Rolling the Genetic Dice: Neutral and Deleterious Smoothened Mutations in Drug-Resistant Basal Cell Carcinoma

Abstract

HHS Public Access Author manuscript Author Manuscript J Invest Dermatol. Author manuscript; available in PMC 2016 February 01. Published in final edited form as: J Invest Dermatol. 2015 August ; 135(8): 2138–2141. doi:10.1038/jid.2015.115. Rolling the genetic dice: neutral and deleterious Smoothened mutations in drug-resistant basal cell carcinoma Scott X. Atwood 1 , Kavita Y. Sarin 1 , Jiang R. Li 1 , Catherine Yao 1 , Nicole M. Urman 1 , Anne Lynn S. Chang 1 , Jean Y. Tang 1 , and Anthony E. Oro 1 1 Program in Epithelial Biology and Department of Dermatology, Stanford University School of Medicine, Stanford, CA, USA Author Manuscript To the editor Author Manuscript Basal cell carcinomas (BCCs) are locally invasive epithelial tumors that are caused by activating mutations in the Hedgehog (HH) pathway, typically through the loss of the receptor Patched1 or by activating the G-protein coupled receptor Smoothened (SMO). Genomic analysis by our group and others have revealed that BCCs are typically diploid and carry a high frequency of non-silent single nucleotide variants (SNVs) compared to other cutaneous and non-cutaneous tumors (Alexandrov et al. 2013; Atwood et al. 2014; Atwood et al. 2015; Jayaraman et al. 2014). Given their high mutational load, how these variants confer selective tumor growth without deleterious effects remains poorly understood. We previously identified and functionally validated nine SMO mutations that drive the majority of drug resistance in BCC through two distinct mechanisms that maintain HH signaling in the presence of drug: induction of constitutive activity or disruption of ligand binding (Atwood et al. 2015). However, SMO mutations with unclear function are frequently found across many HH and non-HH dependent cancers with drug-resistant BCCs bearing the highest rate of recurrent mutations at 66% (Figure 1a). Author Manuscript To determine how these additional SMO mutations promote tumor growth, we identified 28 mutations through our genomic analysis of 44 drug-resistant and 36 sporadic BCC that were either recurrent, found to overlap with the COSMIC database, or were regional-specific (ligand binding pocket or pivot region) and interrogated their ability to promote HH signaling (Figure 1b, c). We expressed wildtype human SMO (SMO-WT) or SMO mutants in Smo-null mouse embryonic fibroblasts (MEFs) to assess the ability of these variants to activate the HH pathway with or without ligand. SMO-W535L is a known constitutively active mutant that was present in many of our tumor samples and significantly increased basal HH activity in the absence of HH ligand as determined by mRNA levels of the HH target gene Gli1 (Figure 2a). No other SMO variant induced constitutive activity, including SMO-WT and the known ligand binding pocket mutant SMO-D473G (Yauch et al. 2009), Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:http://www.nature.com/authors/editorial_policies/license.html#terms Address Correspondence to: Anthony E. Oro, oro@stanford.edu, 269 Campus Drive, CCSR 2140, Stanford, CA 94305, T: (650) 723-7843 F: (650) 725-8762. Conflict of Interest: AO and AC are investigators in Genentech, Novartis, and Eli Lilly clinical trials. JT is a consultant to Genentech.