The NF-κB pathway is aberrantly activated in the majority of lung cancers and is essential in mouse models of lung tumorigenesis 10–13
. NF-κB signaling contributes to tumorigenesis via its promotion of cell proliferation and survival 7–9
. In order for NF-κB to become active, inhibition by IκB must be released. This is achieved through phosphorylation of IκB by the kinase, IKBKB 14
; hence, IKBKB has a critical role in NF-κB activation 6, 14
. In fact, constitutive IKBKB activity has been postulated to drive the aberrant NF-κB activation observed in cancer 8
. Despite what is known about the cascade of protein signaling events that result in NF-κB activation, the genetic mechanisms responsible for the aberrant activation of NF-κB signaling in lung cancer are not well understood. In this study, we hypothesized that genetic disruption and loss of function of the KEAP1 E3-ubiquitin ligase complex, which regulates IKBKB protein levels, is a major mechanism of IKBKB accumulation and consequential NF-κB activation in lung cancer.
Our results provide evidence that somatic E3-ligase complex disruption is a prominent genetic mechanism of NF-κB activation in lung cancer that compromises the ability of cells to degrade the NF-κB activator, IKBKB. We discovered a remarkably high frequency of both genetic disruption and gene expression changes for the genes encoding E3-ligase protein components (KEAP1, RBX1, and CUL3) as well as the gene encoding the complex's oncogenic substrate, IKBKB. We found that genetic disruption of the complex genes alters mRNA expression and results in elevated IKBKB protein levels, demonstrating the consequence of complex disruption. Moreover, we demonstrated evidence of NF-κB activation in complex compromised lung tumors and showed the importance of IKBKB protein expression in driving the lung cancer phenotype.
Although genetic and epigenetic disruption of KEAP1 has been reported in lung cancer before, to our knowledge, this is the first study to comprehensively characterize somatic gene dosage alterations to the CUL3, RBX1, and IKBKB loci in a large cohort of clinical lung tumors. The strikingly high frequency of copy number and gene expression alterations observed in our study highlights the importance of these E3-ubiquitin ligase complex components and also IKBKB in lung cancer. The recurrent nature of DNA copy number alterations at the complex component loci and their effects on gene expression are strong evidence that these genes are targeted for dosage alterations as opposed to passengers of alterations targeting other genes. In addition, the high proportion of disrupted lung tumors observed to have genetic alterations affecting a single component only, at both the copy number (67%, ) and gene expression levels (73%, ), suggests that disruption of only a single complex component is sufficient to compromise complex function and promote NF-κB signaling through abnormal IKBKB accumulation. Interestingly, we observed differential complex component disruption patterns in AC and SCC subtypes (). Although E3-ubiquitin ligase complex disruption occurs in both subtypes, the differences in the component genes preferentially altered suggests that complex disruption is achieved by different means.
Examination of IKBKB protein levels in NSCLC cell lines revealed high expression in lines harboring genetic disruption to at least one complex component or IKBKB, whereas the non-malignant lung line (NHBE) and a line without genetic disruption (H1650) showed very low or undetectable levels. The E3-ligase complex was considered to be genetically intact in H1650 as neither underexpression of KEAP1, RBX1, and CUL3, or overexpression of IKBKB relative to NHBE cells was observed (data not shown). This suggests there are no genetic or epigenetic alterations affecting the complex components or IKBKB in this cell line and the observed IKBKB levels were consistent with H1650 having a functioning E3-ligase complex, supporting our hypothesis. Therefore, in addition to affecting gene expression, copy number losses of the loci coding for complex components and gains of IKBKB appear to influence IKBKB protein expression. A trend towards higher IKBKB expression in lines with more complex components/IKBKB alterations was not evident, suggesting genetic disruption of a single component is sufficient to result in loss of complex function and IKBKB accumulation (). This finding is consistent with the observation that the majority of tumors exhibiting complex disruption have only one complex component altered, further supporting the idea that single component disruption is sufficient to produce an oncogenic effect.
