Background
Transforming growth factor-β (TGF-β) superfamily members signal via membrane-bound heteromeric serine-threonine kinase receptor complexes. Upon ligand binding, receptor activation leads to phosphorylation of cytoplasmic protein substrates of the SMAD family and subsequent accumulation in the nucleus where they act as transcription factors to regulate target gene expression [
1‐
3]. TGF-β acts as a tumor suppressor by promoting cell cycle arrest or apoptosis of normal epithelial cells during early stages of carcinogenesis, while at later stages of tumorigenesis, it functions as a tumor promoter, inducing neoplastic cell invasiveness and metastasis through a process referred to as epithelial to mesenchymal transdifferentiation (EMT), and via modulation of the extracellular tumor microenvironment, production of chemokines and recruitment of immature bone marrow-derived myeloid cells to the invasive front of tumors, and inhibition of anti-tumoral immune defenses [
4‐
8].
Members of the SKI family of proto-oncoproteins are involved in regulation of cellular transformation and differentiation [
9]. SKI was originally identified as the transforming protein (v-ski) of the avian Sloan-Kettering virus, whose overexpression promotes anchorage-independent growth of chicken and quail embryo fibroblasts [
10]. SKI (and SnoN) proteins are also important negative regulators of the TGF-β signaling cascade [
11‐
13]. In the nucleus, SKI proteins repress SMAD ability to transactivate TGF-β target genes by disrupting active heteromeric complexes of SMAD2 or SMAD3 with SMAD4, by recruiting a transcriptional repressor complex containing N-CoR SMRT, Sin3A, and HDAC-1, and by blocking the binding of transcriptional coactivators [
14‐
16]. SKI may also localize in the cytoplasm of tumor cells [
17], where it may interfere with TGF-β signaling by sequestering SMAD proteins and preventing their nuclear accumulation in response to TGF-β, as we demonstrated in the case of SnoN [
18]. The ability of SnoN and SKI to antagonize TGF-β-induced growth arrest is thought to be important for their transforming activity [
19]. Inversely, other reports have shown cell-type specific effects of SnoN as a mediator of TGF-β signaling [
20], and identified ING2 as a mediator of SnoN effects to promote TGF-β-driven transcription [
21], thereby emphasizing the complexity of the interaction between SKI family members and TGF-β signaling. Furthermore, expression levels of SKI family members may be downregulated by TGF-β, as the latter rapidly induces SKI protein poly-ubiquitination and degradation in a SMAD- and proteasome-dependent manner, allowing TGF-β target gene transactivation [
22‐
29].
Consistent with a potential oncogenic role, SKI and SnoN are often expressed at high levels in various human cancers cells derived from melanoma, esophageal cancer, pancreatic cancer and leukemia, due to increased transcription, gene amplification, and/or protein stabilization. Yet, SKI may also exert anti-tumorigenic activities: for example,
Ski+/- mice display an increased susceptibility to chemical-induced tumorigenesis [
30]. The human
SKI gene is located at chromosome 1p36, a potential tumor suppressor locus that is frequently deleted in various human cancers including neuroblastoma, melanoma, colorectal carcinoma and leukemia [
31]. Clearly, the roles of SKI in mammalian tumorigenesis are complex, and more studies are needed in order to define the functions of SKI.
Melanoma cells secrete large amounts of TGF-β: expression of TGF-β1 and β2 is increased in parallel with tumor stage, and all isoforms are expressed in highly aggressive melanoma [
32‐
34]. In melanoma cells, constitutive SMAD signaling occurs in response to autocrine TGF-β secretion [
35], and experimental blockade of TGF-β signaling by SMAD7 overexpression dramatically reduces their tumorigenic and metastatic potential [
36,
37]. Likewise, systemic pharmacologic inhibition of TGF-β signaling in mice prevents experimental melanoma cell metastasis to bone [
38]. Remarkably, it has been reported that melanoma cells express high amounts of SKI protein, which localizes both in the nucleus and in the cytoplasm [
17]. It has been suggested that such high expression of SKI blocks TGF-β transcriptional responses, in particular the induction of p21/WAF, resulting in an inactive TGF-β pathway in melanoma cells and lack of growth inhibitory activity of TGF-β [
39,
40]. SnoN may exert similar functions when SKI is not expressed in some melanoma cell lines [
41]. It is widely accepted that TGF-β is a potent inducer of SKI (and SnoN) degradation [
22‐
29], and we recently demonstrated that in breast cancer cells, TGF-β suppresses the ability of SKI to inhibit tumor metastasis by inducing its degradation via the ubiquitin-proteasome pathway, whereby TGF-β induces the E3 ubiquitin-ligase Arkadia to mediate SKI degradation in a SMAD-dependent manner [
22].
