High-resolution and whole-genome DNA platforms have allowed for reanalysis of tumor samples and identification of cryptic alterations. To identify novel genomic alterations in lung cancers, we used whole-genome tiling path array comparative genomic hybridization (aCGH) to determine copy number changes for 346 NSCLC and SCLC samples (85 cell lines and 261 primary tumors). Significance of recurrent gains or deletions was determined for the lung cancer cell lines using Genomic Identification of Significant Targets in Cancer (GISTIC), a statistical method that calculates significance scores incorporating the amplitude and frequency of copy number alterations at each position in the genome (8
GISTIC analysis identified 5 significant high-level focal copy number amplifications (log2
ratio > 0.8) across the 85 lung cancer cell lines (Figure A): in decreasing order of significance, these were 14q13.2 (q
= 0.0013), 11p13 (q
= 0.015), 1q21.2 (q
= 0.048), 5p15.33 (q
= 0.085), and 7p12.1 (q
= 0.19). Amplifications of 1q21.2, 5p15.33, 7p12.1, and 14q13.2 have been previously reported in lung adenocarcinomas and revealed to harbor known lung cancer oncogenes (ARNT
, and NKX2-1
, respectively), indicative of consistency between array platforms (5
). Although the amplicon associated with 11p13 has been previously reported and is thought to be a predictive marker in lung adenocarcinomas (7
), to our knowledge, the relevance to human lung cancer is not known.
TRAF6 locus amplification occurs frequently in lung cancer.
High-level amplification of 11p13 was the second most significant event in our panel of NSCLC and SCLC cell lines (q
= 0.015277), with the peak amplified region identified by GISTIC spanning an approximately 4-Mb interval (32,126,542–37,251,933 Mb) that contains 26 protein-coding genes (Figure B), none of which have been previously implicated in lung cancer. To identify candidate target genes, we next searched for concomitantly amplified and overexpressed genes within this interval by integrating parallel genetic and gene expression data for the cell lines. Of the 26 genes in the peak region, only TRAF6
(36,467,531–36,488,372 bp) expression was positively and significantly associated with gene amplification (adjusted P
= 0.01) (Figure C, Supplemental Figure 1, and Supplemental Table 1; supplemental material available online with this article; doi:
). Consistent with TRAF6
mRNA overexpression (Supplemental Figure 1), TRAF6 protein was approximately 2-fold higher in lung cell lines with 11p13 amplification (n
= 5) than in samples with diploid 11p13 (n
= 4) (Figure D).
In total, TRAF6 amplification was observed in 17 of 85 (20%) of the lung cancer cell lines, with the vast majority (88%) belonging to the NSCLC subtype (Table and Supplemental Table 2). To confirm the cell line findings, we expanded our analysis to a panel of 261 clinical NSCLC tumor samples. Of these tumors, 24 (9.2%) had a copy number increase of the TRAF6 genomic locus (Table ), highlighting its potential clinical relevance. Furthermore, examination of matched gene expression data for lung tumor samples revealed a significant association between copy number amplification and TRAF6 mRNA overexpression (P = 0.035; Figure E), further validating our cell line findings (Supplemental Figure 1). Thus, our integrative genomic analyses identified TRAF6 as the target of the 11p13 amplicon, underlining a putative oncogenic role for this gene in lung cancer development.
Incidence of 11p13 amplifications in human lung cancer
TRAF6 is a member of the TNF receptor–associated factor (TRAF) family of proteins and is an E3 ubiquitin ligase that catalyzes autologous synthesis of lysine 63–linked (K63-linked) polyubiquitin chains involved in downstream activation of NF-κB (11
). Despite its prominent role in NF-κB activation, TRAF6 has not to our knowledge been directly associated with oncogenesis. To determine whether TRAF6 overexpression leads to malignant cellular transformation, we retrovirally expressed TRAF6 in nontransformed mouse fibroblasts (NIH3T3) and evaluated cell growth and colony formation. Overexpression of TRAF6 resulted in enhanced cell proliferation in serum-deprived conditions (Figure A), formation of anchorage-independent colonies in soft agar comparable in size to colonies transformed with a constitutively active version of RAS, vH-RAS (P
= 0.0047; Figure , B and C), and adoption of a spindle morphology and refractility, consistent with a transformed phenotype and similar to that of vH-RAS–transformed cells (Figure C). Vector-transduced cells were not refractile and grew in monolayers, morphologically resembling parental cells (Figure C). TRAF6- or vector-expressing NIH3T3 cells were injected subcutaneously into NOD/SCID mice and monitored for tumor growth. TRAF6-expressing cells began to form tumors within 4 weeks (P
= 0.011; Figure D), and after 35 days, 80% (9 of 11) of mice developed large tumors (Figure D). Of the mice injected with control cells, only 1 developed a palpable tumor after 8 weeks (Figure D). These results are suggestive of the oncogenic capabilities of TRAF6, including the capacity to malignantly transform cells and form tumors.
TRAF6 overexpression causes malignant phenotype.
