatory vertebral neurons migrate radially along radial glial fibers, Cx43 is highly expressed at the contact points between the migrating neurons and radial fibers, providing dynamic adhesion for cell migration. Cx43 also interacts with intracellular cytoskeletal proteins at these points to enable stable cell migration along the radial fibers.69 Cx43 may serve as a regulator to promote the tangential or radial migration of inhibitory interneurons and assist their precise localization during cortical plate entry.71 Meanwhile, Cx43 exerts complex effects on glioma cell migration and invasion via different domains (channel structure, extracellular loop, and CT) (Fig. 3) (reviewed by Sin et al50,72 and Kameritsch et al73). Fig. 3 View largeDownload slide Cx43 structural pattern and targeted therapy strategies. The second extracellular loop (E2) of Cx43 is the connexon component that shares the least homology with other connexins. Thus, it is a suitable target against which to design specific monoclonal antibodies to inhibit the connexon structure, reduce gap junction communication, and consequently reduce glioma cell invasion. The long CT contains several protein-binding sites. Mimic peptides can be designed to target specific protein-binding sites in the CT, such as the 245–283 amino acid region that binds specifically to c-Src, and these peptides can be linked to transmembrane sequences (such as TAT). Under the guidance of TAT, mimic peptides can enter the cell and bind to c-Src to prevent its activation, thereby inhibiting glioma cell proliferation. Abbreviations: N-term, amino terminus of Cx43; MAbE2Cx43, monoclonal antibodies against the E2 extracellular loop of connexin 43; E1/E2, Cx43 first/second extracellular fragment; C-term (COOH), CT of Cx43; TAT, transactivator of transcription cell penetrating sequence. Fig. 3 View largeDownload slide Cx43 structural pattern and targeted therapy strategies. The second extracellular loop (E2) of Cx43 is the connexon component that shares the least homology with other connexins. Thus, it is a suitable target against which to design specific monoclonal antibodies to inhibit the connexon structure, reduce gap junction communication, and consequently reduce glioma cell invasion. The long CT contains several protein-binding sites. Mimic peptides can be designed to target specific protein-binding sites in the CT, such as the 245–283 amino acid region that binds specifically to c-Src, and these peptides can be linked to transmembrane sequences (such as TAT). Under the guidance of TAT, mimic peptides can enter the cell and bind to c-Src to prevent its activation, thereby inhibiting glioma cell proliferation. Abbreviations: N-term, amino terminus of Cx43; MAbE2Cx43, monoclonal antibodies against the E2 extracellular loop of connexin 43; E1/E2, Cx43 first/second extracellular fragment; C-term (COOH), CT of Cx43; TAT, transactivator of transcription cell penetrating sequence. Despite alternate opinions,74 many results have shown that heterocellular GJIC between glioma and astrocytes or endothelial cells promotes the migration and invasion of glioma. Different substrates including microRNAs and Ca++ can be transferred through these heterocellular channel structures formed by Cx43.75–78 For instance, the inhibition of GJIC reduces the invasive capacity of glioma cells in fresh human glioma biopsy slice cultures.77 After being implanted into the brains of mice, Cx43-expressing glioma cells established functional GJIC with host astrocytes and displayed a more invasive phenotype compared with mock and mutant Cx43-transfected cells.75 However, the roles in the migration and invasion of glioma performed by homocellular GJIC between human glioma cells are more complicated. After the knockdown of Cx43 or the treatment with the gap junction inhibitor 18α-GA, GJIC between adjacent U87MG glioma cells was eliminated as assessed by dye coupling. More importantly, the invasive capacity of these cells increased significantly.78 The same results were found by Aftab et al, that the Cx43-mediated homocellular GJIC between adjacent glioma cells causes these glioma cells to migrate in a slower, more clustered manner.79 The destruction of this adjacent homocellular GJIC promotes the glioma cells to migrate in a faster, single-cell manner.79 It is noteworthy that despite the antimigration effect mediated by adjacent homocellular GJIC, the long-distance information transfer through