We report that despite high levels of SKI protein expression, melanoma cells exhibit strong transcriptional responses to TGF-β. We provide definitive evidence for rapid and efficient dose-dependent degradation of SKI protein in response to exogenous TGF-β, through the ubiquitin-dependent proteasome pathway. Remarkably, SKI antagonism against TGF-β activity primarily occurred when SKI degradation in response to TGF-β was prevented by proteasome blockade. We also report that SKI levels do not correlate with the tumorigenic or metastatic potential of melanoma cells, the latter largely depending upon constitutive TGF-β signaling, and do not correlate with the clinical or pathological stage of human melanoma lesions.
Discussion
The capacity for SKI (and SnoN) to inhibit TGF-β signaling has been extensively described. This has prompted us to consider that SKI proteins may exert tumor promoter activities, by preventing the classical growth inhibitory activity exerted by TGF-β in a variety of non-malignant cell types. Most experimental demonstrations for interference of SKI against TGF-β/SMAD signaling have largely relied on either overexpression or stabilization (by means of proteasome inhibition) of the SKI and SnoN proteins, due to the fact that TGF-β is able to rapidly induce SKI degradation in a proteasome-dependent manner [
22‐
29]. Remarkably, in a number of neoplasms, high SKI and/or SnoN protein levels in tumor cells are observed, concomitant with (a), elevated levels of secreted TGF-β and (b), a great sensitivity of tumor cells to targeted inhibition of TGF-β signaling that strongly interferes with their tumorigenic and metastatic potential. This study was thus initiated in order to clarify the discrepancy in the literature regarding the respective roles played by TGF-β signaling and that of potentially antagonistic SKI proteins in the control of the invasive and metastatic capacities of human melanoma cells.
We, and others, have provided ample evidence that the invasive, tumorigenic and metastatic potential of melanoma cell lines is largely dependent upon autocrine TGF-β signaling. We showed initially that the SMAD cascade is activated in an autocrine fashion in a series of human melanoma cell lines [
35]. We then showed that overexpression of SMAD7 in a highly invasive and metastatic cell line, 1205Lu, inhibits subcutaneous tumor growth as well as incidence and size of osteolytic bone metastases in mice, accompanied with dramatically increased survival [
36,
37]. Consistent with our observations, Lo and Witte [
48] identified intense nuclear immunohistochemical staining of P-SMAD2 in benign nevi, melanoma
in situ, and primary invasive melanoma, suggesting that the tumor cell autonomous TGF-β pathway is hyperactivated in response to autocrine and/or paracrine ligand activity. They demonstrated that tumor cell autonomous hyperstimulation of the TGF-β-SMAD2 pathway is causally related to melanocytic oncogenic progression in the skin and is responsible, at least in part, for the critical switch from radial to vertical growth during human melanoma histogenesis. They showed that this phenomenon requires the collaboration of activated SMADs with an altered genetic or epigenetic cellular context such as PTEN deficiency or MAPK activation [
48]. Considering recent findings showing that (a) TGF-β could act of in SMAD2, SMAD3 and SMAD4-independent manner and present pro-oncogenic activity through enhancement of Ras/Raf tumorigenic transformation [
49], and (b) majority of examined melanoma cells harbor activating mutation in BRAF and NRAS (BRAF V600E in WM793, 1205Lu, 983B) (NRAS Q61R in WM852), it is likely that TGF-βpromotes tumor progression through the enhancement of SKI-independent pathways, possibly MAP kinases [
50]. Our data on Matrigel™ invasion support the hypothesis of uncoupling TGF-β and SKI activities.
