Dysregulation of connexins in cancer: therapeutic opportunities. Multiple stages of connexin life cycle are subject to dysregulation during cancer progression, as exemplified by GJA1 Cx Histone acetylation can be modified by targeting histone acetyltransferase enzymes HATs or histone deacetylases HDACs , typically promoting and repressing transcription, respectively. Moreover, alternative translation initiation, resulting in the synthesis of truncated forms of Cx43, might regulate Cx43 and have important implications for its dysregulation in cancer.
Truncated forms of Cx43, notably the kDa form named GJA1—20k, may be important for the efficient targeting of Cx43 to the membrane. Phosphorylation and other multiple post-translational events, occurring mainly at their C terminus, regulate connexin trafficking and stability at the plasma membrane. Cx43 is also regulated by acetylation, ubiquitination and SUMOylation.
In accordance with the notion that connexins might act as tumour suppressors, the ectopic expression of connexins in cancer cells often partly restores growth control e. Conversely, the experimental depletion of connexins may result in more aggressive cancer cell growth [ 29 ]. In addition to their role in modulating cell proliferation [ 30 ], connexins can either promote or prevent cell death by apoptosis [ 31 ]. Moreover, hemichannels may exchange proapoptotic and survival factors between extracellular and intracellular environments [ 35 ].
There is increasing evidence that connexins can suppress the growth of cancer cells through channel-independent mechanisms [ 22 , 30 , 36 , 37 , 38 , 39 ] Fig. For example, the ectopic expression of the intracellular C terminus CT of Cx43 can in some cases inhibit cell proliferation to a similar extent as full-length protein [ 24 ]. Because connexins present a low level of homology within their CT sequences, the channel-independent regulation of cell growth is expected to vary considerably among different isoforms.
Interactions between connexins and proteins that affect tumour growth and migration. Examples of proteins that interact with specific regions of connexins and may act as therapy targets. Similar mechanisms have been proposed for other proteins associated with the cytoskeleton, such as cadherins, catenins, vinculin, ZO-1 and drebrin. A similar sequestration mechanism may occur with drebrin, ezrin or ZO These proteins, and many others such as Nedd4, also have important roles in regulating Cx43 gap junction plaques, which influence GJIC and therefore may have therapeutic potential.
For instance, c-Src phosphorylation affects the binding of several Cx43 partners. GJIC, gap junction intercellular communication. When assessing the role of connexins in cancer pathogenesis, we must consider that their ability to act as tumour suppressors can vary considerably among tissue types and cancer stages, as well as among connexin isoforms [ 2 ]. Furthermore, there is growing evidence that some connexin isoforms are protumorigenic under certain conditions. For example, connexins can promote the migration and invasion of tumour cells [ 44 , 45 ]. In addition, connexins can form heterologous gap junctions between tumour cells and endothelial cells to facilitate intravasation and extravasation [ 46 , 47 , 48 , 49 ].
Connexins can also nurture metastatic growth and may promote resistance to cancer treatments [ 50 , 51 , 52 , 53 ]. In accordance with this notion, a recent study demonstrated that brain metastatic cancer cells establish gap junctions with astrocytes to promote tumour growth and chemoresistance [ 50 ]. Emerging experimental evidence indicates that connexins play important roles in cancer stem cell CSC biology.
Malignant glioma stem cells GSCs express very low levels of Cx43, and GSC stemness is reduced by its transfection with Cx43 or treatment with peptides containing sequences of Cx43 that interact with c-Src Cx43 mimetic peptides [ 54 , 55 ]. A recent work has found that the expression levels of Cx43 and Cx46 were increased and reduced, respectively, during GSC differentiation and that targeting of Cx46 compromises GSC maintenance [ 56 ].
While Cx43 is almost absent in liver CSCs [ 57 ], the accumulation of cytoplasmic Cx32 has been associated with metastasis and enhanced self-renewal of CSCs in human HuH7 hepatoma cells [ 58 ]. Moreover, Cx43 clearly plays a subtype-dependent role in breast cancer [ 61 ]. Overall, connexins can be both anti- and pro-tumorigenic, acting via processes that include the regulation of CSCs, and this depends on the connexin isoform and tissue type. Understanding this complexity is the key to the development of new and efficient therapies.
