Aberrant Cx43 Expression and Mislocalization in Metastatic Human Melanomas

Abstract

At present, it is unclear if melanocytes contain Cx43 gap junctions and whether Cx43 expression is regulated in melanoma onset and progression. To this end, we cultured pure populations of mouse melanocytes and found that they had no detectable Cx43 and exhibited an inability for dye transfer indicating they were devoid of functional gap junctions. Given the evidence that melanomas acquire the expression of other connexin isoforms during tumor progression, we assessed if Cx43 was also expressed and assembled into gap junctions at any stage of human melanoma onset and progression to distant metastases. Nearly all primary melanomas within the epidermis lacked Cx43. In contrast, nodal metastases expressed low levels of Cx43 which was markedly higher in distant metastases that had invaded vital organs. Importantly, in all stages of melanoma progression, Cx43 could be detected in intracellular compartments but was rarely assembled into gap junctions indicative of functional gap junction channels. Overall, these studies suggest that melanocytes do not form Cx43 homocellular gap junctions and even though Cx43 levels increase during melanoma progression, this connexin rarely assembles into gap junction structures.

Keywords: Cx43; connexin; gap junctional intercellular communication; gap junctions; melanoma..

Figure 1

Primary mouse melanocytes show no evidence of Cx43 expression or GJIC Primary mouse melanocytes stained positive for L-DOPA (black pigment) (A) and were essentially pure across passage P2 and P3 (N = 3, n = 15), as revealed by staining for TRP1 (B; red). (C) Melanocytes exhibited no evidence of Cx43 expression, in comparison to Cx43-rich REKs, and (D) undifferentiated and differentiated primary mouse keratinocytes, which show evidence of Cx43 GJs (arrows) at the junctional membrane (E-cad= E-cadherin). (E) Outlined melanocytes loaded with calcein dye were photobleached and dye recovery was measured every 3 seconds for 450 seconds. In contrast to photobleached REKs (N = 3, n = 30), photobleached melanocytes (N = 3, n = 30) did not receive calcein dye indicating lack of GJIC. (F) Quantified data illustrates fluorescence recovery. Bars represent mean +/- SEM. Scale bar = 20 μm.

Figure 2

Increased intracellular Cx43 in distant human melanoma metastases (A) The majority of images analyzed from primary melanoma (N = 14), and (B) nodal metastases (N = 15) did not show evidence of Cx43 expression as opposed to normal skin where Cx43 gap junctions were abundant (A). In contrast, (C) the majority of images analyzed from melanoma metastases to distant organ sites (N = 7) showed evidence of Cx43 expression, however, this expression was predominately intracellular and did not form gap junction plaque-like structures indicative of potentially functional gap junction channels. Arrows indicate punctate Cx43 gap junction structure at the cell-cell interface. (D) Regardless of the distant organ site of melanoma metastases, tumor cores from lung, femur, pelvic wall, and kidney stained positive for MITF, indicating the tumor was from a melanocytic cell lineage. Bars represent mean +/- SEM. Scale bar = 20 μm.