The functional response of melanoma cells to TGF-β has been addressed by a number of laboratories. For example, it has been shown that TGF-β is a potent inducer of integrins,
IL-8, and
VEGF gene expression [
51‐
54], genes implicated in metastasis and tumoral angiogenesis, respectively. A genome-wide transcriptomic analysis in over a hundred human melanoma cell lines in culture recently identified populations with very distinct gene expression profiles, the most invasive cell lines being characterized by the expression of a number of genes reminiscent of a TGF-β signature [
55].
Comparable levels of expression of SKI although there is almost complete lack of the SKI protein in normal melanocytes as compared to melanoma suggest that degradation of SKI protein in normal melanocytes is far more efficient than in malignant cells and involves an alternative, yet unidentified, TGF-β-independent mechanism of SKI degradation (or translation) and that this mechanism is deregulated in melanoma cells.
The pro-metastatic role of TGF-β extends well beyond melanoma and has been extensively described in other cancers, including, but not restricted to, gliomas, breast, ovarian, colon, or prostate adenocarcinomas [
44,
45,
56‐
58]. The TGF-β pathway is thus considered a prime target for preventive or therapeutic intervention in cancer [
59‐
65]. Remarkably, Nodal, a TGF-β family member that also signals through the SMAD pathway, has been identified as playing a crucial role in melanoma progression and metastasis [
66]. It is thus highly likely that increased availability of TGF-β ligands capable of activating the SMAD pathway will either bypass or overcome the inhibitory action exerted by SKI proteins, despite apparent high expression of the latter.
Our data are consistent with those reported by Medrano and co-workers [
17,
39] that melanoma cells in culture and human melanoma lesions exhibit high SKI protein levels. Yet, we differ significantly regarding the importance of this high of SKI in determining melanoma growth and metastasis. Our data obtained in a large panel of melanoma cell lines suggest that SKI only marginally affects TGF-β signaling: slightly elevated basal expression of some of the classical TGF-β target genes, like
PTHrP and
IL-11, was observed in shSKI-transfected 1205Lu melanoma cells as compared to mock-transfected cells, yet SKI knockdown only marginally affected the response to TGF-β, as estimated both at the level of target gene transcription and cell proliferation. While Reed and colleagues argued that SKI is crucial for the resistance of melanoma cells to TGF-β-induced growth inhibition and subsequent tumor growth [
17,
39], their data were largely obtained with the UCD-Mel-N cell line, and thus could be specific for this cell line or for a subset of melanoma cell lines, and may not be representative of all melanoma cells at large.
Noteworthy, when we initially reported that autocrine SMAD signaling occurs in melanoma cells and is dependent upon secretion and pericellular activation of TGF-β [
35], we did not know the expression status of SKI and SnoN protein in the various cell lines used in our studies. In the present study, we demonstrate that autocrine TGF-β signaling is active despite high levels of SKI and SnoN protein in all melanoma cell lines (11) that we examined, including those from our initial studies. Thus, our data unambiguously demonstrate that the presence of high SKI levels is compatible with active TGF-β signaling, implying that high SKI staining in tumors may not be an indication of an absence of TGF-β-driven disease progression, as exemplified by studies with inhibitors of the TGF-β pathway that efficiently prevent melanoma tumorigenesis and metastasis [
36‐
38]. It is possible that a subgroup of melanomas may reproduce the data obtained by Medrano and co-workers, as a similar observation was reported in a subset of esophageal carcinoma cells that are resistant to TGF-β-induced growth arrest, whereby TGF-β was unable to degrade SnoN [
67].