A number of studies suggest that connexins can be independent tumour markers predicting both better and worse prognoses. Further insights into this seeming contradiction have been provided by recent analytical tools for public databases. Notably, GJA1 encoding Cx43 is featured in the list of 20 genes most significantly associated with an unfavourable prognosis in stomach cancer. Indeed, for most connexins and cancer types, high levels of connexin mRNA expression are associated with a poor prognosis. It is also notable that the mRNA expression of a given connexin-encoding gene can be both favourable and unfavourable, depending on the type of tumour Table 1.
Different connexins can also have differing prognostic prediction results for the same type of tumour e. Cx26 vs Cx32 in renal cancer.
The sub-classification of cancers is also relevant. Several specific studies have also reported a correlation between connexin mRNA levels and survival or phenotypic features. For instance, high levels of Cx43 mRNA in glioma tumours have been correlated with poor survival [ 65 ]. Expression of connexin mRNA in the surrounding tissue may also correlate with a specific tumour phenotype or behaviour. For example, in melanoma, the increased expression of Cx26 and Cx30 at the mRNA level in the surrounding skin keratinocytes is significantly correlated with malignant features such as tumour thickness and, in the case of Cx26, metastasis [ 66 ].
In lung cancer, the reduced expression of Cx43 mRNA in adjacent normal lung tissue due to promoter methylation is significantly correlated with nodal involvement, suggesting that Cx43 could be a marker for micrometastasis in non-small cell lung cancer [ 8 ]. Recently, Cx Mechanistically, Cx Microarray analysis and confirmatory quantitative real-time polymerase chain reaction analysis of breast cancer samples show the upregulation of Cx26 during progression from ductal carcinoma in situ to invasive ductal carcinoma [ 69 ].
At the protein level, Cx26 is detected in the invasive carcinoma foci [ 69 ]. In agreement with these findings, Cx26 was recently found to drive CSC self-renewal in triple-negative breast cancer [ 62 ]. At the DNA level, few studies have provided an insight into the role of connexins in cancer.
Mutations in Cx43 have been described in advanced metastatic colon cancer lesions [ 70 ]. However, current large-scale genetic studies and analytical tools e. Nevertheless, individual genetic studies merit follow-up. For example, the CT polymorphism in GJA4 encoding Cx37 is associated with Helicobacter pylori infection in patients with gastric cancer [ 71 ].
In glioma, GJB6 encoding Cx30 was deleted in However, at the protein level, high Cx30 expression adversely influenced the survival Table 2. A number of immunohistochemical studies of connexins in cancer—most on Cx26 and Cx43—have described specific prognostic associations. As with mRNA-expression studies, connexin protein expression has been associated with both good and poor prognoses. The studies specifically reporting the significant findings related to patient survival are summarised in Table 2. In accordance with mRNA-based studies, the expression of different connexins at the protein level can have an opposing prognostic value depending on cancer type, cancer subtype and connexin isoform.
For example, in the same breast cancer series, elevated Cx43 and Cx30 levels have been associated with improved and worse breast cancer outcomes, respectively [ 64 ]. This observation is supported by additional studies evaluating tumour histology or aggressiveness, but not the overall survival, including those of thyroid cancer [ 72 ] and bladder cancer [ 73 ].
Connexin expression has also been proposed as a diagnostic aid in relation to specific tumour subtypes. An association between the expression level of connexins and metastasis can be found for several cancer types. For instance, Cx43 expression is reduced in primary gastric cancer but increased in matched metastatic lymph nodes [ 75 ]. It should also be noted that, in a number of studies, connexin proteins were overexpressed but mislocalised in the cytoplasm.
Prominent examples include Cx26 in pancreatic [ 76 ] and colon [ 77 ] cancer, and Cx43 and Cx32 in prostate cancer [ 78 ].
Nuclear Cx43 has also been reported in a number of tumours e. It remains to be seen how this mislocalisation correlates with a recent report showing that nuclear Cx43 or the CT alone can act as a direct nuclear transcription factor that induces the expression of N-cadherin, an important regulator of processes including epithelial-to-mesenchymal transition and cell migration [ 80 ]. In summary, connexins are potential prognostic markers in cancer, but a number of limitations need to be addressed before any clinical application.