Individual TRAF6-expressing clones were isolated from soft agar and independently expanded in liquid culture for further analysis. We reevaluated 3 TRAF6 clones (designated C1, C2, and C3) for colony formation in soft agar; these exhibited increased colony-forming ability compared with parental TRAF6-expressing NIH3T3 cells (data not shown). Examination of the TRAF6 clones revealed a pronounced transformed phenotype, including increased refractility, spindle morphology, and multilayered growth (Figure E). All TRAF6 clones exhibited higher TRAF6 protein expression (~2-fold) than that of the parental TRAF6-expressing cells (Figure F), and TRAF6 protein positively correlated with enhanced colony formation (r2 = 0.8032; P = 0.1; Figure G). Consistent with a more aggressive transformed phenotype, tumor size was increased and latency reduced in the TRAF6 clones, approaching that of vH-RAS–induced tumors (Figure , H and I). Therefore, a higher level of TRAF6 expression was associated with malignant transformation.
To investigate the role of TRAF6 in cell survival and oncogenesis in human lung cancer, we used RNA interference (RNAi) to knock down the expression of TRAF6. We chose 6 human lung cancer lines based on 11p13 amplification, KRAS
mutation status, and a RAS
gene expression signature (ref. 12
and Supplemental Table 3). To reduce expression in H1395, H460, and HCC95 cell lines, 3 independent retroviral shRNAs targeting discrete regions of TRAF6
(shT6) were used, resulting in an approximately 50% knockdown of TRAF6
mRNA (Supplemental Figure 2).
RNAi-mediated knockdown of TRAF6 significantly decreased the cell growth of adenocarcinoma lung lines HCC95 and SK-MES-1, which have TRAF6 amplification (P = 0.00036 and P = 0.025, respectively, at day 8; Figure A). TRAF6 depletion also inhibited — albeit not as effectively — growth and viability of cell lines H460 and H2347, without an amplified TRAF6 locus but with a KRAS mutation (P = 0.01 and P = 0.034, respectively; Figure A). Similarly, depletion of TRAF6 in lines H1395 and H520, without a TRAF6 amplification (low TRAF6 protein) or KRAS mutation, had no or moderate effect on cell growth (P = 0.14 and P = 0.006, respectively; Figure A). Depletion of TRAF6 also led to a significant decrease in anchorage-independent colony growth of HCC95 (P = 1.3 × 10–7), SK-MES-1 (P = 0.0009), H2347 (P = 4.9 × 10–5), H460 (P = 2.2 × 10–5), and H520 (P = 4.2 × 10–5) cells, but not of H1395 cells (P = 0.25; Figure , B and C). In support of the growth and colony-forming data, cell viability was significantly reduced in H460 and HCC95 cells after TRAF6 depletion (P = 0.029; Figure D). Given that we observed a TRAF6-dependent cell line that did not have KRAS mutation or TRAF6 amplification (H520), it is possible that other driver mutations affecting RAS or NF-κB signaling occur in this cell line, making it sensitive to TRAF6 inhibition.
Inhibition of TRAF6 impairs growth and tumor formation of lung cancer cells.
We next subcutaneously injected shT6- or vector-expressing lung cancer cells into NOD/SCID mice and monitored tumor growth. Consistent with the colony formation observations, tumor-forming ability was significantly impaired after TRAF6 depletion in lines H460 and HCC95 (P = 0.001 and P = 0.013, respectively); however, TRAF6 knockdown in H1395 cells did not have an effect on tumor formation (Figure , E–G). Overall, these observations demonstrate an oncogenic role of TRAF6 in human lung cancers and suggest that cells with 11p13 amplifications or KRAS mutations may depend on TRAF6 for cell viability and tumor formation.
To determine whether other genes within the 11p13 amplicon are also relevant to the pathogenesis of human lung cancers, we evaluated 3 genes with P values approaching significance in our panel of lung cancers (see Supplemental Table 1). Inhibition of COMMD9 (P = 0.3), CD44 (P= 0.031), and TCP11L1 (P = 0.011) did not suppress colony formation as effectively as did TRAF6 inhibition (P = 0.00021; Supplemental Figure 3), which suggests that TRAF6 is the critical gene within the 11p13 amplicon.