Most critically, Chen and co-workers suggest that SKI should be considered a prime therapeutic target for melanoma treatment [
39], as eliminating SKI protein would unleash the growth inhibitory activity of TGF-β. Such suggestion was recently echoed in a clinical report on the expression of SKI and SnoN in human melanoma lesion at various stages [
47]. While these authors demonstrated that SKI and SnoN expression in melanoma is not associated with disease progression, they extrapolated, without experimental evidence, that SKI and SnoN may mediate the resistance of melanomas to growth inhibition by TGF-β. In our opinion, critical and potentially dangerous issues arise from the assumption that melanoma cells are not responsive to TGF-β: at advanced stages of tumor progression, therapeutic interference with invasion and metastasis, two phenomena that do not require cell proliferation and are largely under the control of TGF-β, is likely to prove essential. Targeting SKI, even though in some instance it may allow some reduction in tumor cell growth, as suggested by Medrano's group, may just do the opposite, as it would eliminate one of the natural defenses that cells have developed to interfere with autocrine TGF-β signals. Noteworthy, discrepancies about the capacity of TGF-β to degrade SKI in melanoma cells have been suggested to be due to the concentrations of TGF-β used in the various studies, and that TGF-β-induced SKI degradation only occurs at "non-physiological" concentrations [
68]. This is not a satisfactory explanation as, if one follows this suggestion, increasing concentrations of TGF-β would eliminate SKI and thus exert its anti-proliferative activity and inhibit tumor progression, in contradiction with experimental evidence that inhibition of TGF-β signaling inhibits melanoma progression and metastasis. Noteworthy, given that TGF-β blockade inhibits metastasis, then whatever active concentration is present is effective to promote metastasis in spite of possible high levels of SKI expression.
Methods
Cells, plasmids and reagents
Melanoma cell lines (1205Lu, WM852, WM983B, SK28, MNT1, Dauv-1, Fo-1, WM239A, WM1341D, SK-mel501, SK-mel888) have been described previously [
35,
42,
69‐
71]. NHEM (Normal Human Epidermal Melanocytes) were purchased from Promocell (Heidelberg, Germany) and cultured in ready-to-use medium, also supplied by Promocell. All cells were grown at 37°C in a humidified atmosphere of 5% CO2. The reporter plasmids (CAGA)
9-MLP-luc [
72] and 2.4 kb p21/WAF1 promoter luciferase reporter construct [
73] were gifts from Drs. Sylviane Dennler (INSERM U697, Paris, France) and Bert Vogelstein (Johns Hopkins University, Baltimore, MD), respectively. The pRL-TK vector was from Promega (Madison, WI). pSuper vector expressing
SKI shRNA has been described previously [
22]. Human recombinant TGF-β1, purchased from R&D System Inc. (Minneapolis, MN) is referred to as TGF-β throughout the text. The ALK5/TβRI inhibitor SB431542, and the proteasome inhibitor leu-leu-leu-al (MG132) were both from Sigma-Aldrich (St. Louis, MO). Acetyl-leu-leu-norleu-al (ALLN) was purchased from CalBiochem (San Diego, CA).
Immunoblotting experiments
Protein extraction and Western blotting were performed as previously described [
74]. Anti-SMAD3 and anti-Actin antibodies were purchased from Zymed (San Francisco, CA) and Sigma-Aldrich, respectively. The rabbit anti-phospho-SMAD3 antibody [
75] was a generous gift from Dr. Edward Leof (Mayo Clinic College of Medicine, Rochester, MN). Anti-c-SKI, anti-SnoN, anti-SMURF2, anti-HSP60 and secondary anti-mouse and anti-rabbit horseradish peroxidase-conjugated antibodies were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Anti-Arkadia was obtained from Abnova Gmbh (Heidelberg, Germany).
Cell transfections and luciferase assays
Melanoma cells were seeded in 24-well plates and transfected at approximately 80% confluency with the polycationic compound Fugene (Roche Diagnostics, Indianapolis, IN) in fresh medium containing 1% FCS. TGF-β and/or inhibitors were added 4 h after transfections. Following a 16-h incubation, cells were rinsed twice with PBS and lysed in passive lysis buffer (Promega). pRL-TK (Promega) was co-transfected to assess transfection efficiency. Luciferase activities were determined with a Dual-Glo luciferase assay kit according to the manufacturer's protocol (Promega). Each experiment was repeated at least three times using triplicate dishes in each of them for each experimental condition.
