Early efforts looking for Cx mutations in cancer specimens found that 4 of 7 primary human hepatic angiosarcomas carried a proline-to-serine substitution in codon 319 of the Cx37 gene (Saito et al., 2000). The same genetic variant was found in DNA extracts from adjacent non-tumor tissue, suggesting that it corresponded to a germline mutation or a single nucleotide polymorphism. The amino acid 319 is located in the cytoplasmic tail of the protein that contains diverse regulatory motifs. Of note, Cx37 has been shown to form functional hemichannels (Wong et al., 2006) and its expression reduced the proliferation of cultured rat insulinoma cells (Burt et al., 2008). In follow-up studies it was shown that introduction of Cx37 mutations that abrogate both the intercellular channel and hemichannel functions (T154A and C61A-C65A) failed to suppress the proliferation (Good et al., 2011, 2012). The inablity of the mutant protein to mimic the effect of the wild type Cx37 in cell proliferation indicates that the passage of molecules through Cx37-based channels is required to influence cell growth/survival. In addition, association between the C1019T polymorphism of the Cx37 gene, gastric adenocarcinomas and H. pylori infection was identified in a retrospective Chinese cohort (Jing et al., 2012).

Another study looking for Cx mutations in sporadic colorectal cancers found 2 frameshift Cx43 mutations in 3 of 6 studied tumors (a single nucleotide deletion A311V in 2 samples and a single nucleotide insertion I358N in 1 tumor) (Dubina et al., 2002). No alterations in Cx32 gene were identified. The Cx43 variants were mapped to the carboxy-terminal tail of the peptide and were not detected in DNA extracts from peripheral blood samples, confirming their somatic nature. However, such mutations have not been mechanistically characterized or further reported in larger tumor series.

Using a high-resolution array-based CGH platform to screen human hepatocellular carcinomas, deletions in the region encoding the Cx40.1 gene were identified (Zender et al., 2008). In support of a tumorigenic effect of this variant, an in vivo, xenograft-based RNAi screen showed that Cx40.1 downregulation using shRNA elicited a prominent acceleration of tumor growth. Little is known about the properties of human Cx40.1. For instance, it is expressed in the human heart (Söhl and Willecke, 2004), but its channel forming capacity remains to be proven.

Deletions of an 817 kb area at 6q14-21 containing the Cx62 gene were found in DNA extracts from 21 of 55 human prostatic adenocarcinomas using a high-resolution single nucleotide polymorphism array (Liu et al., 2007). However, the levels of Cx62 mRNA from the ONCOMINE database were comparable between tumor samples and normal controls, suggesting that another transcript located in this deleted segment such as MAP3K7 might have tumor suppressive actions.

Aggressive oral squamous cell carcinomas are predominantly associated with alcohol and tobacco use and are extremely rare in children. Interestingly, the case of a 6-year old girl was reported with psoriasiform dermatitis and sensorineural hearing loss that presented with a high grade, 3.3 cm palatal and nasal squamous cell carcinoma (Rednam et al., 2011). Due to the clinical findings the patient was screened and the single nucleotide Cx26 mutation F142L was found. Although there was no demonstration of causal relationship between the tumor and the Cx26 variant, other Cx26 mutations associated with excessive keratinocyte proliferation and syndromic hearing loss have been shown to produce hemichannels with increased function and altered permeability (Sánchez et al., 2010; Mese et al., 2011).