Standardisation is also difficult in terms of reliable antibodies and evaluation parameters such as the quantification of subcellular connexin localisation e. The complex and multifunctional role of connexins in cancer provides a wide spectrum of therapeutic opportunities and challenges. Increasing numbers of drugs, peptides and RNAi approaches are available to inhibit or enhance GJIC, hemichannel activity or connexin protein-signalling activity.
Below, we highlight a selection of in vivo studies demonstrating that the cancer phenotype is altered as a direct consequence of such an experimental targeting of connexins. GJIC can be augmented either by enhancing the permeability of existing gap junctions or by increasing the expression of connexins and thereby elevating the number of open gap junction channels [ 81 ].
Many fungal and plant-based compounds, as well as an increasing number of synthetic chemical compounds, either increase GJIC or prevent the loss of GJIC in response to cellular exposure to tumour promoters, which is often associated with reduced tumour growth in vivo. Compounds shown to modify connexins or GJIC and affect the tumour phenotype in vivo are summarised in Table 3.
For example, docetaxel increases Cx43 expression in murine salivary gland carcinoma to reduce tumour growth [ 83 ]. This has implications for combinatorial effects in cancer therapy. However, because connexins under specific conditions can facilitate malignant features, in particular, metastasis or growth of metastases [ 2 ], there are situations in which the blockade of GJIC or other functions of connexins may offer distinct therapeutic opportunities.
The inhibition of GJIC can be achieved through a number of different mechanisms Table 3 and recent comprehensive reviews [ 5 , 84 ]. Despite their non-specificity, many chemical gap junction channel inhibitors have been extensively used, even in vivo. For instance, the GJIC blocker oleamide a fatty-acid derivative reduces the formation of pulmonary and hepatic metastatic foci and increases the overall survival of mice injected intravenously with MDA-MB breast cancer cells [ 85 ].
Mechanistically, oleamide-induced loss of heterologous GJIC between tumour cells and endothelial cells was suggested to interfere with cancer-cell extravasation. Inhibition of heterologous GJIC was achieved either by the knockdown of Cx43 expression or by the use of the gap junction channel inhibitors tonabersat or meclofenamate, which pass the blood—brain barrier [ 50 ].
Tonabersat has been used as a migraine prophylaxis drug, whereas meclofenamate is an FDA-approved anti-inflammatory drug for oral administration. Mechanistically, these GJIC inhibitors seem to inhibit tumour growth by blocking a cGAMP-mediated signalling cascade that orchestrates paracrine signalling between host and tumour cells [ 50 ].
Recently, a novel role of Cxbased gap junctions was also identified in breast cancer bone metastasis [ 86 ]. Gap junctions were found to mediate calcium flow from osteogenic cells to the cancer cells. Blocking such heterocellular intercellular calcium transfers from the osteogenic niche to cancer cells, by gap junction inhibitors carbenoxelone and arsenite trioxide or by the ablation of Cx43 in either the osteogenic cells or the cancer cells, prevented bone metastasis progression in mice [ 86 ].
Thus, many of the studies reporting the inhibitory effects on tumour growth upon inhibition of GJIC mechanistically relate this effect to a loss of heterologous GJIC between tumour cells and cells in their microenvironment as reviewed in [ 50 , 85 ] and discussed elsewhere in this manuscript. The use of peptides arguably provides for a more specific targeting compared with the more general chemical GJIC inhibitors and may reduce potential adverse effects. Cxblocking antibodies also reduce tumour growth in murine models.
Notably, the intravenous administration of a monoclonal antibody targeting the second extracellular loop of Cx43 reduces glioma growth and survival of experimental animals when used alone [ 88 ] or in combination with standard cancer therapy approaches [ 89 ]. The same antibody has been used as a guidance system to deliver diagnostic markers or therapeutic compounds, such as cisplatin, to Cxpositive high-grade gliomas [ 90 , 91 ].
Several lines of evidence suggest that Cx43 hemichannels are involved in promoting tumour growth. Antibodies against the second extracellular loop domain of Cx43, which blocks hemichannels [ 94 ], reduce glioma tumours generated with C6 cells in rats [ 89 ]. On the other hand, Cx43 hemichannels in osteoblast bone cells may provide an intrinsic self-defence mechanism against breast cancer metastasis [ 95 ]. Among the other molecules released by Cx43 hemichannels in osteoblasts, ATP acts as a paracrine signal that triggers an inflammatory cascade to inhibit the migration, invasion and anchorage-independent growth of breast cancer cells [ 96 ].
