Varieties of transforming growth factor-β (TGF-β) antagonists have been developed to intervene with excessive TGF-β signalling activity in cancer.,Activin receptor-like kinase5 (ALK5) inhibitors antagonize TGF-β signalling by blocking TGF-β receptor-activated Smad (R-Smad) phosphorylation.,Here we report the novel mechanisms how ALK5 inhibitors exert a therapeutic effect on a mouse B16 melanoma model.,Oral treatment with a novel ALK5 inhibitor, EW-7197 (2.5 mg/kg daily) or a representative ALK5 inhibitor, LY-2157299 (75 mg/kg bid) suppressed the progression of melanoma with enhanced cytotoxic T-lymphocyte (CTL) responses.,Notably, ALK5 inhibitors not only blocked R-Smad phosphorylation, but also induced ubiquitin-mediated degradation of the common Smad, Smad4 mainly in CD8+ T cells in melanoma-bearing mice.,Accordingly, T-cell-specific deletion of Smad4 was sufficient to suppress the progression of melanoma.,We further identified eomesodermin (Eomes), the T-box transcription factor regulating CTL functions, as a specific target repressed by TGF-β via Smad4 and Smad3 in CD8+ T cells.,Thus, ALK5 inhibition enhances anti-melanoma CTL responses through ubiquitin-mediated degradation of Smad4 in addition to the direct inhibitory effect on R-Smad phosphorylation.
S-phase kinase protein 2 (Skp2), an F-box protein, targets cell cycle regulators via ubiquitin-mediated degradation.,Skp2 is frequently overexpressed in a variety of cancers and associated with patient survival.,In melanoma, however, the prognostic significance of subcellular Skp2 expression remains controversial.,To investigate the role of Skp2 in melanoma development, we constructed tissue microarrays and examined Skp2 expression in melanocytic lesions at different stages, including 30 normal nevi, 61 dysplastic nevi, 290 primary melanomas and 146 metastatic melanomas.,The TMA was assessed for cytoplasmic and nuclear Skp2 expression by immunohistochemistry.,The Kaplan-Meier method was used to evaluate the patient survival.,The univariate and multivariate Cox regression models were performed to estimate the harzard ratios (HR) at five-year follow-up.,Cytoplasmic but not nuclear Skp2 expression was gradually increased from normal nevi, dysplastic nevi, primary melanomas to metastatic melanomas.,Cytoplasmic Skp2 expression correlated with AJCC stages (I vs II-IV, P<0.001), tumor thickness (≤2.00 vs >2.00 mm, P<0.001) and ulceration (P = 0.005).,Increased cytoplasmic Skp2 expression was associated with a poor five-year disease-specific survival of patients with primary melanoma (P = 0.018) but not metastatic melanoma (P>0.05).,This study demonstrates that cytoplasmic Skp2 plays an important role in melanoma pathogenesis and its expression correlates with patient survival.,Our data indicate that cytoplasmic Skp2 may serve as a potential biomarker for melanoma progression and a therapeutic target for this disease.