Epigenetic regulation of Cxs is a well-described process (reviewed in Vinken et al., 2009; Oyamada et al., 2013). Reduction in the expression of diverse Cxs with concomitant promoter hypermethylation has been shown in multiple human neoplasms and tumor cell lines. Downregulation of Cx32 gene expression by promoter hypermethylation was shown using bisulphite modified DNA-based PCR and a restriction enzyme-based assay in HK-2 and Caki-2 human renal cell carcinoma cells (Hirai et al., 2003). Treatment of cells with the demethylating agent 5-Aza-2′-Deoxycytidine restored Cx32 expression, confirming that Cx32 expression was suppressed. Similarly, downregulation of Cx43 in nasopharyngeal carcinoma CNE-1 cells was found to be associated with GJA1 (Cx43) gene hypermethylation (Yi et al., 2007). A high frequency of Cx30, Cx36, and Cx37 promoter hypermethylation was also detected in colorectal carcinoma samples, as well as in Cx30, Cx36, and Cx45 in colon cancer cell lines (Sirnes et al., 2011). However, correlation between reduced transcript levels and the presence of promoter methylation was found only for Cx45. A more recent study identified Cx45 promoter hypermethylation in 33% of 485 colorectal carcinomas and a positive association with the actionable oncogenic BRAF exon 15 mutation (e.g., V600E) was noted (Ahmed et al., 2011).

Low Cx43 protein levels in human non-small cell carcinomas were associated with Cx43 CpG island promoter methylation and with heavy tobacco use (Jinn and Inase, 2010). Strikingly, the presence of Cx43 promoter hypermethylation in the non-tumor peritumoral lung tissue, but not in the tumor tissue proper, was significantly associated with lymph node positivity in non-small cell cancer patients (Chen et al., 2003). The biological determinants of this association are not clear. Reduced Cx26 protein and mRNA levels in a variety of lung carcinomas (including small-cell neuroendocrine carcinomas) and cell lines was also correlated with promoter hypermethylation and reverted by 5-Aza-2′-Deoxycytidine (Chen et al., 2005). Cx26 promoter hypermethylation was also found in 11 of 20 human invasive breast carcinomas and in cell lines (Tan et al., 2002). However, the absence of Cx26 expression in human squamous esophageal carcinoma cells was not correlated with the presence of Cx26 promoter hypermethylation, suggesting the presence of a different silencing mechanism (Loncarek et al., 2003). Using methylation specific PCR, we found Cx32 promoter hypermethylation in 4 of 9 frozen samples from human gastric adenocarcinomas (Schalper et al., unpublished observation, Figure 1). The 246 bp methylation-specific band was detected exclusively in the tumor and not in non-tumor areas of the samples (Figure 1A). However, we found no clear relationship between the presence of Cx32 promoter hypermethylation and Cx32 protein levels as detected by immunohistochemistry (Figures 1B,C). Taken together, these findings suggest that although more frequent in cancer tissues, promoter hypermethylation in Cxs genes is not unequivocally related with reduced/absent protein.

To our knowledge, regulation of Panx genes by epigenetic control has not been reported. Using the NIH PROSCAN webtool (http://www-bimas.cit.nih.gov/molbio/proscan/), we evaluated a 1000 bp region upstream of the initiation codon of the human Panx1 gene. We identified a 434 bp region with diverse transcription factor binding motifs and a significant probability of being a promoter region (Promoter score = 88.86; Promoter cutoff = 53.00). In the same genomic region and using the EMBL-EBI Cpgplot tool (https://www.ebi.ac.uk/Tools/seqstats/emboss_cpgplot/) we detected a 554 bp region with >50% C/G nucleotides, consistent with a CpG island. The latter suggests that human Panx1 has a CpG nucleotide-rich promoter region and could be modulated by hypermethylation.

Few events associated with histone modifications have been shown to affect Cx26 and Cx43 genes in human neoplasms. Li and collaborators (Li et al., 2013) recently found an inverse relationship between Cx26 and JARID1B (also known as KDM5B) histone demethylase protein levels in transitional cancer cell lines and advanced human bladder tumors. Overexpression of JARID1B was associated with reduced Cx26 protein in HT1376 and T24 cells, indicating that this demethylase represses Cx26 expression.

In androgen dependent (LNCaP) and androgen-independent human prostate cancer cell lines (DU145 and PC3), treatment with the histone deacetylase inhibitor Trichostatin A prominently increased Cx43 mRNA and protein expression. The intercellular transfer of the fluorescent dye Lucifer yellow was also increased by Trichostatin A, indicating increased gap junction mediated intercellular communication (Hernandez et al., 2006). Similar findings were observed in cultured non-malignant human peritoneal mesothelial cells (Ogawa et al., 2005).