Consequently, the antibody blockade of osteocyte Cx43 hemichannels increases bone metastasis in mice [ 95 ]. Collectively, these data suggest that Cx43 hemichannel activity in healthy tissue cells may have a beneficial effect by preventing metastasis, whereas hemichannels in tumour cells may favour their growth. Connexins interact with a wide variety of proteins that, independent of channel activity, can affect cancer cell phenotype, including cell growth, migration and differentiation Fig.
Strategies that mimic, promote or disrupt some of these specific interactions could be used in cancer therapy. The fact that CxCT is a disordered region favours the use of mimetic peptides that can restore or interfere with its functions. In tumour cells with low levels of Cx43, the restoration of Cx43 function may reduce cell proliferation. In glioma cells, this occurs via the inhibition of the oncogenic activity of c-Src [ 99 ].
In contrast, some proteins may favour cell growth or migration upon interaction with CxCT. Other Cxinteraction partners regulate the Cx43 protein level or subcellular localisation. Disruption of these interactions may have therapeutic potential. The bystander effect underpins a key therapeutic strategy in anticancer approaches and significantly enhances suicide gene therapy strategies [ ]. A well-established suicide gene therapy approach involves the viral transduction of the herpes simplex virus HSV thymidine kinase TK gene into cancer cells [ ]. The viral TK enzyme phosphorylates the nucleoside analogue ganciclovir GCV , which causes chain termination during DNA replication and leads to tumour cell death.
This led to the hypothesis that the induction of connexin expression or re-establishment of GJIC might potentiate the bystander effect [ ], a claim substantiated early on, both in vitro [ , ] and in vivo [ ]. This theory is supported by many studies e. However, other studies reported no evidence for a GJIC-mediated bystander effect [ ]. The bystander effect is important in a wide range of circumstances other than suicide gene therapy.
However, the strength and type of the signal may have distinct and opposing effects: 1 toxic signals kill neighbouring cells which may be tumour cells or healthy cells or 2 toxic signals are diluted into neighbouring cells, favouring the survival of the targeted cells. Importantly, this implicates GJIC in coordinating cellular and tissue responses to carcinogens, radiation and chemotherapies and leads to the possible strategies via both enhanced or blocked GJIC for either protecting undamaged non-targeted cells against such signals or potentiating the signals in order to kill tumour cells.
Application of this knowledge to other aspects of connexin cancer biology is important. Within the field of cancer therapy, there is a general consensus on the need for rational combinatorial targeted therapy. Synergistic effects on cancer cell growth as a result of enhanced connexin expression or GJIC in combination with drugs targeting other cellular processes have been described in many studies [ , , , , , ] Table 3.
For instance, kanglaite, a natural plant seed compound that upregulates Cx43 expression, sensitises colorectal cancer cells to Taxol [ ]. Simvastatin a statin induces GJIC and enhances the effect of platinum-based chemotherapeutic drugs [ ]. In addition, protection can be provided against the non-desired cytotoxic effects of cisplatin on healthy cells, including reproductive testicular Sertoli cells [ ]. It also poses a therapeutic challenge. The putative positive and negative effects such therapies can provide in relation to tumour growth must therefore be carefully assessed in order to avoid a possible worsening rather than an improvement of the clinical outcome.
The therapeutic response of cancer cells can also be affected by the direct overexpression of connexins, in an isoform-specific manner. For instance, Cx43 overexpression can enhance the sensitivity to common chemotherapeutic drugs such as doxorubicin, fluorouracil and oxaliplatin in human gastric cancer cells [ ], etoposide, paclitaxel and doxorubicin in glioma cells [ ], and artesunate in MCF-7 breast cancer cells [ ]. Cx32 potentiates the cytotoxicity of vinblastine and Src inhibitors in renal cell carcinoma cells [ ], whereas Cx26 increases the effect of cisplatin in human bladder cancer cells [ ] and of doxorubicin in prostate cancer cells [ ].