tumor microtubes (TMs),49,80 which is also dependent on the expression of Cx43 at the intersection of TMs, plays a significant role in promoting glioma invasion. In the peritumoral area, many astrocytoma and GBM cells can form long and stable TMs and cross-link with each other to form a multicellular microtube network that promotes glioma cell migration,49 proliferation, and chemoresistance.80 The expression of Cx43 is particularly high at the junctions of this TM network, and calcium waves can propagate via these crossings from one TM to another. The inhibition of Cx43 expression significantly decreases the long-term stabilization of TMs and the tumor volume while significantly prolonging the survival time of mice harboring glioma.49 Interestingly, this TM-like structure also exists between glioma cells and astrocytes.81 In addition to the channel structure–dependent GJIC, the adhesive function mediated by the extracellular loop of Cx43 plays an important role in the regulation of glioma cell invasion (reviewed by Kameritsch et al73). In vitro cultured glioma cells expressing Cx43 aggregated with astrocytes, whereas connexin-free control cells aggregated to a lesser extent.75 Antibodies against the extracellular loops of Cx43 significantly reduced this aggregation between Cx43-expressing glioma cells and astrocytes.75 Intracellular free C-terminal Cx43 protein also affects glioma cell invasion,74 especially in glioma cells expressing low levels of Cx43 primarily in the cytoplasm or around the nucleus, such as GL261 and LN18.82–84 The CT domain of Cx43 contains a large number of phosphorylation sites that can interact with cytoskeletal proteins, such as α-tubulin, β-tubulin, actin, development regulatory brain protein (drebrin), and cortactin.73,85–87 By interacting with these cytoskeletal proteins, the Cx43 CT can regulate the cytoskeleton and alter cellular structures, such as pseudopodia formation, to promote cell migration.83 More interestingly, when the Cx43 channel structure and CT are simultaneously expressed at high levels, cells exhibit migration that is regulated mainly by the Cx43 channel structure.79 The shRNA-mediated downregulation of full-length Cx43 and the simultaneous transduction of the TrCx43 mutant (it can form the channel structure but lacks many phosphorylation and protein-protein interaction sites due to the truncation of the Cx43 tail at amino acid 242) do not significantly affect the migration of U118 cells.79 However, glioma cells transfected with both Cx43 shRNA and the Cx43 T154A mutant (a dominant negative channel mutant that can block gap junction intercellular communication) exhibit a significantly higher migration rate compared with wild-type glioma cells.79 In short, glioma cells expressing high levels of Cx43 in the peritumoral region in vivo utilize the Cx43 channel structure to form membrane TM structures or heterocellular gap junctions with adjacent astrocytes to promote their own migration, whereas the inhibition of the channel structure can significantly inhibit the invasion of glioma cells. However, for glioma cells expressing low levels of Cx43, which are located at the center of the tumor, the channel structure and the extracellular loop–mediated antimigration effects and Cx43 CT–mediated pro-migration effects were weakened at the same time. Thus, these glioma cells migrated in a faster, single cell manner. As mentioned above, the GSCs of the heterogeneous glioma population express low levels of Cx43, whereas Cx43 expression is relatively high in differentiated glioma cells. Thus, we can speculate that differentiated glioma cells expressing high levels of Cx43 can form heterocellular gap junctions with adjacent astrocytes and long-distance membranous microtube network structures at the edge of the glioma, invade the surrounding area as clusters, and provide support to GSCs (which express low levels of Cx43). Conversely, GSCs expressing low levels of Cx43 may utilize single-cell invasion, which is faster, but they may require the support of TM structures formed by differentiated glioma cells expressing high levels of Cx43 or the guidance of other support structures, such as the ependyma, blood vessels, and white matter fibers. Osswald et al found that the TM networks consisting of glioma cells expressing high levels of Cx43 included small quantities of GSCs that remained stationary for a prolonged time and did not form significant membranous