Post-transcriptional modulation by small non-coding RNAs of Cx genes has also been reported in human tumors. Li et al. (2012a,b) found an inverse relationship between miR-20a and Cx43 protein and mRNA expression in human prostate tumors and cell lines. In this model, downregulation of miR-20a caused a 4-fold increase in Cx43 expression and reduced the cell proliferation. Using a luciferase reporter assay, the miR-20a antagomir increased the luciferase activity using the wild type Cx43 sequence, but was ineffective with Cx43 mutated at the miR-20a binding site at the 3′UTR region. The latter demonstrates that Cx43 is a direct target of miR-20a. Using a similar approach, miR-221/222 was shown to bind Cx43 mRNA, reduce Cx43 expression and promote cell growth and invasion in the human glioblastoma multiforme cell line U251 (Hao et al., 2012). Finally, transfection of miR-218 in nasopharyngeal carcinoma cells reduced Cx43 expression and was associated with increased apoptosis. Moreover, miR-218 transfection reduced the tumor growth after intramuscular implantation of C666-1 cells in SCID mice (Alajez et al., 2011). Although likely to occur, regulation of Cxs expression by lncRNAs has not been reported yet. In addition and to our knowledge, modulation of Panxs by non-coding RNAs has not been communicated.

The cellular NAD⁺ homeostasis is tightly regulated in all organisms and contributes to the maintenance of the energetic balance, intracellular redox potential and signal transduction. Tumor cells undergo metabolic adaptations to support growth and survival, including a pronounced shift from oxidative phosphorylation toward more NAD⁺ and lactate production through aerobic glycolysis, a phenomenon known as the Warburg effect (Chiarugi et al., 2012). The net cellular NAD⁺ content is the result from its synthesis by various enzymatic cascades involving niacin (vitamin B3), tryptophan, aspartic acid and reutilization of intracellular nicotinamide-related compounds, as well as NAD⁺ degradation through enzymatic hydrolysis, ribosylation and deacetylation. CD38 is the major mammalian NAD⁺ glycohydrolase and ADP-ribosyl cyclase, thus directly participating in NAD⁺ metabolism and production of second messengers (Chiarugi et al., 2012). However, the catalytic domain of CD38 is located in the extracellular domain, apart from its cognate intracellular substrate.

The first experimental evidence for the involvement of Cx43 hemichannels in transmembrane NAD⁺ transport came from studies using cultured NIH3T3 fibroblasts, HeLa cells and proteoliposomes (Bruzzone et al., 2001). In these models, NAD⁺ efflux required (and was paralleled by) Cx43 expression. Moreover, the NAD⁺ release was increased by lowering the extracellular Ca²⁺ concentration and prominently blocked by beta-glycyrrhetinic acid, La³⁺ and a Cx43 monoclonal antibody, thus implicating Cx43 hemichannels (Bruzzone et al., 2001). Shortly after, the same group convincingly demonstrated the involvement of Cx43 hemichannels in the [Ca²⁺]_i-dependent, cADPR-induced cell proliferation in co-cultured mouse NIH/373 fibroblasts with or without CD38 expression (Franco et al., 2001; De Flora et al., 2004). In this model, the efflux of NAD⁺ through activated hemichannels allowed the interaction of this intracellularly produced dinucleotide with the extracellular catalytic segment of CD38, giving a mechanistic response to this topographical paradox. After its production by CD38-mediated NAD⁺ ribosylation, the ubiquitous second messenger cADPR can re-enter the cells to induce IP₃-pathway independent-/ryanodine receptor induced Ca²⁺ transients and ultimately mediate cell cycling and proliferation. Later studies by another research group extended these observations and showed that cADPR re-uptake is mediated by Cx43 hemichannels through bidirectional NAD⁺ extrusion/cADPR import upon FCγ receptor stimulation in cultured murine J774A.1 macrophages (Song et al., 2011) (Figure 2, green colored arrows and text).

