Ed Stavnezer

The Protooncogene Ski Controls Schwann Cell Proliferation and Myelination

Schwann cell proliferation and subsequent differentiation to nonmyelinating and myelinating cells are closely linked processes. Elucidating the molecular mechanisms that control these events is key to the understanding of nerve development, regeneration, nerve-sheath tumors, and neuropathies. We define the protooncogene Ski, an inhibitor of TGF-beta signaling, as an essential component of the machinery that controls Schwann cell proliferation and myelination. Functional Ski overexpression inhibits TGF-beta-mediated proliferation and prevents growth-arrested Schwann cells from reentering the cell cycle. Consistent with these findings, myelinating Schwann cells upregulate Ski during development and remyelination after injury. Myelination is blocked in myelin-competent cultures derived from Ski-deficient animals, and genes encoding myelin components are downregulated in Ski-deficient nerves. Conversely, overexpression of Ski in Schwann cells causes an upregulation of myelin-related genes. The myelination-regulating transcription factor Oct6 is involved in a complex modulatory relationship with Ski. We conclude that Ski is a crucial signal in Schwann cell development and myelination.

SKI knockdown inhibits human melanoma tumor growth in vivo.

The SKI protein represses the TGF‐β 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‐β 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‐β signaling including high levels of nuclear Smad3 and p21Waf1, 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‐β‐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‐β‐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‐β pathway and its deficiency restores TGF‐β tumor suppressor and apoptotic activities in spite of the likely presence of oncogenic mutations in melanoma tumors.

Ski Regulates Muscle Terminal Differentiation by Transcriptional Activation of Myog in a Complex with Six1 and Eya3

Overexpression of the Ski pro-oncogene has been shown to induce myogenesis in non-muscle cells, to promote muscle hypertrophy in postnatal mice, and to activate transcription of muscle-specific genes. However, the precise role of Ski in muscle cell differentiation and its underlying molecular mechanism are not fully understood. To elucidate the involvement of Ski in muscle terminal differentiation, two retroviral systems were used to achieve conditional overexpression or knockdown of Ski in satellite cell-derived C2C12 myoblasts. We found that enforced expression of Ski promoted differentiation, whereas loss of Ski severely impaired it. Compromised terminal differentiation in the absence of Ski was likely because of the failure to induce myogenin (Myog) and p21 despite normal expression of MyoD. Chromatin immunoprecipitation and transcriptional reporter experiments showed that Ski occupied the endogenous Myog regulatory region and activated transcription from the Myog regulatory region upon differentiation. Transactivation of Myog was largely dependent on a MEF3 site bound by Six1, not on the binding site of MyoD or MEF2. Activation of the MEF3 site required direct interaction of Ski with Six1 and Eya3 mediated by the evolutionarily conserved Dachshund homology domain of Ski. Our results indicate that Ski is necessary for muscle terminal differentiation and that it exerts this role, at least in part, through its association with Six1 and Eya3 to regulate the Myog transcription.

Peroxisome Proliferator-activated Receptor γ (PPARγ) Mediates a Ski Oncogene-induced Shift from Glycolysis to Oxidative Energy Metabolism

Background: Oncogenic transformation is usually accompanied by increased aerobic glycolysis, lipid metabolism, and glutamine catabolism. Results: Ski-transformed fibroblasts shift from aerobic glycolysis to oxidative metabolism of fatty acids, mitochondrial biogenesis, and glutamate catabolism. Conclusion: The Ski-induced metabolic alterations require the activity of the lipid-activated transcription factor PPARγ. Significance: The metabolic program accompanying oncogenesis may be more varied than currently appreciated. Overexpression of the Ski oncogene induces oncogenic transformation of chicken embryo fibroblasts (CEFs). However, unlike most other oncogene-transformed cells, Ski-transformed CEFs (Ski-CEFs) do not display the classical Warburg effect. On the contrary, Ski transformation reduced lactate production and glucose utilization in CEFs. Compared with CEFs, Ski-CEFs exhibited enhanced TCA cycle activity, fatty acid catabolism through β-oxidation, glutamate oxidation, oxygen consumption, as well as increased numbers and mass of mitochondria. Interestingly, expression of PPARγ, a key transcription factor that regulates adipogenesis and lipid metabolism, was dramatically elevated at both the mRNA and protein levels in Ski-CEFs. Accordingly, PPARγ target genes that are involved in lipid uptake, transport, and oxidation were also markedly up-regulated by Ski. Knocking down PPARγ in Ski-CEFs by RNA interference reversed the elevated expression of these PPARγ target genes, as well as the shift to oxidative metabolism and the increased mitochondrial biogenesis. Moreover, we found that Ski co-immunoprecipitates with PPARγ and co-activates PPARγ-driven transcription.