Somatic activating mutations of KRAS
are the most frequent genetic events in NSCLCs (1
). Despite the prominence of MAPK pathway activation by RAS (13
), NF-κB is also essential for KRAS
-mediated lung cancers in mouse models (2
). The importance of NF-κB is further supported by reports of constitutive NF-κB activation in more than 50% of human lung cancers (3
). Despite high levels of NF-κB activation in human lung tumors and a critical role in RAS-mediated lung cancer formation in vivo, the mechanisms leading to constitutive NF-κB activation in human lung cancers remains unknown. Because TRAF6 is an upstream activator of NF-κB, we wanted to investigate whether TRAF6 is a key activator of NF-κB in human lung cancer. Therefore, 8 lung adenocarcinoma cell lines were evaluated for NF-κB activation. The lung lines with TRAF6
amplification and overexpression displayed increased NF-κB activation, as determined by κB-site luciferase activity (11p13 amplification, 8.9 ± 5.9; no 11p13 amplification, 3.5 ± 1.5; P
= 0.1), and by a shift toward nuclear translocation of the NF-κB subunit, p65 (Figure , A and B). Inhibition of TRAF6 in HCC95 (11p13 amplification) resulted in a significant decrease of NF-κB activation (P
= 0.0014; Figure C). Inhibition of TRAF6 in H460 cells, which have a KRAS
mutation, similarly resulted in a significant decrease of NF-κB activation (P
= 0.027; Figure D). These findings suggest that increased TRAF6 expression is a potential cause of constitutive NF-κB activation in human lung cancers.
TRAF6 is upstream of NF-κB in human lung cancer cells.
We observed that TRAF6 inhibition reduced NF-κB activation and tumor formation of lung cancer cells with KRAS mutations (Figures and ); therefore, we wanted to investigate whether TRAF6 is also an essential link in RAS-mediated tumorigenicity. To investigate a relationship between mutant KRAS and TRAF6 amplifications, we examined an additional 705 human cancer cell lines. As expected, samples with TRAF6 gain and/or amplification had significantly higher mRNA levels of this gene than did cell lines without TRAF6 copy number increase (P < 0.0001). In this panel of cell lines, KRAS mutations were enriched in samples with TRAF6 amplifications (17%; 21 of 125) compared with samples diploid at 11p13 (11%; 68 of 632; P = 0.043, Fisher exact test; Table ). Therefore, there was an association between KRAS mutation and TRAF6 copy number change, further supporting the interaction between these genes during tumorigenesis.
Incidence of KRAS mutations and TRAF6 amplifications
E3 ligase–mediated autoubiquitination is a measure of TRAF6 activation. To determine whether RAS also activates TRAF6, we overexpressed constitutively active KRAS
or vH-RAS, subjected TRAF6 to IP, and measured TRAF6 polyubiquitination. As shown in Figure A, expression of KRAS
activating mutant (V12) or vH-RAS resulted in autoubiquitination of TRAF6; however, an inactive KRAS
mutant (N17) did not, validating previous findings (17
). Inhibition of mutant KRAS
abrogated TRAF6 autoubiquitination in an NSCLC cell line with mutant KRAS
(Figure B). These findings suggest that TRAF6 activation may occur in human lung cancer through active KRAS
, as previously proposed in other cellular contexts (17
TRAF6 is necessary for RAS-mediated oncogenesis.
RAS dependence on TRAF6 was evaluated by overexpressing vH-RAS, a constitutively active version of RAS (18
), in Traf6–/–
mouse embryonic fibroblasts (MEFs; Figure C) or in NIH3T3 cells coexpressing a dominant-negative TRAF6 (T6DN) construct. In contrast to Traf6+/+
cells (Figure , D and F), vH-RAS–mediated anchorage-independent growth was completely abrogated in Traf6–/–
cells (Figure , D and F) and significantly reduced in NIH3T3 coexpressing T6DN (Figure , E and F; P
= 0.04). Similarly, the ability of vH-RAS–expressing cells to form tumors was completely abolished in the absence of TRAF6 (Figure , G and H) and was significantly impaired when TRAF6 was blocked with T6DN (P
= 0.053; Figure I). These findings suggest that RAS-mediated anchorage-independent colony growth, tumor formation, and NF-κB activation are dependent on TRAF6. Given the prominent role of TRAF6 in human lung cancers, we hypothesized that the TRAF6
locus is also frequently amplified in other human cancers. By aCGH, we identified frequent amplification of the TRAF6
locus in a panel of 124 additional human cancer cell lines (Table ). In addition, we investigated whether TRAF6
amplifications exist in a broader analysis of human cancer cell lines. In total, 757 cancer cell lines were analyzed, of which 126 (16.6%) displayed TRAF6
copy number gain and/or amplification. In a subset of 705 of these cell lines that had matching expression data, samples with TRAF6
gain and/or amplification had significantly higher mRNA levels of this gene compared with cell lines without TRAF6
copy number increase (P
< 0.0001, Mann-Whitney U
test), confirming the findings from the lung cancer analyses. Beroukhim et al. also reported amplification of the TRAF6
locus as a somatic and frequent event in several human cancer types (19
). In our analysis, breast cancers exhibited amplification of 11p13 in approximately 30% of samples. In these cell lines, TRAF6
mRNA overexpression also correlated with amplification of 11p13 (Supplemental Figure 4). Overexpression of T6DN in MCF7, a breast cell line with high levels of TRAF6
expression, abrogated anchorage-independent colony growth in vitro (P
= 0.023; Supplemental Figure 4). In contrast, SKBR3, a breast cell line with lower levels of TRAF6
, were not sensitive to TRAF6
= 0.32; Supplemental Figure 4).
Incidence of 11p13 amplifications in human cancer cell lines