Although CD38 is expressed in various mammalian tissues including brain, prostate and muscle, it is most prominently found in lymphoid/blood cells (Deaglio et al., 2008). CD38 is frequently upregulated and has been indicated as a prominent oncogenic and adverse prognostic factor in hematologic malignancies including multiple myeloma and chronic lymphocytic leukemia. Moreover, humanized monoclonal antibodies targeting CD38 (e.g., daratumumab, MOR03087, and SAR650984) are under evaluation in early phase clinical trials to treat patients with advanced hematological B-cell malignancies (www.clinicaltrials.gov; trial identifiers NCT01615029, NCT00574288, NCT01749969, NCT01084252 and NCT01421186). As an integral component of the CD38-Cx43-cADPR axis and NAD⁺ degradation pathway, it is tempting to speculate that expression of Cx43 in neoplastic cells could serve as a prognostic/predictive biomarker for such compounds and modulation of Cx43 hemichannels may itself represent a novel anti-tumor therapeutic target, especially in CD38 expressing B cell malignancies.

Cx43 was shown to be expressed in isolated human B cells from tonsil and peripheral blood and Cx-based channels participate in immunoglobulin production and B cell maturation (Oviedo-Orta et al., 2000, 2001). In addition, Cx43 defective knockout mice have lower IgM-positive B cells than their wild type littermates and the defect is more pronounced in the homozygous than in the heterozygous animal, supporting a gene-dosage effect (Montecino-Rodriguez and Dorshkind, 2001). Although some authors have ascribed the functional effects of lymphocyte Cx43 to their participation in the pool of intercellular channels located at the cell-cell contacts between activated immune cells (e.g., “the immunological synapse”), the involvement of hemichannels in these processes has not been excluded. This is particularly relevant in immune cells that are expected to spend a considerable part of their lifespan detached from other cells. In this regard, the reduction in immunoglobulin levels observed by Oviedo-Orta and collaborators in primary human lymphocytes was triggered by traditional Cx-channel blockers and by a Cx43 mimetic peptide corresponding to the second extracellular loop (e.g., Gap27) (Oviedo-Orta et al., 2001), known to inhibit Cx43 hemichannels (Evans et al., 2006; Wang et al., 2012). Notably, additional short peptides corresponding to the intracellular domains of Cx43 (e.g., Gap18 and Gap20) were ineffective in this model.

More recently, Cx43 was shown to be required for activation and migration of cultured murine B cells (Machtaler et al., 2011). In this study, B cell responses were independent of intercellular gap junction channels and immunofluorescence experiments of Cx43 in the murine B lymphoma cell lines WEHI 231 and A20, and in primary murine B splenocytes, showed a predominant homogenous membranous distribution. The latter indicates a lack of preferential clustering/enrichment of Cx43 in areas of cell-cell contact. A similar diffuse membranous Cx43/CD38 co-localization staining pattern was observed in cultured J774A.1 macrophages (Song et al., 2011).

Surprisingly few studies have explored the expression of Cxs in native human lymphoid tissues and human lymphoid neoplasms. For instance, Cx43 was detected in germinal centers of fresh samples from human tonsil and spleen (Krenacs et al., 1997). Of note, Cx43 protein and mRNA were predominantly located in cells with irregular shape and co-expressing CD21 and CD35, consistent with follicular dendritic cells. Only scarce, more rounded cells with lymphocyte appearance and of undefined lineage were found to carry the Cx43 transcript in this study.