microtube connections with the surrounding glioma cells expressing high levels of Cx43.49 Therefore, the Cx43 channel structure plays a key role in glioma cell invasion, and the destruction or inhibition of this channel structure may be an effective method for the inhibition of glioma cell invasion. Treatment Strategy for Targeting Cx43 In summary, the inhibition of Cx43 expression in invading glioma cell clusters at the periphery of gliomas can reduce the invasive capacity of glioma cells and may be beneficial for surgical resection. The upregulation of Cx43 in GSCs can inhibit GSC self-renewal, which inhibits the proliferation and expansion of the glioma cell population. Thus, combining the 2 strategies may be a promising approach for glioma treatment (Fig. 4). Fig. 4 View largeDownload slide Cx43 plays an important role in glioma cell invasion. Many glioma cells at the periphery of a glioma express high levels of Cx43. These cells connect with each other to form a membranous microtube network structure and promote the migration of glioma cells. A small number of quiescent GSCs expressing low levels of Cx43 are present in these membranous microtube network structures, and these cells may migrate with the help of the membranous microtube structures. The distal region of tumor foci contains small numbers of GSCs expressing low levels of Cx43, and these cells exhibit a scattered distribution along the structure of white matter fibers and constitute the origins of distant glioma recurrence. Fig. 4 View largeDownload slide Cx43 plays an important role in glioma cell invasion. Many glioma cells at the periphery of a glioma express high levels of Cx43. These cells connect with each other to form a membranous microtube network structure and promote the migration of glioma cells. A small number of quiescent GSCs expressing low levels of Cx43 are present in these membranous microtube network structures, and these cells may migrate with the help of the membranous microtube structures. The distal region of tumor foci contains small numbers of GSCs expressing low levels of Cx43, and these cells exhibit a scattered distribution along the structure of white matter fibers and constitute the origins of distant glioma recurrence. Antibody Treatment Targeting the Cx43 Channel Structure in Glioma Cells Based on the features of glioma invasion at the leading edge—① glioma cells form TM structures via Cx43 channels to promote migration and ② glioma cells and adjacent astrocytes or endothelial cells interact via Cx43 channels to promote invasion—inhibiting Cx43 channel structures at the edge of the glioma is a viable treatment strategy.88 The antibody MAbE2Cx43 targeting the second extracellular loop of the Cx43 was generated. This antibody disrupts the functional gap junction between cells by specifically binding to the extracellular loop of Cx43 (Fig. 4).89,90 Isotope-labeled MAbE2Cx43 entered the rat brain 72 hours after being injected into glioma-bearing rats, and their concentration was much higher in the right brain, which harbored a glioma, compared with the left normal brain (0.272% vs 0.005%). This antibody was specifically enriched in brain tissue around the glioma, and a further analysis of the distribution of fluorescence-labeled antibodies showed that MAbE2Cx43 was primarily distributed in peritumoral glioma cells and reactive astrocytes (ie, at the glioma invasion leading edge).88,89 More importantly, MAbE2Cx43 alone or in combination with other drugs can prolong the survival of glioma-bearing animals. Specifically, after treatment with a single dose of MAbE2Cx43 (5 mg/kg), the median survival time of C6 tumor-bearing rats was 39.5 days, which is higher than that of rats treated with a single dose of temozolomide (TMZ) (median survival time, 34 days) or radiotherapy alone (36 Gy, median survival time, 38 days). Overall, MAbE2Cx43 combined with radiotherapy prolonged survival to 60 days.4 In addition, because Cx43 is overexpressed in glioma cells at the leading edge of invasion, Cx43 monoclonal antibodies (mAbs) can be used to treat gliomas in combination with cisplatin nanogel. In this approach, cisplatin is enriched in tissues at the periphery of gliomas, which enhances the efficacy and prolongs survival time. Furthermore, the concentration of cisplatin in normal tissue is decreased, which reduces side effects. The median survival time of the glioma-bearing animals was prolonged by treatment with