Both channel-dependent and -independent mechanisms have been suggested to underlie this effect [ , ]. In other contexts, connexins can have an inverse effect with respect to drug sensitivity. Reduced Cx43 expression is associated with increased drug sensitivity in glioma cells [ 52 , , , ], whereas the upregulation of Cx26 is associated with gefitinib resistance in lung cancer cells [ ]. The specific inhibition of Cxmediated GJIC in GSCs attenuates proliferation, self-renewal and tumour growth and synergises with temozolomide to induce apoptosis [ ].
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There is also in vivo evidence to support this dichotomy in relation to therapeutic efficacy. For instance, shRNA-mediated knockdown of Cx43 or inhibition of GJIC by meclofenamate and tonabersat strongly potentiates the effect of carboplatin-based chemotherapy on brain metastases from breast and lung carcinoma cells [ 50 ].
Moreover, the efficacy of tumour necrosis factor-related apoptosis-inducing ligand therapy is enhanced when combined with the GJIC inhibitor carbenoxolone in an intracranial glioma model [ ]. The combined use of tumour necrosis factor-related apoptosis-inducing ligand and carbenoxolone could offer a favourable alternative for the treatment of glioma, particularly considering the low cytotoxic nature of carbenoxolone. Notably, carbenoxolone, as well as peptides targeting Cx43, attenuates cancer-induced bone pain [ ], highlighting another facet of cancer care in which connexins can be further explored.
That GJIC between normal cells and tumour cells heterologous GJIC can inhibit the growth of tumour cells was shown in the mid s by Loewenstein and colleagues [ ].
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This phenomenon has since been demonstrated in many cancer types and model systems, as recently reviewed [ ]. Connexins and the tumour stroma. GJIC can occur between cancer cells or in a heterocellular manner between cancer cells and nearby cells such as noncancerous epithelial tissue cells and stromal cells, including cancer-associated fibroblasts, immune cells and vascular and lymphatic endothelial cells. In addition, there is crosstalk via the hemichannel release of autocrine and paracrine signals. These signals influence tumour growth both positively and negatively in a context-dependent manner and help to regulate apoptosis, proliferation, invasion, intravasation and extravasation.
In addition, connexins are thought to be implicated in other communication forms, as a part of tunnelling nanotubes microtubes or extracellular vesicle function e. Other tumours, or parts of tumours, are devoid of GJIC and may or may not express connexins at high levels in the cytoplasm or nucleus, thus escaping the direct GJIC with surrounding cells.
This may be associated with a reduced polarity and cell—cell adhesion. The benefits and drawbacks of maintained GJIC are likely tissue and stage dependent. An understanding of this complex network of signals is essential to move forward with additional therapeutic strategies of targeting connexins in cancer. Tumour cells deliver antigenic peptides through gap junctions to dendritic cells, and this cross-presentation is associated with enhanced immune-mediated tumour elimination.
Presentation of antigenic peptides between melanoma cells and dendritic cells increases the melanoma-specific T-cell response [ ]. The infection of melanoma tumours with Salmonella bacteria induces Cx43 expression, which enhances the antigen presentation into infiltrating dendritic cells, activating an antitumour response [ ]. Cxmediated GJIC may also improve the immune surveillance of tumours via the activation of natural killer cells [ , ].
These studies and others, as reviewed recently [ ] are of particular interest in relation to immunotherapy and tumour vaccines [ ]. Connexins also regulate many other functions within the immune and lymphatic systems, which awaits an additional analysis [ ]. Angiogenesis, the formation of new capillaries from pre-existing blood vessels, is required for cancer progression [ ]. Overexpression of Cx43 in melanoma and breast cancer cells suppresses tumour angiogenesis [ 28 , ].
In accordance with these findings, Cx43 knockdown in melanoma cells increases vessel density [ ]. The silencing of Cx43 in breast cancer cells results in increased vascular endothelial growth factor expression and decreased thrombospondin expression [ 29 ]. In contrast, the overexpression of Cx26 or Cx43 in breast cancer cells is associated with the upregulation and secretion of IL-6 and MCP-1, which inhibit endothelial cell tube formation in vitro and tumour vascularisation in vivo [ 28 ].
