EWI-2 negatively regulates TGF-β signaling leading to altered melanoma growth and metastasis

Abstract

In normal melanocytes, TGF-β signaling has a cytostatic effect. However, in primary melanoma cells, TGF-β-induced cytostasis is diminished, thus allowing melanoma growth. Later, a second phase of TGF-β signaling supports melanoma EMT-like changes, invasion and metastasis. In parallel with these "present-absent-present" TGF-β signaling phases, cell surface protein EWI motif-containing protein 2 (EWI-2 or IgSF8) is "absent-present-absent" in melanocytes, primary melanoma, and metastatic melanoma, respectively, suggesting that EWI-2 may serve as a negative regulator of TGF-β signaling. Using melanoma cell lines and melanoma short-term cultures, we performed RNAi and overexpression experiments and found that EWI-2 negatively regulates TGF-β signaling and its downstream events including cytostasis (in vitro and in vivo), EMT-like changes, cell migration, CD271-dependent invasion, and lung metastasis (in vivo). When EWI-2 is present, it associates with cell surface tetraspanin proteins CD9 and CD81 -molecules not previously linked to TGF-β signaling. Indeed, when associated with EWI-2, CD9 and CD81 are sequestered and have no impact on TβR2-TβR1 association or TGF-β signaling. However, when EWI-2 is knocked down, CD9 and CD81 become available to provide critical support for TβR2-TβR1 association, thus markedly elevating TGF-β signaling. Consequently, all of those TGF-β-dependent functions specifically arising due to EWI-2 depletion are reversed by blocking or depleting cell surface tetraspanin proteins CD9 or CD81. These results provide new insights into regulation of TGF-β signaling in melanoma, uncover new roles for tetraspanins CD9 and CD81, and strongly suggest that EWI-2 could serve as a favorable prognosis indicator for melanoma patients.

Novel impact of EWI-2, CD9, and CD81 on TGF-β signaling in melanoma

“…EWI-2 was discovered to be a negative regulator of TGF-β signaling based on a combination of EWI-2 knockdown and overexpression experiments, soluble TGF-β titration experiments, TGF-β inhibition experiments, and unbiased gene expression analyses in melanoma cells. 5 In a key experiment illustrating this point, ablation of EWI-2 in SK-Mel-28 cells markedly enhanced phosphorylation of Smads 2 and/or 3, major downstream mediators of canonical TGF-β signaling. 6 EWI-2 is well positioned to play a key role in orchestrating TGF-β signaling transitions in melanoma cells.…”

“…6 EWI-2 is well positioned to play a key role in orchestrating TGF-β signaling transitions in melanoma cells. 5 EWI-2 expression is absent from primary melanocytes (which are growth-inhibited by TGF-β), present in primary tumors (which have diminished TGF-β signaling), and then diminished in metastatic melanomas (in which TGF-β signaling has reappeared) [Fig. 1A].…”

“…Consistent with EWI-2 serving as a temporary repressor of TGF-β signaling on primary tumor cells, ablation of EWI-2 caused (i) increased TGF-β-dependent cytostasis and (ii) increased TGF-β–dependent invasion, metastasis, and EMT-like changes in melanoma cells. 5 The signaling pathways and transcriptional events responsible for selective upregulation of EWI-2 expression on primary melanoma cells remain to be determined.…”

“…Furthermore, in the absence of EWI-2, CD9 becomes free to associate with TβR2 in a CD81-dependent manner. 5 Thus, CD9 (and likely also CD81) is well-positioned to support TβR1–TβR2 association and subsequent downstream Smad2/3 activation, leading to cytostasis, invasion, EMT, and metastasis. Conversely, when EWI-2 is present, nearly all ( ~ 80%) of it is associated directly with CD9 and CD81.…”

“…Conversely, when EWI-2 is present, nearly all ( ~ 80%) of it is associated directly with CD9 and CD81. 4,5,7 This direct EWI-2 sequestration of CD9 and CD81 makes them unavailable to support TGF-β–initiated TβR1–TβR2 receptor association (Fig. 1Bi).…”