Rexhep Uka1, Christian Britschgi1, Anja Krättli1, Claudia Matter1, Daniela Mihic2, Michal J. Okoniewski3, Marco Gualandi1, Roger Stupp1, Paolo Cinelli1, Reinhard Dummer1, Mitchell P. Levesque1, Olga Shakhova1
- Department of Medical Oncology and Hematology, University Hospital Zurich, University of Zurich, Wagistrasse 14, 8952, Schlieren, UK
- Department of Surgical Pathology, University Hospital Zurich, University of Zurich, Schmelzbergstrasse 12, 8091, Zurich, UK
- Scientific IT Services ETH Zurich, ETH Zurich, Weinbergstrasse 11, 8092, Zürich, UK
Despite advances in the systemic treatment of patients with metastatic melanoma using immune checkpoint and tyrosine kinase inhibitors (TKI), the majority of stage IV melanoma patients eventually succumb to the disease. We have previously identified the transcription factor Sox10 as a crucial player in melanoma, yet the underlying molecular mechanisms mediating Sox10-dependent tumorigenesis remain largely uncharacterized. Here, we show that MEK and RAF inhibitors do not suppress levels of SOX10 protein in patient-derived cells in vitro, as well as in melanoma patients in vivo. In a search for pharmacological inhibitors of SOX10, we performed a mass spectrometry-based screen in human melanoma cells. Subsequent analysis revealed that SOX10 directly interacts with β-catenin, which is a key mediator of canonical Wnt/β-catenin signaling. We demonstrate that inhibitors of glycogen synthase kinase 3 alpha/beta (GSK3α/β) efficiently abrogate SOX10 protein in human melanoma cells in vitro and in melanoma mouse models in vivo. The mechanism of action of GSK3-mediated SOX10 suppression is transcription-independent and relies on the presence of a proteasome degradable form of β-catenin. Taken together, we provide evidence that activation of canonical Wnt signaling has a profound effect on melanoma growth and is able to counteract Sox10-dependent melanoma maintenance both in vitro and in vivo.
CovalX Technology Used
To understand the molecular mechanisms of SOX10 protein (which plays an important role in melanoma) dependent tumorigenesis, this article uses patient-derived cells in vitro and melanoma patients in vivo to show that MEK and RAF inhibitors do not suppress levels of SOX10 protein.
CovalX nLc Orbitrap mass spectrometry service was used to search for pharmacological inhibitors of SOX10 in human melanoma cells from the different ratios of recombinant SOX10 protein and whole cell extracts isolated from the M010817 primary melanoma culture. On top of instrumentation, CovalX also provided dialysis of the protein extract and SOX10, cross-link experiments, and trypsin proteolysis services.
It was found that SOX10 directly interacts with β-catenin during neural crest development, and by activating WNT signaling, SOX10 expression can be terminated and melanoma formation can be inhibited by three independent inhibitors of GSK3α/β (CHIR99021, LY2090314, and AZD1080) in a panel of human melanoma cell cultures.