Endothelial connexins also play important roles in tumour angiogenesis. The knockdown of endothelial Cx37, Cx40 or Cx43 or the pharmacologic inhibition of GJIC diminishes the angiogenic sprouting of endothelial cells in in vitro studies [ ]. A recent work using a combination of different in vitro, ex vivo and in vivo models has shown that targeting of endothelial Cx40 decreases tumour growth by reducing angiogenesis and improving vessel perfusion [ 87 ].
This was demonstrated both in mouse endothelial-specific Cx40 knockout models and by the injection of the specific peptide 40 Gap27 that binds to the extracellular loop of Cx40 and blocks channel activity [ ]. Because endothelial and myoendothelial gap junctions are fundamentally important for a coordinated vessel response over longer distances [ , ], a therapeutic approach focused on vascular connexins would be conceivable through the specialised targeting of tumour vessels in combination with anti-angiogenic strategies.
There is also evidence of direct communication between tumour cells and endothelial cells Fig. Notably, endothelial cells deliver miR—5p to colon cancer cells via gap junctions, causing the upregulation of Cx43 expression and reduced angiogenesis [ ]. Moreover, the inhibition of GJIC by carbenoxolone blocks the interchange of specific cancer-associated microRNAs between human microvascular endothelial cells and a human glioma cell line [ ]. As mentioned, Cx26 may facilitate extravasation into the endothelium during metastasis [ 47 ], and Cx43 enhances attachment and diapedesis into endothelial cells in models of breast cancer [ 48 , ] and melanoma metastasis [ ].
Heterocellular Cxmediated GJIC between gastric cancer cells and mesothelial cells may facilitate diapedesis during peritoneal metastasis [ 75 ]. Using zebrafish and chicken embryo models of brain metastasis, Stoletov et al. RNAi-mediated depletion of Cx26 and Cx43 in melanoma and breast cancer cells, respectively, or pharmacological inhibition of GJIC using carbenoxolone, was found to inhibit brain colonisation by blocking tumour cell extravasation and blood vessel co-option [ ]. Such crosstalk between cancer cells and blood vessels may also occur via hemichannels, such as the one through ATP release, which ultimately can stimulate angiogenesis [ ].
There is also evidence that GJIC facilitates cancer cell migration through the lymphatic endothelium [ ]. Thus, blockade of connexin-mediated heterocellular communication is emerging as a potential viable strategy for reducing metastasis. Indeed, the Cx43 channel blocker oleamide has antimetastatic properties in the MDA-MB breast cancer cell line, presumably due to the inhibition of extravasation into the endothelium [ 85 ].
Connexins can also elicit pro-tumourigenic effects in brain tumours. Cx43 expression and heterologous GJIC between malignant glioma cells and reactive astrocytes enhance cell invasion into the brain parenchyma [ , , ], which may in part occur through microRNA-mediated signalling [ ]. Glioma cells can also become more resistant to chemotherapy- or radiotherapy-induced cell death through both homocellular and heterocellular GJIC pathways [ 51 , 53 , ], as well as via Cxspecific GJIC-independent mechanisms [ ]. GJIC between reactive astrocytes and melanoma cells protects against chemotherapy through the sequestration of calcium [ 34 ].
In summary, GJIC between tumour cells and astrocytes promotes colonisation, resistance to chemotherapy and survival of tumour cells in the brain [ 34 , 50 , , ]. Targeting of this axis may provide clinical benefits. Rous sarcoma virus-infected fibroblasts display a loss of GJIC, and its oncogene pp60 v-src causes Cx43 tyrosine phosphorylation and reduced Cx43 levels [ ].
Human cytomegalovirus is not recognised as a tumour virus. However, human cytomegalovirus proteins have been detected at high levels in gliomas and can downregulate Cx43 and GJIC [ ]. These data suggest that a range of viruses inhibit GJIC. In , McNutt and Weinstein recognised that gap junction plaques are lost in cervical cancers [ ]. Subsequently, cervical cancers were shown to be caused by HPVs [ ]. Reduced connexin expression can occur in preneoplastic cervical lesions [ ], perhaps in part due to the loss of epithelial differentiation. E6 alters Cx43 trafficking to the plasma membrane either through its ability to alter cell signalling pathways or through its interaction with the Cx43 partner protein Dlgh1 [ 42 , ].