Aiming to characterize Cx43 expression in native human lymphoid tissues and lymphoid malignancies, preliminary experiments from our group using chromogenic immunohistochemistry detected low levels of Cx43 protein in formalin fixed-paraffin embedded samples from 4 non-tumor lymph nodes and absence of signal in a set of 22 malignant B cell lymphomas (13 Diffuse large B cell lymphomas [DLBCL] and 9 classical Hodgkin lymphomas [CHL]) (Figure 3). While distinctive Cx43-positive plaques were evident toward the intercalated discs of human myocardiocytes used as positive control (Figures 3A,B), only focal labeling of Cx43 in occasional germinal centers from reactive-type lymph nodes was seen in cells with morphology consistent with endothelial cells, macrophages and follicular dendritic cells (Figures 3C,D). The latter finding is somewhat consistent with previous observations by Krenacs and collaborators (Krenacs et al., 1997) and points to the possibility that Cx43 expression in lymphocytes might be too low to be detected by immunohistochemistry in unstimulated lymph nodes (e.g., lower than in myocardium). Alternatively, other Cxs known to be present in lymphocytes such as Cx40 (Oviedo-Orta et al., 2001) or Panxs could represent the main hemichannel forming pool in native human lymphoid tissues and lymphomas. Of note, Panx1 hemichannels have been previously shown to be expressed in T lymphocytes and to allow the passage of ATP that is structurally related with NAD⁺ and has a similar molecular mass (MW NAD⁺ = 663.42 vs. ATP = 551.14) (Woehrle et al., 2010). In addition, we found membranous-like Panx1 expression in samples from high grade, DLBCL that showed prominent co-localization with the pan-B cell marker CD20 in confocal stacks (Shoji et al., unpublished observation). Future studies using more sensitive methods and functional assays will be required to clarify this.

The presence of hemichannels in tumor cells even without CD38 expression might confer on them growth/migration advantages through increased autocrine/paracrine NAD⁺ signaling and the possible interaction with CD38 present in the membrane of adjacent non-tumor cells (e.g., stromal cells). In addition, relatively low increases in the extracellular NAD⁺ concentration (in the micromolar range) through cell death/lysis or release through activated hemichannels can exert local immune suppressive effects through activation of ionotropic P2X₇ purinergic receptors in immune cells, leading to effector cytotoxic T cell death and expansion of immune suppressive regulatory CD4+/CD25+/FoxP3+ T cells (Tregs) (Scheuplein et al., 2009; Hubert et al., 2010). The latter effect could favor tumor progression by shielding cancer cells from immune-mediated cell killing (Figure 2). Indeed, increasing studies have found elevated Cx43 and/or Cx26 to be associated with more aggressive (e.g., shorter survival) and disseminated disease in diverse human malignancies and in vitro cancer models (Kanczuga-Koda et al., 2006; Tate et al., 2006; Naoi et al., 2007; Kyo et al., 2008; Cronier et al., 2009; Bui et al., 2011; Lamiche et al., 2012; Stoletov et al., 2013; Tang et al., 2013; Ghosh et al., 2014). In addition, a recent study demonstrated that increased post-treatment Cx43 levels were significantly associated with response to neoadjuvant chemotherapy in human breast carcinomas (Teleki et al., 2013) and high tumor Cx43 expression by immunohistochemistry was predictive of response to platinum-based chemotherapy in non-small cell lung cancer (Du et al., 2013). Notably, many of these studies reported predominant cytoplasmic location of Cxs and failed to detect a corresponding increase in the intercellular gap junctional communication. Recently available antibodies capable of selectively detecting (and blocking) Cx hemichannels through recognition of extracellular loop sequences (Riquelme et al., 2013a) might shed light on the relative contribution of hemichannels and intercellular channels in these responses.

ATP is responsible for energy storage and transfer in the form of renewable high-energy phosphoryl bonds. In addition to its role in cell metabolism, intracellular ATP participates in the maintenance of electrochemical gradients through the fueling of pumps, active vesicular transport and also serves as a coenzyme and mediator for signal transduction and DNA synthesis. The cellular levels of ATP are determined by its constant production occurring mostly through oxidation of a variety of carbon-based substrates (e.g., aerobic cellular respiration); and degradation by hydrolysis into the lower level nucleotide phosphoforms ADP and AMP.

