SKI knockdown inhibits human melanoma tumor growth in vivo

Abstract

The SKI protein represses the TGF-beta tumor suppressor pathway by associating with the Smad transcription factors. SKI is upregulated in human malignant melanoma tumors in a disease-progression manner and its overexpression promotes proliferation and migration of melanoma cells in vitro. The mechanisms by which SKI antagonizes TGF-beta signaling in vivo have not been fully elucidated. Here we show that human melanoma cells in which endogenous SKI expression was knocked down by RNAi produced minimal orthotopic tumor xenograft nodules that displayed low mitotic rate and prominent apoptosis. These minute tumors exhibited critical signatures of active TGF-beta signaling including high levels of nuclear Smad3 and p21(Waf-1), which are not found in the parental melanomas. To understand how SKI promotes tumor growth we used gain- and loss-of-function approaches and found that simultaneously to blocking the TGF-beta-growth inhibitory pathway, SKI promotes the switch of Smad3 from tumor suppression to oncogenesis by favoring phosphorylations of the Smad3 linker region in melanoma cells but not in normal human melanocytes. In this context, SKI is required for preventing TGF-beta-mediated downregulation of the oncogenic protein c-MYC, and for inducing the plasminogen activator inhibitor-1, a mediator of tumor growth and angiogenesis. Together, the results indicate that SKI exploits multiple regulatory levels of the TGF-beta pathway and its deficiency restores TGF-beta tumor suppressor and apoptotic activities in spite of the likely presence of oncogenic mutations in melanoma tumors.

Figure 1. Stable downregulation of endogenous SKI by overexpression of 783-nt SKI ds-RNA impairs anchorage-independent growth in human melanoma cell lines

A) Upper panel: Western blot showing SKI protein levels in pooled clones of UCD-Mel-N expressing empty vector control (EV), RNAi-SKI and SKI transfected into RNAi-SKI cells (rescue experiment). Lower panel: Colony growth in the absence of substratum (agar growth) and quantification of colony inhibition (The mean ± SE of two independent experiments is shown). B) SKI levels in A375 melanoma cells transfected with control (EV) and ds-RNAi-SKI vectors (RNAi-SKI). Colonies were quantified as described above. C) Immunofluorescence analysis showing that down-regulation of SKI significantly enhances the nuclear localization of Smad2/3 after treatment with 0.6ng/ml TGF-β for 2 hrs (see Material and Methods for details). Smad2/3 proteins were detected by an anti-Smad2/3 Ab and a Texas red-conjugated secondary Ab. DNA was stained by DAPI.

Figure 2. Downregulation of SKI prevents growth of melanoma xenografts by restoring signatures of active TGF-β-growth inhibitory signals

A) Downregulation of SKI generated minimal UCD-RNAi-SKI tumor xenograft growth compared to the robust growth of xenografts displaying endogenous levels of SKI-EV. B) Histopathological assessment of time-matched SKI-EV and RNAi-SKI tumors. Photomicrographs of hematoxylin and eosin (H&E)-stained sections of SKI-EV (left side, panels a, c) show a large tumor nodule with multiple zones of tumor cell necrosis. Tumor cells have a large epithelioid or spindle-shaped appearance and a high mitotic rate. H&E-stained sections of UCD-RNAi-SKI (right side, panels b, d) show a small tumor nodule with prominent central necrosis (star). These tumor cells have a small epithelioid appearance and a low mitotic rate (Supplemental Table 1). C) Immunohistochemical detection of upregulated Smad3 and p21WAF-1 in the small RNAi-SKI knockdown tumors. An anti-SKI antibody confirmed that SKI was still downregulated in the RNAi-SKI tumors compared to SKI-EV controls (top row, panels a, b). Expression of Smad3 (middle row, panels c, d) was seen along the stroma (S) and edges (Te) but not in the middle (Tm) of SKI-EV tumors expressing endogenous SKI compared to the prominent nuclear and cytoplasmic localization of Smad3 observed in RNAi-SKI tumor cells. Expression of p21WAF-1 (bottom row, panels e, f) was observed in only a few scattered SKI-EV tumor cells compared to the prominent nuclear localization throughout the rim of viable RNAi-SKI tumor cells surrounding zones of necrosis (star). H&E-stained sections, original magnification 33× (top row), 132× (bottom row). Immunohistochemical labeling was performed as described in the Materials and Methods, original magnification 66× (all panels).

Figure 3. Endogenous SKI is required for Smad3 linker phosphorylations induced by TGF-β in melanoma cells

A) Western blot of total cell extracts using specific anti-pSmad2C (Ser 465–467), anti-pSmad3C (Ser423–425), anti-pSmad2L (S250–255) and anti�Smad3L (S208–213) antibodies. Arrows indicate the dramatic increase in pSmad3L (S208–213) in cells displaying endogenous levels of SKI. B) These amino acid residues are minimally phosphorylated in normal human neonatal (NHM) and adult melanocytes (AHM) compared to the robust phosphorylation induced by TGF-β in wild-type UCD-Mel-N melanoma cells. Cells were treated with 0.6ng/ml TGF-β for 1 hr. C) A Western blot shows that overexpression of SKI in RNA-SKI UCD-Mel-N melanoma cells (rescue experiment) re-established strong Smad3L phosphorylations (arrows) after TGF-β treatment. Cells were treated with 0.6ng/ml TGF-β for 1 hr.

Figure 4. SKI is necessary for preventing TGF-β-mediated down-regulation of c-MYC and for up-regulation of PAI-1

A) Levels of p-ERK, pJNK and c-MYC in normal melanocytes, UCD-Mel-N and A375 melanoma cell lines and derivatives expressing RNA-SKI. Protein extracts (50 µg/ml) were analyzed by western blots with antibodies indicated in the figure. The arrowheads indicate downregulation of c-MYC in RNAi-SKI melanoma cells. Cells were treated with 0.6ng/ml TGF-β for 1 hr. B) Western blot of total cell extracts prepared after TGF-β treatment. The arrow indicates robust pSmad3L phosphorylation that peaked at 2 hrs after TGF-β treatment. Black lines indicate significant changes in pSmad3L phosphorylation, PAI-1 and p21^Waf-1 expression between RNAI-SKI and EV control cells. Cells were treated with 0.6ng/ml TGF-β for the times indicated in the figure.

Figure 5. A working model suggesting that SKI stimulates the pro-oncogenic pathway of TGF-β in melanoma cells (data modified from (Matsuzaki, 2006))

TGFβ binds TGF-βRII, which in turns recruits the low affinity receptor (TGF-βRI) to activate the pathway (Groppe et al., 2008). Complex cross-talks between TGF-β, RAS, JNK, CDK2, and CDK4, a protein overexpressed in a subset of melanomas displaying amplification of the CDK4 gene (Muthusamy et al., 2006), result in Smad3 phosphorylations at Thr¹⁷⁹ and Ser²⁰⁸ (Matsuzaki et al., 2009; Wang et al., 2009). The model suggests that by enhancing Smad3L phosphorylations, SKI becomes a major regulator of the switch of TGF-β from a growth inhibitor to a pro-oncogenic protein in human malignant melanoma.