Connexins are responsible for the bystander immunity to viruses [ ], and so viruses may have evolved means to inactivate this response by regulating connexins, a feature that could be exploited therapeutically. Tunnelling nanotubes TNTs are thin actin-based membrane bridges that connect cells over distances of up to several cell diameters [ ]. These structures allow for the intercellular transfer of microRNAs, proteins and cytoplasmic organelles, including mitochondria.
The presence of connexin channels interposed in the nanotube connections permits long-distance electrical coupling between cells [ , ]. TNTs are formed between various cancer cell types in vitro [ ]. TNTs between malignant cells and stromal cells may be involved in chemoresistance [ ] and tumour—stromal crosstalk [ , ]. Recently, Osswald et al. Cxcontaining gap junctions within this network were suggested to turn the tumour into a syncytium of interconnected cells that is highly resistant to radiation therapy, presumably by distributing calcium between cells to prevent apoptosis upon radiation-induced release of intracellular calcium [ ].
Extracellular vesicles EVs , which constitute microvesicles, apoptotic bodies and exosomes, are membrane-based structures that can carry and deliver bioactive molecules, including proteins and nucleic acids, from one cell to another. This form of cell-to-cell communication can occur over very long distances and efficiently cross the blood—brain barrier. EVs released by tumour cells and cancer-associated fibroblasts affect cancer progression by transferring molecules influencing tumour initiation, angiogenesis, metastasis and drug resistance [ ].
Functional Cx43 channels were recently identified in the membrane of EVs [ ]. In melanoma, Cx32, Cx43 and Cx45 were detected in these structures [ ]. Connexins facilitate the transfer or exchange of EV contents with target cells, possibly by improving fusion events with cells [ ]. This may be exploited therapeutically to improve drug delivery. However, in one mouse model, delivery of doxorubicin as a chemotherapeutic agent was not improved in the presence of Cx43 in EVs, although it strikingly reduced cardiotoxicity [ ]. In addition, Cx43 phosphorylation through extracellular signal-regulated kinase signalling induces exosome release upon traumatic brain injury [ ].
Thus, it seems clear that connexins are involved in the formation and function of EVs, but the therapeutic implications await further works. Substantial knowledge on how gap junctions contribute to cancer has accumulated since the seminal work by Loewenstein and Kanno demonstrated a loss of electrical coupling in liver cancer more than 50 years ago [ 7 ].
Connexins predict the prognosis of a number of cancers, although the lack of established protocols and confirmatory independent studies has limited their clinical utility. Several promising studies using an expanded set of tools to modulate connexin or gap junction function have demonstrated potent antitumoral effects. As we decipher their cancer type- and stage-specific roles, significant progress towards targeting of connexins and gap junctions in a patient-specific therapeutic setting can be expected.
Nevertheless, there are significant challenges that remain to be addressed. The fact that connexins can be both anti- and pro-tumorigenic is of particular concern. For example, would enhancing GJIC or connexin expression in a primary tumour run a risk of more efficient metastatic spread or growth? Does inhibition of GJIC in patients with metastasis increase the risk of further tumour dissemination or re-activated tumour growth in sites harbouring dormant tumour cells?
Currently, this clinical problem can only be addressed by hypothetical risk assessment and thus further research, including an extensive use of in vivo models and a careful follow-up of ongoing clinical trials, is required. Currently, only a few of the 21 connexin isoforms have been characterised in terms of their role in cancer.
Additional studies are necessary to elucidate the GJIC-dependent and -independent mechanisms by which the various connexins positively or negatively affect cell growth, differentiation, invasion and other important cancer-associated features. In this context, more specific tools will be required to target the different functions of connexins. Further efforts need to be devoted to the identification of more specific connexin inhibitors. The use of specific peptides and peptidomimetics have shown a great promise towards this.
However, their side effects must be carefully addressed particularly if applied systemically due to the critical functions of several connexins in various organs. Another important challenge will be to dissect the molecular basis of the regulation of the various connexin isoforms at the transcriptional, translational and post-translational levels and to define how the dysregulation of these processes contributes to aberrant levels or subcellular localisation of connexins during various stages of cancer progression.
