Frequent concerted genetic mechanisms disrupt multiple components of the NRF2 inhibitor KEAP1/CUL3/RBX1 E3-ubiquitin ligase complex in thyroid cancer

Production of reactive oxidative species (ROS) by thyroid cells is essential to normal hormonogenesis and growth of the thyroid gland [1, 2]. However, increased production of ROS is damaging to cells, and therefore pathways controlling cellular defensive mechanisms in response to ROS, such as the NFE2-related factor 2 (NRF2) pathway are of particular importance to the thyroid. Paradoxically, constitutive activation of this pathway occurs in several tumor types affecting multiple oncogenic functions [3] (Figure 1A). Recently, the NRF2 pathway was found activated in papillary thyroid carcinoma (PTC) [4].

Activating mutations in the NRF2 gene, NFE2L2, are not common in PTC and genetic mechanisms underlying NRF2 activation remain to be elucidated for this tumor type [4]. NRF2 protein levels are negatively regulated by the KEAP1/CUL3/RBX1 E3 ubiquitin-ligase complex [5] (Figure 1A). Disruption of any complex component is sufficient to disrupt complex function and activate NRF2, highlighting the importance of simultaneously examining alterations to multiple complex components [6].

Since tumor genomes are disrupted by multiple genetic mechanisms, integration of multi-‘omics data for individual tumors represents a powerful approach for discovering genes frequently altered. Therefore, we simultaneously investigated DNA sequence mutations, gene DNA copy number, and DNA methylation affecting key components of the KEAP1/CUL3/RBX1 complex and the NFE2L2 gene using a cohort of 310 PTC tumors from The Cancer Genome Atlas (TCGA) project (Additional file 1). We hypothesize that component gene disruption (DNA level) of the KEAP1/CUL3/RBX1 complex is a frequent event in PTC and may explain activation of NRF2 previously observed in PTC. We report a remarkably high frequency of DNA disruption to NRF2 inhibitor complex components and gene expression patterns in PTC tumors concordant with NRF2 activation.

Abundance of physiological ROS results in oxidative stress which is damaging to cells and can lead to malignant transformation. Paradoxically, the NRF2 pathway, which is responsible for protecting cells against ROS damage, is constitutively activated in multiple cancer types and associated with many cancer hallmarks [11, 12]. Over-activation of this pathway has recently been described in PTC [4]. Given recent findings and the importance of ROS to normal thyroid hormonogensis, elucidation of the molecular mechanisms underlying NRF2 deregulation in PTC is relevant. Here, we provide evidence of DNA mechanisms that likely promote stabilization and activation of NRF2 in PTC.

We answered questions pertaining to NRF2 activation utilizing a large panel of PTCs with multi-‘omics data from the TCGA data portal. Consistent with previous reports, activating mutations or copy number gains/amplifications targeting NFE2L2 are uncommon in PTC [4]. Moreover, while BRAF V600E mutations were common, these were not associated with increased NRF2 expression in PTC, as described for other cancers [8], indicating that NRF2 pathway activation in PTC likely occurs through other mechanisms (Figure 1B).

The KEAP1/CUL3/RBX1 complex is a key negative regulator of NRF2. We applied a multi’omics approach to interrogate DNA disruptions to KEAP1/CUL3/RBX1 complex components (Figure 1A). In a previous study, Ziros et al., found no correlation between KEAP1 and NRF2 protein expression levels, arguing against decreased KEAP1 levels as mechanisms for NRF2 activation [4]. Based on our results, alterations affecting other complex components, such as CUL3 and RBX1, might be also contributing to increased expression of NRF2 observed in PTC. We found that DNA copy number loss and promoter hypermethylation frequently affect individual components, that when considered together affect a remarkably high proportion of PTCs (80%). This phenomenon has been observed by our group and others in lung cancer [6, 13]. However, the frequency by which multiple complex components were simultaneously disrupted, and the molecular mechanisms affecting individual components, were strikingly different for PTC (Figure 1C-D), compared to other tumor types.

Promoter hypermethylation is the predominant mechanism affecting KEAP1 and CUL3 in PTC. Silencing of KEAP1 by hypermethylation has been described in lung cancer [14], and associated with stabilized NRF2 and increased expression of NRF2 target genes in colorectal cancer [15]. While KEAP1 mutations are frequent in other cancers, they are not in PTC. However, germ-line KEAP1 mutations have been identified in a family with multinodular goiter [16], alluding to the importance of this gene to normal thyroid physiology. Interestingly, segmental DNA losses is almost the exclusively the type of alteration affecting RBX1. This phenomenon has been also observed at a similar frequency in lung tumors, with a concurrent complex function disruption [6].

Gene set enrichment analysis (GSEA) revealed that not only were genes containing the consensus NRF2-binding motif over-expressed in PTCs, but overall, the most significantly enriched gene sets corresponded to functions related to NRF2, including the NRF2 activator c-JUN and the NRF2 target competitive binders, BACH1 and 2 (Figure 2B). Bach1, competes with NRF2 protein for binding to target genes, leading to inhibited expression of NQO1 and HO-1 for example [17‐19]. These results indicate that in addition to DNA disruption to NRF2 complex inhibitors, the NRF2 pathway is also likely activated by increased activity of NRF2 activators and decreased activity of NRF2 gene target transcriptional repressors in PTCs.

ETV5 was the putative NRF2 transcriptional target most highly upregulated in tumors compared to controls. ETV5 is an oncogenic member of the ETS transcription factor family, associated with cell proliferation and metastasis in various cancers [20], and a known target of thyroid-specific transcription factor FOXE1. Although we did not detect overexpression of FOXE1 in PTCs at the mRNA level, further exploration of ETV5 in the context of NRF2 signaling is warranted.

Taken together, we found that i) DNA level disruption of NRF2 inhibitory complex components occurs at an extremely high frequency in PTC, ii) primarily by promoter hypermethylation, iii) NRF2 transcriptional targets are overexpressed in PTC, and iv) activation of NRF2 in PTC is complex and likely involves activation and inhibition of multiple NRF2 activators and repressors.

A major caveat of this study is the lack of protein assessment. However, the data presented here provide rationale to interrogate the role of the NRF2 pathway in PTC at the protein level, and elucidate the target molecules driving PTC phenotypes. In summary, we present compelling evidence that activation of the NRF2 pathway is extraordinarily, selectively activated in PTC and provide further rationale for exploration of this pathway as a therapeutic target in PTC.