cisplatin alone (31 days for the free cisplatin-treated group vs 15 days for the control group), but signs of toxicity in the mice treated with cisplatin were significant. Conversely, the MAbE2Cx43 antibody combined with cisplatin nanogel not only increased the median survival time compared with the cisplatin group (42 days for the group treated with cisplatin nanogels with Cx43 mAbs vs 31 days for the free cisplatin-treated group) but also reduced cisplatin toxicity.5,91 Targeted Treatment for GSCs with Low Expression of Cx43 Although GSCs are not numerous and account for only a small proportion of the glioma cell population, they can self-renew, exhibit multipotency, are resistant to TMZ chemotherapy, and can mediate the repopulation process. These cells are considered to be the root cause of glioma chemotherapy failure and malignant recurrence.2 Therefore, strategies that target GSCs are an important direction of glioma treatment to be explored. As mentioned above, Cx43 expression is much lower in GSCs than in differentiated glioma cells and is closely correlated with the malignant phenotype of GSCs. Moreover, the upregulation of Cx43 expression in GSCs inhibits GSC self-renewal and promotes GSC differentiation. In GSCs, the Wnt/β-catenin signaling pathway is overactive and plays an important role in GSC self-renewal and the maintenance of malignant phenotypes.92 The upregulation of Cx43 in GSCs can also promote the complexation of Cx43 with E-cadherin, which reduces the activity of the Wnt/β-catenin signaling pathway and inhibits the self-renewal and other malignant phenotypes of GSCs.51 In addition, the upregulation of Cx43 may also promote GSC differentiation, resulting in the loss of GSC properties and the attenuation of their tumorigenicity.3 The therapeutic significance of Cx43 upregulation in the targeted treatment of cancer stem cells has been shown for other types of tumors; the small molecule sulforaphane can upregulate Cx43 expression via posttranslational regulatory mechanisms, and upregulated Cx43 can suppress tumor stem cell markers (c-Met and CD133), improve GJIC, and ultimately inhibit the malignant phenotype of pancreatic ductal cancer stem cells.93 This behavior suggests that Cx43 may serve as a candidate for targeted GSC therapy.47,51 Because the Cx43 CT plays a central role in suppressing the expansion of the glioma cell population and tumor repopulation, directly introducing the Cx43 CT into GSCs, which express low levels of Cx43 or no Cx43, may exert an important therapeutic effect. To this end, Gangoso et al synthesized a mimetic peptide containing amino acids 245–283 of the Cx43 CT and a transmembrane sequence of HIV.3 This mimetic peptide can enter GSCs and specifically inhibit c-Src Tyr-416 (Y416 c-Src) phosphorylation, thereby reversing the malignant phenotype of GSCs, which endogenously express low levels of Cx43 (Fig. 4).3 Specifically, this peptide inhibits Id-1 and Sox2 expression, causes a switch in expression from N-cadherin to E-cadherin, reduces GSC spheroid formation, and facilitates the differentiation of GSC into oligodendrocyte cells (O4 upregulation).3 Discussion and Perspectives Based on the important role of Cx43 in gliomas, a treatment strategy targeting the different domains of Cx43 warrants further exploration. Glioma cells at the peritumoral invasion front express high levels of Cx43, which can form heterocellular channels and a special TM network that facilitates the migration of glioma cells and may even provide structural support for the invasion of GSCs. Monoclonal antibodies designed against the extracellular domain of Cx43 can effectively block the intercellular channel–mediated communication among glioma cells and between glioma cells and normal brain tissue cells, thus inhibiting glioma invasion. In addition, Cx43 utilizes a large number of protein-binding sites on its CT to regulate its stability, activity, and phosphorylation status and to regulate other proteins via protein-protein interactions; these activities negatively regulate glioma cell population expansion and proliferation. However, Cx43 expression is low or absent in GSCs, the key subgroup regulating tumor population expansion and reconstruction. Therefore, introducing a Cx43-CT mimetic peptide into the GSC cell subset with low or absent Cx43 expression may inhibit these cells and prevent the expansion of the glioma cell population. However, the strategies described