Although a variety of connexins are involved in GJIC in different cell types, the expression of Cx43 in vascular endothelial cells and in many tumor cell lines indicates a primary role for this protein in the heterologous gap junctional communication during tumor cell-endothelial cell interaction [23
]. Functional GJIC between tumor cells and vascular endothelial cells mediated via Cx43 has been demonstrated [9
]. Consequently, it was also shown that quantitative and qualitative changes in connexin expression are associated with tumor progression, proliferation, invasion, and metastasis [9
]. Cx43 enhanced the adhesiveness and mediated the invasion in malignant gliomas [20
]. In addition, the increased expression of specific connexins correlated with increased invasiveness of lung squamous cell carcinoma or NIH3T3-Ras cells [29
] and Cx43 expression was upregulated in urethane-induced mouse lung adenomas [31
]. Increased levels of Cx43 mRNA was found in hepatocellular carcinoma tissues [32
] and in the stroma of multiple intestinal neoplasia-mice adenomas [23
]. Expression of Cx43 in mammary carcinoma cell lines lacking endogenous Cx43 enabled the formation of heterocellular GJIC with microvascular endothelial cells and increased their diapedesis [12
]. Increased cytosolic and plasma membrane expression of Cx43 in lymph node metastases of breast cancer was demonstrated [11
]. Cx43 was detectable in normal lung, smaller size tumor, and larger size mouse lung tumors [24
]. Our study shows a role for Cx43 in breast cancer metastasis to the lung in a syngeneic, murine experimental metastasis model.
Although connexins are classically considered as gap-junctional proteins, non-junctional roles for connexins in hemichannels with ion-channel-like functions or possessing channel-independent, signaling-type functions have received considerable attention recently [33
]. Adhesion-mediated establishment of functional GJIC between lung-metastatic B16F10 melanoma cells and endothelium has been shown to be dependent on the expression of Cx43 in both cell partners [35
The major conclusion of this study is that gap junctional communication via Cx43 facilitates metastatic homing by increasing the arrest of cancer cells in the lung vasculature. The reduced attachment of cancer cells with the dominant-negative G138R that allows formation of gap-junction plaques but not of functional junctions suggests a positive correlation between the loss of intercellular communication and the number of adherent tumor cells in the lungs. The idea that the formation of functional gap junctions between a cancer cell and the endothelial cell is critical for tumor cell adhesion to the pulmonary endothelium is novel. Normal gap junction plaques are found in the lateral membranes of cells, below the level of the tight junction belt. In the case of heterologous gap junction formation with adherent cells, the apical surface of the endothelial cells comes into contact with the surface of cancer cells (Figure ). Therefore, a role for connexins in heterologous adhesion or in establishment of functional GJIC would require the presence of connexins at the apical surface of endothelial cells, as demonstrated for Cx43 in Figure . Very high concentration of Cx43 has been demonstrated at the apical surface of epithelial cells [36
]. The residual coupling between Cx43G138R expressing tumor cells and endothelial cells expressing wild-type Cx43 in comparison to 18β-glycyrrhretinic acid treated cells (Fig red and blue bars) is mediated through heterotypic Cx43G138R-Cx43WT channels. These channels were recently described as strongly inhibited but not completely abolished in their function [37
]. The decreased attachment efficiency with the G138R mutation might also be the result of alteration in conformation of the Cx43 protein. The C61S mutant does not express the mutated Cx43 in the membrane; therefore, it did not alter the cell adhesion mediated by endogenous Cx43. The increased adhesion of cancer cells with overexpression of wild-type Cx43 further confirms the role for Cx43 in tumor cell attachment efficiency as a result of enhanced GJIC.
The marked upregulation of Cx43 in tumor cell-endothelial cell contact areas, whether in preexisting 'homing' vessels or in newly formed tumor vessels, indicates that Cx43 can serve as a marker of micrometastases and tumor vasculogenesis. This raises the possibility of a role for Cx43 in the early incorporation of endothelial cells into pre-hypoxia size tumors as seeds for vasculogenesis.
Cx43 has been shown to enhance angiogenesis in vitro
]. Tube formation by human umbilical vein endothelial cells cocultured with Cx43-transfected malignant glioma cells or with naturally Cx43-expressing malignant glioma cells was significantly increased compared with tube formation by endothelial cells alone [5
]. In addition, upregulation of Cx43 in the hypertrophic myocardium in mice suggested its role in cardiac angiogenesis [38
]. These reports are consistent with our finding of upregulation of Cx43 in cancer cell-endothelial cell contact areas. This selective upregulation of Cx43 can be exploited as a marker for sites of intravascular metastases as well as for vasculogenic loci within metastatic tumors. Previously, it had been shown that Cx43 mRNA in normal tissue surrounding lung tumor may act as a molecular marker of nodal micrometastasis in non-small cell lung cancer [39
The association between cancer and the lack of communication between cancer cells set the stage for a role of dysfunctional gap junctions in carcinogenesis nearly 40 years ago [40
]. Since then, both increased and decreased levels of various connexins shown in different models of primary and metastatic tumors had generated multiple interpretations [41
]. The difficulty in developing a unified hypothesis regarding the role of connexins in primary and secondary carcinogenesis may be associated with a multiplicity of contradictory functions that the metastatic cell has to perform during intravasation and distant localization. A recent review predicted that "a gain of function (of gap junctions) may characterize the metastatic stage" [41
]. Our results confirm this prediction and we hope that they will set the stage for a renewed assessment of the role of connexins in metastatic tumorigenesis and tumor vasculogenesis.