In addition to the prominent intracellular function, extracellular ATP serves as a pleiotropic intercellular messenger through its interaction with ionotropic (P2X₁–P2X₇) and metabotropic (P2Y₁, P2Y₂, P2Y₄, P2Y₆, P2Y₁₁, P2Y₁₂, P2Y₁₃, and P2Y₁₄) P2 purinergic membrane receptors (Falzoni et al., 2013). Examples of this include its function as neurotransmitter, proliferation stimulating agent, pro-inflammatory mediator and signaling factor between immune cells. In particular, extracellular ATP has been recognized to have a key role in the tumor-host interactions (reviewed in Di Virgilio, 2012). Despite its prominent extracellular role, the routes for cellular ATP release in both tumor and non-tumor cells are not well characterized. Diverse mechanisms have been proposed including passive transmembrane diffusion, vesicular release/exocytosis, cellular lysis and extrusion through specific chloride channels, ABC transporters, P2X₇ receptors and Cx/Panx hemichannels (Di Virgilio, 2012; Baroja-Mazo et al., 2013; Falzoni et al., 2013; Orellana et al., 2013). The first direct indication of ATP release occurring through Cx hemichannels came from experiments showing that the propagation of Ca²⁺ waves in cultured astrocytes required the presence of extracellular ATP and Cxs expression, but not of functional intercellular channels (Cotrina et al., 1998). In addition, cultured C6, HeLa and U373 cells showed UTP-induced ATP release only after transfection with Cx26, 32, or 43 and the responses were mimicked by removal of extracellular Ca²⁺ (Cotrina et al., 1998). Later on, numerous studies confirmed the permeability of Cx hemichannels to ATP in diverse cell types and using an extensive repertoire of experimental conditions including the removal of extracellular divalents (Arcuino et al., 2002; Stout et al., 2002; Stout and Charles, 2003; Gomes et al., 2005; Pearson et al., 2005; Zhao et al., 2005; Bahima et al., 2006), mechanical stimulation (Arcuino et al., 2002; Stout et al., 2002; Gomes et al., 2005; Zhao et al., 2005; Richter et al., 2014), increased [Ca²⁺]_i (Braet et al., 2003a,b; De Vuyst et al., 2009), hypoxia/ischemic like conditions (Faigle et al., 2008; Clarke et al., 2009), membrane depolarization (Kang et al., 2008; Nualart-Marti et al., 2013), application of bacterial lypopolysaccharide (De Vuyst et al., 2007), treatment with amyloid beta peptide (Orellana et al., 2011), exposure to hypotonic stress (Lu et al., 2012), after spinal cord injury (Huang et al., 2012), in activated polymorphonuclear granulocytes (Eltzschig et al., 2006), induced by gamma irradiation (Ohshima et al., 2012) and after air-stimulation of cultured keratinocytes (Barr et al., 2013). Hemichannels formed by Panx1 have also been consistently shown to allow the passage of ATP in diverse cell types and experimental models (Bao et al., 2004; Locovei et al., 2006; Huang et al., 2007; Reigada et al., 2008; Schenk et al., 2008; Buvinic et al., 2009; Iglesias et al., 2009; Qiu and Dahl, 2009; Ransford et al., 2009; Chekeni et al., 2010; Garré et al., 2010; Murata et al., 2010; Sridharan et al., 2010; Woehrle et al., 2010; Seminario-Vidal et al., 2011; Dolmatova et al., 2012; Iglesias and Spray, 2012; Li et al., 2012a,b; Lohman et al., 2012; Sandilos et al., 2012; Xia et al., 2012; Xiao et al., 2012; Pinheiro et al., 2013; Riquelme et al., 2013b). Moreover, Panx1 hemichannels are functionally coupled to P2X and P2Y receptors activation (Locovei et al., 2006; Pelegrin and Surprenant, 2006; Wang et al., 2013a,b).