here targeting different domains of Cx43 use either antibodies or mimetic peptides to achieve therapeutic purposes, and the application of these therapeutics in the clinic is inconvenient and limited. Future research should focus on integrating these 2 strategies. For example, we can construct a therapeutic fusion peptide consisting of a single-chain soluble antibody against the Cx43 extracellular domain + linker sequence (which can be cleaved by matrix metalloproteinases) + Cx43-CT mimetic peptide + transmembrane sequence (Fig. 5). When a therapeutic fusion peptide reaches the glioma, it can be cleaved into 2 segments by local high concentrations of matrix metalloproteinases. For Cx43-expressing glioma cells, the single-domain soluble antibody moiety binds to the extracellular domain of Cx43, destroying the heterocellular gap junction channel and inhibiting TM network–mediated invasion. For the GSC subgroup expressing low levels of Cx43 or in which Cx43 expression is absent, the Cx43-CT mimetic peptide can enter cells under the guidance of the transmembrane sequence and bind to c-Src, Hsc70, and PKC, thus inhibiting the proliferation and self-renewal and other malignant phenotypes of GSCs. Our lab is performing these experiments. Moreover, we are also using a similar strategy to create chimeric antigen receptor (CAR)–modified T cells (Fig. 6) in an effort to apply this strategy for the treatment of glioma. However, before solving the problem of fusion peptides or new CAR T cells passing through the blood–brain barrier to reach brain lesions, intraoperative or intrathecal administration may be a method worth trying. In addition, the disruption of glioma connexins by small molecules, as summarized by Osswald et al, is also a promising strategy.94 The orally bioavailable modulators of gap junctions meclofenamate and tonabersat disrupt the astrocyte gap junctional network and attenuate established brain metastasis.95 Fig. 5 View largeDownload slide Schematics of the therapeutic fusion peptide for the treatment of glioma. This fusion peptide consists of a single-domain soluble antibody recognizing the Cx43 extracellular domain linked to a mimetic peptide of the Cx43 CT, which is followed by a transmembrane sequence in which the linker comprises a specific amino acid sequence cleavable by matrix metalloproteinases. When this fusion peptide enters the body, it can be cleaved into 2 parts by endogenous matrix metalloproteinases generated locally in tumors. The first part is the single-domain soluble antibody recognizing the Cx43 extracellular domain, which can bind specifically to the Cx43 extracellular domain in glioma cells expressing high levels of Cx43 to destroy the gap junction channel and thereby inhibit glioma invasion mediated by the membrane microtube structure. The second fragment is the Cx43-CT mimetic peptide with a transmembrane sequence, which can enter glioma cell subsets (such as GSCs) expressing low levels of Cx43 under the guidance of the transmembrane sequence and mimic the function of the endogenous Cx43 CT to inhibit the proliferation and self-renewal of cells. Abbreviations: scFv, single chain antibody fragment; TAT, transactivator of transcription cell penetrating sequence. Fig. 5 View largeDownload slide Schematics of the therapeutic fusion peptide for the treatment of glioma. This fusion peptide consists of a single-domain soluble antibody recognizing the Cx43 extracellular domain linked to a mimetic peptide of the Cx43 CT, which is followed by a transmembrane sequence in which the linker comprises a specific amino acid sequence cleavable by matrix metalloproteinases. When this fusion peptide enters the body, it can be cleaved into 2 parts by endogenous matrix metalloproteinases generated locally in tumors. The first part is the single-domain soluble antibody recognizing the Cx43 extracellular domain, which can bind specifically to the Cx43 extracellular domain in glioma cells expressing high levels of Cx43 to destroy the gap junction channel and thereby inhibit glioma invasion mediated by the membrane microtube structure. The second fragment is the Cx43-CT mimetic peptide with a transmembrane sequence, which can enter glioma cell subsets (such as GSCs) expressing low levels of Cx43 under the guidance of the transmembrane sequence and mimic the function of the endogenous Cx43 CT to inhibit the proliferation and self-renewal of cells. Abbreviations: scFv, single chain