The identification of solutions to these research challenges will set the stage for new diagnostic and therapeutic advances. Laird DW. Life cycle of connexins in health and disease. Biochem J. Gap junctions and cancer: communicating for 50 years. Nat Rev Cancer. Saez JC, Leybaert L. Hunting for connexin hemichannels. FEBS Lett. An update on minding the gap in cancer.
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Clin Cancer Res. Downregulation of connexin 26 in human lung cancer is related to promoter methylation. Int J Cancer. DNA methylation analyses of the connexin gene family reveal silencing of GJC1 Connexin45 by promoter hypermethylation in colorectal cancer. Variable promoter region CpG island methylation of the putative tumor suppressor gene Connexin 26 in breast cancer.
Cell Mol Neurobiol. Suppression of CX43 expression by miRa in the progression of human prostate cancer. Cancer Biol Ther. Autoregulation of connexin43 gap junction formation by internally translated isoforms. Cell Rep. Internal translation of the connexin 43 transcript.
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Cell Commun Signal. Internal ribosomal entry site IRES activity generates endogenous carboxyl-terminal domains of Cx43 and is responsive to hypoxic conditions. J Biol Chem. Altered translation initiation of Gja1 limits gap junction formation during epithelial-mesenchymal transition. Mol Biol Cell. The effects of connexin phosphorylation on gap junctional communication. Int J Biochem Cell Biol. Leithe E. Regulation of connexins by the ubiquitin system: Implications for intercellular communication and cancer.
Biochim Biophys Acta. Involvement of gap junctions in tumorigenesis: transfection of tumor cells with connexin 32 cDNA retards growth in vivo. Negative growth control of HeLa cells by connexin genes: connexin species specificity. Cancer Res. Retroviral delivery of connexin genes to human breast tumor cells inhibits in vivo tumor growth by a mechanism that is independent of significant gap junctional intercellular communication.
Connexin43 suppresses proliferation of osteosarcoma U2OS cells through post-transcriptional regulation of p The gap junction-independent tumor-suppressing effect of connexin Growth retardation in glioma cells cocultured with cells overexpressing a gap junction protein. Transfection with different connexin genes alters growth and differentiation of human choriocarcinoma cells.
Exp Cell Res. Gap junction genes Cx26 and Cx43 individually suppress the cancer phenotype of human mammary carcinoma cells and restore differentiation potential. Connexins act as tumor suppressors in three-dimensional mammary cell organoids by regulating differentiation and angiogenesis. Down-regulation of Cx43 by retroviral delivery of small interfering RNA promotes an aggressive breast cancer cell phenotype. Aasen T. Connexins: junctional and non-junctional modulators of proliferation.
Cell Tissue Res. Multiple and complex influences of connexins and pannexins on cell death. Gap junctional communication promotes apoptosis in a connexin-type-dependent manner. Cell Death Dis. Gap junction intercellular communication propagates cell death in cancerous cells.
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Reactive astrocytes protect melanoma cells from chemotherapy by sequestering intracellular calcium through gap junction communication channels. Paracrine signaling through plasma membrane hemichannels. Differential effect of subcellular localization of communication impairing gap junction protein connexin43 on tumor cell growth in vivo. Olbina G, Eckhart W. Mutations in the second extracellular region of connexin 43 prevent localization to the plasma membrane, but do not affect its ability to suppress cell growth.
Mol Cancer Res. Connexin43 acts as a colorectal cancer tumor suppressor and predicts disease outcome. A novel route for connexin 43 to inhibit cell proliferation: negative regulation of S-phase kinase-associated protein Skp 2. Connexin controls cell-cycle exit and cell differentiation by directly promoting cytosolic localization and degradation of E3 ligase Skp2. Dev Cell. A functional interaction between the MAGUK protein hDlg and the gap junction protein connexin 43 in cervical tumour cells.
Kotini M, Mayor R. Connexins in migration during development and cancer. Dev Biol. Defamie N, Chepied A.
Mesnil M. Connexins, gap junctions and tissue invasion. Now, Dr. Naznin Virji-Babul at the Djavad Mowafaghian Centre for Brain Health is recruiting participants for a study that aims to turn the standard on its head. Jump to Navigation. Search form Search. Clinic for Alzheimer Disease and Related Disorders. Study saves B.