(A–C) NIH3T3 cells transduced with vector (MSCVpac) or FLAG-tagged TRAF6 were selected in puromycin and assessed (A) for growth in 2% serum by BrdU incorporation and (B) for colony formation in 0.3% soft agar (1 × 10⁴ cells plated). (C) Morphology of vector- and TRAF6-expressing NIH3T3 cells is shown in agar and in liquid culture. Original magnification, ×20 (agar); ×40 (liquid). (D) NIH3T3 cells transduced with vector or TRAF6 were subcutaneously injected (2.5 × 10⁵ cells/flank) into NOD/SCID mice and assessed for tumor formation by measuring tumor volume. (E) Individual colonies formed by TRAF6-expressing NIH3T3 cells were extracted from soft agar, expanded in liquid culture, and reanalyzed for soft agar colony formation. Representative cells are shown. Original magnification, ×20 (agar); ×40 (liquid). (F) 3 TRAF6 clones (C1–C3), along with 2 independently created parental TRAF6 cells and vector-expressing cells, were evaluated for FLAG-TRAF6 expression by IB (anti-FLAG). Densitometric values for FLAG-TRAF6 protein relative to actin are shown below. (G) Colony-forming frequency (no. colonies/1 × 10³ cells plated) is shown relative to expression of FLAG-TRAF6 for TRAF6 clones (n = 3) and parental TRAF6 cells (n = 2). (H and I) NIH3T3 cells transduced with vector, TRAF6, vH-RAS, or TRAF6 clone C1 were subcutaneously injected (2.5 × 10⁵ cells/flank) into NOD/SCID mice and assessed for tumor formation by measuring (H) tumor mass at day 15 and (I) tumor volume. *P < 0.05.

(A) NF-κB activity was measured by κB-site luciferase assays in human lung cancer cells with (HCC95, H526, H2171, HTB58) and without (H2170, H2347, H520, HTB117) amplification of the TRAF6 locus (11p13). (B) Nuclear NF-κB (p65) was IB in nuclear extracts from 7 human lung cancer cell lines with (H2170, H2347, H520) or without (H2170, H2347, H520, HTB117) amplification at the TRAF6 locus (11p13). (C) H460 (diploid at 11p13) and HCC95 (amplification of 11p13) were evaluated for κB-site luciferase activity and for TRAF6 protein expression. (D) NF-κB activity was measured by κB-site luciferase assays after TRAF6 inhibition. *P < 0.05.

(A) Copy number data for 85 lung cancer lines is shown by genomic location (rows). False discovery rates (q values; 0.25 cutoff for significance shown by green line) and scores for amplicons are plotted at each genome position. Individual chromosomes are denoted by white and gray shading; black boxes indicate significant high-level amplifications. (B) Copy number status of chromosome 11p for 2 representative lung cancer lines. The minimally amplified region spanning chromosome 11p13 to 11p12 (32,126,542–37,251,933) and known genes are shown at right. Normalized log₂ signal intensity ratios were plotted and visualized by SIGMA² (32). Vertical lines denote log₂ signal ratios from –1 to +1; copy number gains (red) are to the right of 0. Each dot represents a single probe. (C) A subset of 66 lung cancer samples was analyzed to identify genes whose expression correlated with gene amplification. P values (–log₂) for genes within the minimally amplified region on 11p13 are shown as a histogram. Values above the dashed line denote P ≤ 0.05. (D) IB for TRAF6 and actin in 5 lung cancer lines diploid at 11p13 (N, no 11p13 amplification; H2171, H1395, H2347, H520, and H460) and 4 lung cancer lines with an 11p13 amplification (amp; HCC95, H526, H2171, and SK-MES-1). Densitometric values for TRAF6 protein relative to actin are shown below. (E) TRAF6 mRNA levels were evaluated in lung tumor samples with (n = 13) and without (n = 31) amplification of 11p13. Significance was determined by Mann-Whitney U test. *P < 0.05.

(A) Growth curves of cell lines with TRAF6 inhibition (shT6) is shown relative to vector-expressing (pLKO.1 control) cells. (B and C) Soft agar colony formation for the indicated cell lines after TRAF6 inhibition. Original magnification, ×20. (D) Cell lines H460 and HCC95 were transduced with virus encoding shT6. After TRAF6 inhibition, cell survival was evaluated by flow cytometry for Annexin V expression. HCC95 cells transduced with vector or shT6 were also cultured in liquid media, and morphological assessment confirmed that shT6-expressing cells exhibited features of increased cell death, such as cell detachment and shrinkage and membrane blebbing. Original magnification, ×40. (E–G) Cell lines H1395 (E), H460 (F), and HCC95 (G), transduced with vector (pLKO.1-control) or shT6, were subcutaneously injected into NOD/SCID mice. Tumor volume is shown at the experimental endpoint. *P < 0.05.

(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.