antibody fragment; TAT, transactivator of transcription cell penetrating sequence. Fig. 6 View largeDownload slide Schematic representation of a Cx43 fusion peptide CAR-modified T cell killing a glioma cell. This CAR consists of the Cx43 carboxyl-terminal mimetic peptide connected to a transmembrane peptide, which is linked to a single-domain soluble antibody recognizing the second extracellular loop (E2) of Cx43 via a linker (containing a matrix metalloproteinase-specific cleavage site) plus the T-cell transmembrane structure TM (CD28) and intracellular cascade signal 4-1BB-CD3zeta. After T cells modified by this receptor enter the body, the receptor can be digested into 2 parts by the local high concentrations of endogenous matrix metalloproteinases in the tumor. After the Cx43 carboxyl-terminal mimetic peptide containing the transmembrane sequence (TAT) is released from the T-cell surface, it enters glioma cell subpopulations expressing low levels of Cx43, such as GSCs, under the guidance of the transmembrane sequence. The peptide mimics the function of the endogenous Cx43 CT to inhibit GSC proliferation and self-renewal; the Cx43-E2 single-domain CAR remains on the T-cell surface, directing T cells to specifically recognize glioma cells expressing high levels of Cx43 and transducing the signal into T cells via the transmembrane (TM) structure. This signal activates the intracellular signaling cascade 4-1BB-CD3zeta, which triggers the targeted killing of glioma cells expressing high levels of Cx43 by T cells. Abbreviations: Cx43 CT, CT of Cx43; Cx43-E2 single chain antibody, single chain antibody against the E2 extracellular loop of connexin 43; VH, heavy chain; VL, light chain; TM, transmembrane region; TAT, transactivator of transcription cell penetrating sequence. Fig. 6 View largeDownload slide Schematic representation of a Cx43 fusion peptide CAR-modified T cell killing a glioma cell. This CAR consists of the Cx43 carboxyl-terminal mimetic peptide connected to a transmembrane peptide, which is linked to a single-domain soluble antibody recognizing the second extracellular loop (E2) of Cx43 via a linker (containing a matrix metalloproteinase-specific cleavage site) plus the T-cell transmembrane structure TM (CD28) and intracellular cascade signal 4-1BB-CD3zeta. After T cells modified by this receptor enter the body, the receptor can be digested into 2 parts by the local high concentrations of endogenous matrix metalloproteinases in the tumor. After the Cx43 carboxyl-terminal mimetic peptide containing the transmembrane sequence (TAT) is released from the T-cell surface, it enters glioma cell subpopulations expressing low levels of Cx43, such as GSCs, under the guidance of the transmembrane sequence. The peptide mimics the function of the endogenous Cx43 CT to inhibit GSC proliferation and self-renewal; the Cx43-E2 single-domain CAR remains on the T-cell surface, directing T cells to specifically recognize glioma cells expressing high levels of Cx43 and transducing the signal into T cells via the transmembrane (TM) structure. This signal activates the intracellular signaling cascade 4-1BB-CD3zeta, which triggers the targeted killing of glioma cells expressing high levels of Cx43 by T cells. Abbreviations: Cx43 CT, CT of Cx43; Cx43-E2 single chain antibody, single chain antibody against the E2 extracellular loop of connexin 43; VH, heavy chain; VL, light chain; TM, transmembrane region; TAT, transactivator of transcription cell penetrating sequence. The channel function of Cx43 is primarily mediated by the 4 transmembrane domains and 2 extracellular loops. In addition to targeting the second extracellular loop, studies need to be performed to determine the effects on Cx43 channel function of targeting the 4 transmembrane structures and the first extracellular loop. In addition, the Cx43 CT, which interacts with c-Src, E-cadherin, zonula occludens 1, cytoskeleton proteins, Hsc70, and PKC, contains many protein-binding sites. Our previous study showed that some sequences of the Cx43 CT could specifically bind to protein kinase B (Akt) and extracellular signal-regulated kinase (ERK) 1 and 2, thus inhibiting Akt and ERK1/2 phosphorylation in GSCs. This finding suggests that the CT of Cx43 can act as a platform for the accumulation of proteins in various signaling pathways, resulting in more complex interactions and a wider regulation of cells.9,32–34,96 Future studies need to explore strategies