Figure 5. TRAF6 is necessary for RAS-mediated oncogenesis.

(A) Extracts from HEK293 cells transfected with the indicated vectors were IP with an anti-TRAF6 antibody, then IB for polyubiquitination with ubiquitin or TRAF6 antibodies. RAS and tubulin were determined by IB on the pre-IP lysates. (B) Extracts from H460 cells (KRAS mutation) transduced with the indicated shRNA vectors were IP with an anti-TRAF6 antibody, then IB with anti-TRAF6 antibodies. Actin was IB on the pre-IP lysates. A proposed model of KRAS-mediated activation of TRAF6 in lung cancer is also shown. (C) Traf6^–/– or Traf6^+/+ MEFs were transduced with a retroviral vector encoding vH-RAS (MSCVpac) and IB for RAS. (D) Soft agar colony formation for vH-RAS–expressing Traf6^–/– or Traf6^+/+ MEFs (1 × 10⁴ cells plated). (E) Soft agar colony formation for vH-RAS–expressing NIH3T3 cells transduced with T6DN or vector control (MIY). (F) Representative colonies from D and E. Original magnification, ×20. (G and H) vH-RAS–expressing Traf6^–/– or Traf6^+/+ MEFs were subcutaneously injected into NOD/SCID mice. (G) Representative tumors (red outline) on the dorsa. (H) Tumor volume at the experimental endpoint. (I) vH-RAS–expressing NIH3T3 cells transduced with T6DN or vector control were subcutaneously injected into NOD/SCID mice. Tumor volume is shown at the experimental endpoint. (J) Model of TRAF6-sensitive lung cancers. For TRAF6 inhibition–sensitive lung cancers, TRAF6 inhibition reduced proliferation, anchorage-independent growth, tumor formation, and NF-κB activation. *P < 0.05.