Response to the letter by Reed et al.

Dear Sir, We wish to thank Medrano and co-workers for their detailed summary on the role of SKI in melanoma. We are fully aware of their data published over the years on the possible role of SKI in melanoma. The same applies to the extensive literature on the biology of SKI and SNO proteins as well as on the role of TGF-b in tumorigenesis and melanoma progression and metastasis. In our recent review (Javelaud et al., 2008), we did not mean to question the data by Medrano’s group, and clearly reported their demonstration that antisense-mediated downregulation of SKI levels allows p21 induction and growth inhibition by TGF-b. Since SKI was not the main subject of the review on TGF-b in cutaneous melanoma, the topic was only succinctly covered. Yet, we wanted to open readers’ mind to the possibility that the role of SKI during tumorigenesis may require further analysis and understanding, taking into account the pro-metastatic role of TGF-b. Smad-dependent transcriptional responses of melanoma cells to TGF-b have been characterized before the SKI hypothesis was brought (Rodeck et al., 1999), were shown to contribute to their aggressive and metastatic nature (Javelaud et al., 2005; Javelaud et al., 2007). In accordance with earlier findings by Medrano and others (Sun et al., 1999; Reed et al., 2003), we recently found elevated SKI levels in a panel of 10 human melanoma cell lines. In these cell lines, TGF-b induces a strong Smad-dependent transcriptional response (Javelaud et al., 2007), as well as a rapid, proteasome-mediated, degradation of SKI protein (Mauviel et al., 2007). The same was also found in breast cancer cells (Le Scolan et al., 2008), in which we identified the E3 ubiquitin ligase Arkadia as critical to mediate TGF-beta-induced degradation of SKI in a SMAD-dependent manner, consistent with recent results from other laboratories (Levy et al., 2007; Nagano et al., 2007). Of note, other ubiquitin ligases, Smurf2 and anaphase-promoting complex (APC), have also been identified as mediators of SnoN degradation in response to TGF-b (Bonni et al., 2001; Stroschein et al., 2001; Wan et al., 2001). We also found that reducing SKI expression in breast and lung cancer cells with specific shRNAs does not affect tumor cell growth, but enhances tumor metastasis in vivo (Le Scolan et al., 2008). Thus, in these cells, SKI seems to have an anti-tumorigenic activity, suggesting that the ability of TGF-b to induce SKI degradation may represent an additional mechanism contributing to its pro-metastatic activity. We raised the possibility that SKI expression in melanoma cells, even though it may potentially attenuate the effects of low TGF-b concentrations, may not antagonize TGF-b if the latter is present in large amounts. Consistent with this hypothesis, it has been reported that, unless stabilized by mutagenesis, overexpressed SnoN proteins only moderately attenuate the ability of cells to undergo cell cycle arrest in response to TGF-b, likely because of their rapid degradation following TGF-b stimulation (Stroschein et al., 1999). In the context of a tumor, it is very difficult to assess TGF-b concentration precisely. The latter may vary considerably within a tumor from one site to the next, allowing SKI degradation in some areas of the tumor while other regions may still exhibit detectable levels of SKI, compatible with Medrano’s results in vivo (Reed et al., 2001). This, in turn, may result in variable responsiveness to TGF-b depending on specific areas of the tumors. Why melanoma cells simultaneously exhibit high SKI expression together with constitutively active TGF-b signaling in melanoma cells is highly intriguing. Cultured 1205Lu melanoma cells, for example, exhibit high SKI protein levels together with constitutive Smad transcriptional activity (Rodeck et al., 1999; Mauviel et al., 2007). Pharmacologic inhibition of TGF-b signaling in these cells results in further elevation of SKI protein levels (Javelaud et al., unpublished observations), suggesting that SKI protein levels are controlled by autocrine TGF-b signaling. Understanding the TGF-b ⁄ SKI axis will also require investigations that take into account other signaling pathways potentially activated in melanoma that may or may not contribute to SKI stabilization and affect TGF-b responsiveness.