to more rationally inhibit GSCs by utilizing the large number of protein-binding sites on the Cx43 CT. Of course, such studies require a more in-depth understanding of the mechanisms mediating interactions with the Cx43 CT. Assessing the expression and functional status of Cx43 in the GSC subpopulation relies mainly on surface makers—for example, CD133. However, it should be noted that CD133 is not only expressed in GSCs but can also be expressed in other cells, such as normal neural stem cells. A series of relatively specific markers of GSCs,97,98 such as PBK, CENPA, KIF15, DEPDC1, CDC6, DLG7, KIF18A, EZH2, HMMR, and cadherin-19, which have been identified recently, should be used to investigate the expression and functional status of gap junction proteins in heterogeneous glioma cell populations. In addition, compared with the data generated from primary culture models (primary glioblastoma cells kept in serum-free medium, neurosphere-forming conditions), data from glioma cell line models (U251, U87, C6, etc, kept in serum-containing medium, monolayer conditions) should be presented very differently. These culture conditions might actually explain much of the contradictory findings regarding the relationship between Cx43 and glioma invasiveness. In future studies, the simultaneous detection of the expression and the functional status of gap junction proteins in glioma cell lines, primary glioma cells, human specimens, and mouse spontaneous tumors, as a recent series of studies have performed, may be a better strategy.49,95 In addition to Cx43, other connexins, especially Cx46, play important roles in the development and progression of glioma.47,99,100 The expression of Cx46 was negatively correlated with Cx43 expression in gliomas. The expression of Cx46 is high in GSCs, whereas Cx43 is low in this subpopulation; in contrast, in the non-GSC subgroup with high Cx43 expression, Cx46 expression was low or absent. More importantly, the expression of Cx46 and the GJIC mediated by Cx46 were closely related to the self-renewal and tumorigenicity of GSCs, and the survival time of patients with high Cx46 expression was significantly shortened.47 Therefore, the combined targeting of different connexins is worth exploring in the future. However, as shown in Fig. 2, the expression levels of Cx43 and Cx46 might be different in distinct GSCs; thus, the molecular subtypes of GSCs need to be evaluated prior to the application of this strategy. In addition, the gap junction channels formed by different connexins have obvious selectivity for the passage of substances. For example, adenosine passage is approximately 12-fold greater through channels formed by Cx32 compared with channels formed by Cx43. In contrast, ATP passage is 300-fold greater through channels formed by Cx43.101 Thus, the therapeutic implications of this selectivity are worth considering in the development of connexin-targeting strategies for glioma therapy. In summary, Cx43 is a multidomain transmembrane protein involved in the multifaceted regulation of many biological characteristics of gliomas via the channel structures formed by Cx43 and the long CT of Cx43. We believe that these findings and progress from related research will result in strategies targeting Cx43 that have promising applications in the clinical treatment of glioma. Funding This study was supported by grants from the National Key Research and Development Program of China (2016YFA0202104 to S-C.Y.), the National Natural Science Foundation of China (81572880 and 81172071 to S-C.Y., 81302193 to Q-K.Y.), the Outstanding Youth Science Foundation of Chongqing (CSTC2013JCYJJQ10003 to S-C.Y.), the Postgraduate Education Foundation of Chongqing (YJG153062 to S-C.Y.), and the Key Clinical Research Program of Southwest Hospital (SWH2016ZDCX1005 to S-C.Y.). Acknowledgments We thank Miss Ying Ji for assistance with preparing schematic diagrams. Conflict of interest statement The authors declare that they have no conflicts of interest. References 1. Siegel R , Naishadham D , Jemal A . Cancer statistics, 2013 . CA Cancer J Clin . 2013 ; 63 ( 1 ): 11 – 30 . 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Google Scholar CrossRef Search ADS PubMed © The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

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Neuro-OncologyOxford University Press

Published: Nov 2, 2017

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