Intracellular Ca²⁺ is recognized as key second messenger and master regulator of cell fate. Altered cytosolic Ca²⁺ can be associated with cell death and cell proliferation, depending on the cell type, concentration and biological context. The intracellular Ca²⁺ balance is tightly regulated and active mechanisms have evolved to keep its levels in the low nanomolar range under resting conditions. The extracellular levels of Ca²⁺ are several orders of magnitude higher (e.g., milimolar range), creating a prominent inward concentration gradient. A complex signaling system administrates dynamic intracellular Ca²⁺ reservoirs within organelles such as the endoplasmic reticulum, mainly through the function of endomembranous ion channels, Ca²⁺ ATPases and binding to Ca²⁺ rich proteins (Berridge et al., 2003). Also, this system allows the functional coupling between intracellular Ca²⁺ stores and extracellular signals through activation of plasma membrane receptors. Such surface receptors include the dimeric tyrosine kinase growth factor-type receptors (e.g., EGFR, FGFR, PDGFR, etc.) and a wide array of seven-transmembrane domain G-protein coupled receptors (e.g., P2Y purinergic receptors, peptide hormone receptors, chemokine receptors, neurotransmitters, etc.). In addition, the influx of Ca²⁺ through ligand-activated channels also participates in the intracellular Ca²⁺ homeostasis. Intracellular Ca²⁺ signaling and the generation of intercellular Ca²⁺ waves have been involved in key cancer-related process including escape from apoptosis, perpetuation of growth, cell migration, metastasis and promotion of angiogenesis (Monteith et al., 2007; Parkash and Asotra, 2010; Monteith et al., 2012). In particular, Ca²⁺ increases have been shown to favor cell proliferation through altered transcription and enhance cell motility through remodeling of actin cytoskeleton and focal adhesions (Lee et al., 2011; Monteith et al., 2012). Moreover, remodeling of Ca²⁺ signaling is a feature of diverse cancers and consists frequently in altered expression and function of Ca²⁺ channels and pumps (Roderick and Cook, 2008; Monteith et al., 2012). For instance, purinergic receptors, TRP channels, store operated Ca²⁺ channels (e.g., ORAI and STIM) and Ca²⁺ pumps are deregulated in diverse human tumors (Lee et al., 2011; Chen et al., 2013). Intracellular effectors commonly involved in the pro-oncogenic effects of Ca²⁺ include protein kinases (e.g., PKC), phosphatases (e.g., calcineurin), transcription factors (e.g., NFAT) and Ca²⁺-sensitive signaling proteins (e.g., calmodulin).

As discussed, the activation of hemichannels is linked to the intracellular Ca²⁺ balance by mediating the autocrine/paracrine signaling through transmembrane exchange of nucleotides and Ca²⁺-modifying second messengers (reviewed in Wang et al., 2013a,b) (Figure 2, blue arrows and text). In addition, early observations suggested that hemichannels activated by metabolic inhibition induced massive Na⁺ and Ca²⁺ entry in cultured rabbit myocardiocytes (Li et al., 2001). In support of this notion, recent data using reconstituted systems support that hemichannels formed by Cx26 (Fiori et al., 2012), Cx32 (Sánchez et al., 2009) and Cx43 (Schalper et al., 2010) allow Ca²⁺ influx (reviewed in Orellana et al., 2012). Notably, increasing the [Ca²⁺]_i with endogenous ligands (e.g., FGFs) or a Ca²⁺ ionophore enhances the activation of hemichannels formed by Cx32 and Cx43 (De Vuyst et al., 2006, 2009; Schalper et al., 2008a; Garré et al., 2010). Also, autocrine/paracrine signaling by additional (non-nucleotide) hemichannels permeants such as IP₃, glutamate and prostaglandins involve increased intracellular Ca²⁺ (Schwartz and Alford, 2000; Meves, 2006; Gossman and Zhao, 2008; Traynelis et al., 2010), suggesting the possibility of hemichannels-mediated Ca²⁺ influx/Ca²⁺-increase/Ca²⁺ influx loops (see Figure 2). To our knowledge, Panx hemichannels have not yet been proven to be Ca²⁺ permeable. However, they can also be activated by increased [Ca²⁺]_i and the permeability properties of Panx1 hemichannels suggest that they are likely to allow the passage of ions. Taken together, these data suggest that under specific pro-tumorigenic circumstances (e.g., altered signal transduction and tissue microenvironment) activated hemichannels could favor cancer progression by directly increasing intracellular Ca²⁺-dependent tumor cell proliferation and motility.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.