PDF file - 113K, Supplementary Table S1. Oral squamous cell carcinoma- patient and tumor characteristics. Supplementary Table S2. Lung squamous cell carcinoma -patient and tumor characteristics. Supplementary Table S3. Thyroid Carcinoma -patient and tumor characteristics. Supplementary Table S4. Lung neuroendocrine carcinoma -patient and tumor characteristics. Supplementary Table S5. Sequences of the real-time PCR primers for SCCRO5 and housekeeping gene GAPDH (in 5'-3' direction) Supplementary Table S6. Sequences of the DNA mutation analysis primers for SCCRO5 Supplementary Table S7. Sequences of the primers used for SCCRO5 PCR for deletion and mutagenesis.
ARTICLE ABSTRACTPurpose: To determine mechanisms by which SCCRO5 (aka DCUN1D5) promotes oncogenesis.Experimental Design:SCCRO5 mRNA and protein expression were assessed in 203 randomly selected primary cancer tissue samples, matched histologically normal tissues, and cell lines by use of real-time PCR and Western blot analysis. SCCRO5 overexpression was correlated with survival. The effect of SCCRO5 knockdown on viability was assessed in selected cancer cell lines. Structure–function studies were performed to determine the SCCRO5 residues required for binding to the neddylation components, for neddylation-promoting activity, and for transformation.Results: In oral and lung squamous cell carcinomas, SCCRO5 mRNA levels corresponded with protein levels and overexpression correlated with decreased disease-specific survival. Knockdown of SCCRO5 by RNAi resulted in a selective decrease in the viability of cancer cells with high endogenous levels, suggesting the presence of oncogene addiction. SCCRO5 promoted cullin neddylation while maintaining conserved reaction processivity paradigms involved in ubiquitin and ubiquitin-like protein conjugation, establishing it as a component of the neddylation E3. Neddylation activities in vitro required the potentiating of neddylation (PONY) domain but not the nuclear localization sequence (NLS) domain. In contrast, both the NLS domain and the PONY domain were required for transformation of NIH-3T3 cells.Conclusions: Our data suggest that SCCRO5 has oncogenic potential that requires its function as a component of the neddylation E3. Neddylation activity and nuclear localization of SCCRO5 are important for its in vivo function. Clin Cancer Res; 20(2); 372–81. ©2013 AACR.