Given our hypothesis, we focused on measuring gene dosage alterations that could account for disruption of the E3-ubiquitin ligase complex and its downstream consequences. However, it is conceivable that other genetic and epigenetic mechanisms could also contribute to silencing of the E3-ubiquitin ligase complex components thereby contributing to the observed accumulation of IKBKB and aberrant NF-κB signaling. Mutations and hypermethylation of KEAP1
have been described and these events can result in downregulation of KEAP1
expression 15, 17, 18, 21
. The COSMIC (Catologue of Somatic Mutations in Cancer) database has compiled mutation status for thousands of genes in cancer genomes 31
. As KEAP1
mutations are known to exist in lung cancer (albeit at low frequencies), we searched COSMIC for reported mutations in RBX1, CUL3
. No RBX1
mutations have been reported in over 170 cancer samples analyzed, while CUL3
mutations have been identified in three of 173 and 5 of 660 cancer samples, respectively, of which only one IKBKB
mutation was in lung cancer. Thus, the rarity of mutations in these genes in lung cancer specifically, suggests they are unlikely to be mutated and unlikely to play a role in complex disruption in the lung cancer cell lines we assessed. Furthermore, we have shown that DNA alterations at the KEAP1
, and IKBKB
loci are associated with concurrent expression changes, providing evidence that gene dosage alterations are a significant mechanism driving complex disruption at the genetic level.
We have conclusively demonstrated elevated IKBKB protein expression in NSCLC cell lines with complex disruption, however, measuring this effect directly in tumor tissue sections was not a straightforward task due to the extent of heterogeneity in tumor staining intensity across and within individual tumors. The variation in tissue distribution of stained cells is exemplified by two tumor samples shown in Supplemental Digital Content 7B
(genetic complex disruption) and 7C (no genetic complex disruption), and likely reflects the heterogeneous nature of lung tumor specimens. Due to this innate tumor heterogeneity, unlike cell lines, we were unable to conclude whether or not there was a significant correlation between E3-ligase complex disruption and IKBKB protein levels in vivo
To investigate the direct consequence of complex disruption on IKBKB accumulation and NF-κB activity, we performed siRNA knockdowns on the individual complex coding genes (KEAP1, RBX1, and CUL3). Consistent with our hypothesis, we observed elevated levels of activated IKBKB and NF-κB upon complex component disruption in HBEC cells (). Since phospho-NF-κB is an indicator of active NF-κB signaling, these results clearly illustrate the functional consequence of E3-ligase complex disruption. Our work provides evidence to support the hypothesis that genetic loss of the complex component encoding genes causes downregulation in their expression, and that loss of expression of these genes results in increased levels of activated IKBKB and NF-κB.
A number of reports have detailed the critical role of IKBKB protein in driving NF-κB activation, and the importance of IKBKB to cancer cell viability is emphasized by the development of IKBKB inhibitors as a strategy for tempering NF-κB signaling 8, 14, 22
. We found that IKBKB inhibition reduced NSCLC cell viability and that cells without complex or IKBKB
disruption, which we hypothesized to be less dependent on IKBKB expression for growth, were indeed more insensitive to IKBKB inhibition, as were cells with high endogenous levels of IKBKB protein (). In addition to cell experiments to verify the importance of E3-ligase complex disruption in lung cancer, we analyzed the expression levels of several NF-κB target genes in tumors with complex disruption to measure its effect on NF-κB activity. Despite the possibility that other mechanisms could also contribute to the transcription of the NF-κB target genes, we observed a significant increase in the expression of NF-κB target genes in complex compromised tumors (). We also observed elevated expression of NF-κB target genes following knockdown of the E3-ligase complex components. Together, these findings support our hypotheses and demonstrate the biological significance of complex disruption and subsequent IKBKB overexpression in lung cancer biology.
Collectively, our analyses have revealed remarkably frequent genetic disruption and aberrant expression not only of KEAP1, but all members of the KEAP1 E3-ubiquitin ligase complex and IKBKB in lung cancer. We have shown that IKBKB protein expression is elevated in NSCLCs with genetic loss of KEAP1, CUL3 or RBX1 or gain of IKBKB, and that knockdown of complex components leads to an accumulation of active IKBKB and NF-κB, thereby demonstrating the functional consequence and significance of complex disruption. We have also provided evidence of NF-κB activity, a downstream effect of IKBKB accumulation, in complex disrupted tumors and cell lines. Interestingly, it appears that AC and SCCs of the lung acquire copy number alterations to different components of the E3-complex or IKBKB which suggests the genetic mechanisms of complex disruption that promote NF-κB activation may be subtype specific. Our findings suggest that prominent genetic disruption to the E3-ubiquitin ligase complex and its oncogenic substrate, IKBKB, play a major role in driving the aberrant NF-κB activation that is characteristic of lung tumorigenesis.