Dock180 protein is one of the dedicator of the cytokinesis superfamily which plays a role as a GEF for Rho GTPases . Moreover, Rac1 is a member of the Rho GTPases which control signal transduction pathways in eukaryotic cells and contributes to cell migration and invasion resulting from a variety of stimuli [10, 24]. Eleven members of the Dock family have been identified, including Dock-A (Dock180, Dock2, Dock5), Dock-B (Dock3, Dock4), Dock-C (Dock6-8), and Dock-D (Dock9-11) . While most other Dock members use the conserved Docker domain, Dock180 uses the characteristic Dbl homology domain to regulate the GDP/GTP exchange on Rho GTPases . Furthermore, Elmo1 is necessary for the nucleotide exchange in Rac1 as well as Dock180 .
Elmo1, initially known as a mammalian homolog of C. elegans Ced-12, is essential for the regulation of cell migration and the engulfment of dying cells . Also, Elmo1 interacts with Crk and Dock180 functionally to promote phagocytosis and morphological changes, including filopodia formation associated with cell motility [16, 26, 27]. Moreover, Elmo1, which inhibits the ubiquitination of Dock180 , attaches to Dock180 and functions as an unconventional bipartite GEF for Rac1 . Previous studies reported that the small GTPase RhoG interacted with Elmo1 directly. Rac1 can be activated by a complex including Dock180 and Elmo1, resulting in cell migration, phagocytosis, and neurite outgrowth [28, 29]. Also, Dock180 and Elmo1 are known to be conserved proteins contributing to multiple biological processes associated with cell migration [26, 30, 31].
The upregulation of Dock180 protein has been reported to be involved in the invasion of some cancers, including human glioma and ovarian cancer [19, 32, 33]. Moreover, it has been suggested that overexpression of Elmo1 also may induce cell migration and invasion in human cancers, such as hepatocellular carcinoma and breast cancer [17, 34]. However, the effects of Dock1 and Elmo1 and the relationship between them have not been studied well in melanoma. In the present study, we extended these observations to both ex vivo and in vitro human melanoma models and found that Dock1 and Elmo1 were important for promoting cancer cell proliferation and migration. Our results showed that Dock180 and Elmo1 were overexpressed in melanoma tissues compared to normal skin tissues using Western blotting and IHC staining. We also found that the inhibition of Dock180 and Elmo1 suppressed proliferation and increased apoptosis in melanoma cells. These findings suggest that both Dock180 and Elmo1 may have a role in the carcinogenesis of melanoma.
To clarify the relationship between apoptosis and the silencing of Dock180 and Elmo1 in melanoma cells, we investigated changes in apoptosis-related proteins using Western blotting. Our data showed that silencing of Dock180 and Elmo1 increased pro-apoptotic proteins, including Bax, PUMA, and cleavage of caspase-3 and PARP, while it decreased anti-apoptotic proteins, including Bcl-2. Consistent with a previous report that Dock180 and Elmo1 inhibited apoptosis in endothelial cells , these findings support that overexpression of Dock180 and Elmo1 may protect melanoma cells from apoptosis via regulating pro-apoptotic and anti-apoptotic proteins.
In addition, we demonstrated that inhibition of Dock180 and Elmo1 attenuated the invasive behavior of melanoma cell lines. Some studies have reported that both Dock180 and Elmo1 were associated with cell motility in cancers. It has been suggested that changes in gene and protein expression of Dock1, Elmo1, and Rac1 may be responsible for the migratory and invasive behavior of glioma cells [36–39]. Wang et al.  reported that Dock180 and Elmo1 synergistically activated Rac1 and promoted cell motility in ovarian carcinoma. Elmo1 expression was associated with lymph node and distant metastasis, whereas knockdown of Elmo1 impaired metastasis to the lung in breast cancer . Consistent with the findings of these previous studies, our data suggest that Dock180 and Elmo1 may promote cancer progression by enhancing cell migration and invasion in melanoma.
Furthermore, we investigated the interaction between Dock180 and Elmo1 in G361 cells. In our studies, Dock180 influenced the expression of Elmo1 in melanoma cells while Elmo1 didn’t affect the expression of Dock180. Moreover, some studies have reported that Dock180 and Elmo1 were associated with Rac1. In breast cancer cells transfected with si-Dock180 and si-Elmo1 respectively, inhibition of Dock180 suppressed expression of Elmo1 and GTP-bound Rac1 (Rac1-GTP) while inhibition of Elmo1 reduced expression of Dock180 and Rac1-GTP . However, in glioma cell transfected with si-Dock180 and si-Elmo1 respectively, although downregulation of Elmo1 affected expression of Dock180 and Rac1-GTP, Dock180 inhibition reduced expression of Rac1-GTP except Elmo1 protein . Moreover, downregulation of Elmo1 decreased Rac1 expression in hepatocellular carcinoma cells treated with si-Elmo1 . Our data demonstrated that the expression of Rac1 was significantly changed by silencing Elmo1 rather than silencing Dock180 in G361 cells. Collectively, these results indicate that the interaction between Dock180, Elmo1, and Rac1 may vary depending on the cancer type.
In addition, some studies have reported that Dock180 and Elmo1 may be involved in activation of the MEK/ERK1/2 and/or PI3K/AKT pathways in glioma cells, endothelial cells, and macrophages [18, 35, 42]. Consistent with previous reports, silencing of Dock180 and Elmo1 inhibited phosphorylation of ERK and AKT in melanoma cells. These results demonstrate that both Dock180 and Elmo1 can cross-communicate via the MEK/ERK1/2 and/or PI3K/AKT pathways, which are important for cancer progression, including cell survival, proliferation, and migration in melanoma cells.
Drugs such as BRAF inhibitors have been known to extend overall survival of patients with melanoma having a BRAF mutation [43–46]. According a previous report, BRAF mutant melanoma cells such as G361 are intrinsically resistant to BRAF inhibitors . In addition, SK-MEL-2, one of human melanoma cell lines expressing wild-type BRAF, is also resistant to BRAF inhibitors . In our experiments, we found that vemurafenib treatment reduced more significantly cell viability of melanoma cells transfected with Dock180- and Elmo1-specific siRNA alone or in combination compared to si-Ctrl. These findings suggest that Dock180 and Elmo1 may be associated with drug resistance in melanoma.
Collectively, the results of the present investigation, together with the findings in previous studies, suggest that cell proliferation and migration are regulated by Dock180 and Elmo1. Furthermore, our study showed that both Dock180 and Elmo1 were overexpressed in melanoma tissues and might be associated with cell proliferation and migration via the downregulation of apoptosis-related proteins and the activation of MEK/ERK1/2 and/or PI3K/AKT pathways in human melanoma cells. Moreover, our results demonstrated that both Dock180 and Elmo1 might be related to drug resistance in melanoma. Therefore, Dock180 and Elmo1 might be potential targets for the development of therapeutics to treat and/or prevent melanoma.