RNA extraction and gene expression analysis
Total RNA was isolated using an RNeasy™ kit (Qiagen GmbH, Hilden Germany). Genomic DNA contaminations were eliminated by DNAse I treatment. One microgram of RNA from each sample was reverse transcribed using the Thermoscript kit (InVitrogen) following the manufacturer's protocol. The resulting cDNAs were then processed for either semi-quantitative or real-time PCR using SYBR Green technology. In the latter case, reactions were carried out in a 7300 Real-time PCR System (Applied Biosystem) for 40 cycles (95°C for 15 sec/60°C for 1 min) after an initial 10-min incubation at 95°C, using the following primer sets: SKI (sense, 5'-gagaaattcgactatggcaacaag-3'; antisense, 5'-gtcatctgttttgggtcttatgga-3'; IL-11 (sense, 5'-actgctgctgctgaagactc-3'; antisense, 5'-ccacccctgctcctgaaata-3'); PTHrP (sense, 5'-tttacggcgacgattcttcc-3'; antisense, 5'-ttcttcccaggtgtcttgag-3'); GAPDH (sense, 5'-gctcctcctgttcgacagtca-3'; antisense, 5'-accttccccatggtgtctga-3'). Data were analyzed using Applied Biosystems Sequence Detection Software (version 1.2.1).
Matrigel™ invasion assays
Tissue culture Transwell
® inserts (8-μm pore size, Falcon, Franklin Lakes, NJ) were coated for 3 h with 10 μg of growth factor-reduced Matrigel™ (Biocoat, BD Biosciences, San Jose, CA) in 100 μl of PBS at 37°C. After air-drying the chambers for 16 h, the Matrigel™ barrier was reconstituted with 100 μl DMEM for 24 h at 37°C. The chambers were then placed into 24-well dishes containing 750 μl of W489 medium supplemented with 0,1% FCS. Cells (5 × 10
4) were added to the upper well of each chamber in 500 μl of serum-free W489 medium. After a 24 h-incubation period, the number of invading cells was counted by bright-field microscopy at ×200 in six random fields. Additional details of the procedure may be found in [
37].
Cell proliferation assays
Melanoma cells were plated in 12-well plates at an initial density of 5000 cells/well. Cell growth was measured after 72 h in 1% FCS, with or without TGF-β (10 ng/ml), by counting the cells after trypsinization using a Malassez cell. For each experimental condition, duplicate dishes were counted. Experiments were performed at least three times with similar results.
Human tissues
Formaldehyde-fixed and paraffin-embedded naevi (n = 12), primary cutaneous melanomas (n = 37), cutaneous and lymph node metastases (n = 17 and 10, respectively) from adult patients were obtained from the pathology archives of the Radboud University Nijmegen Medical Centre, and re-evaluated by an expert pathologist (see corresponding Results section for details). Tissues were obtained according to local ethical guidelines and approved by the local regulatory committee.
Immunohistochemistry
Paraffin-embedded 4 μm sections on superfrost slides (Menzel-Glaser, Braunscheig, Germany) were de-waxed in xylene, rehydrated through graded alcohol baths, then rinsed with PBS. After quenching of endogenous peroxidases, an antigen retrieval step (sodium citrate 10 mM, pH 6.0 for 10 min. at 95°C) was performed. Tissue sections were subsequently pre-incubated with 20% normal goat serum in PBS, followed by an overnight incubation with rabbit polyclonal SKI antibody (H-329, 1 μg/ml, Santa Cruz Biotechnology Inc.) or affinity-purified rabbit polyclonal anti-phospho-Smad3C (P-SMAD3C) antibody [
75] in PBS containing 1% bovine serum albumin overnight at 4°C. For detection of SKI, the Powervision system (Immunovision Labs, Brisbane, CA) was used as a secondary reagent with 3-amino-9-thylcarbazole served as a chromogen. For detection of P-SMAD3C, a biotin-avidin peroxidase complex was generated according to standard procedures (Vector, Burlingame, CA) and developed with 3,3'-diaminobenzidine. Counterstaining was performed with hematoxylin. Samples with nuclear SKI appear purple and were scored positive in case positivity was detected in at least 10% of melanocytic cells.
Statistical analyses
Data were entered in a computerized database and analyzed using SPSS software (version 16.0.1 for Windows). The binomial test was used to analyze frequency of SKI expression in nevi. The Mann-Whitney U test was used to correlate SKI expression and tumor thickness. The correlation between SKI expression and the level of invasion was determined by the Pearson Chi-square test. Fisher exact test for small sample numbers was used to determine the correlation between SKI expression in cutaneous and nodal metastases.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
DJ, VIA and ELS performed gene expression and protein studies in vitro. LVK performed the immunohistochemical studies on human samples as well as statistical analyses. KL and AM contributed to the design of the study and drafted the manuscript. All authors read and approved the manuscript.