Targeting neddylation E2 for anticancer therapy, putting new wine into new bottles?

The cancer cell is capable of hijacking normal protective mechanisms to adapt to the extremely harsh intracellular and extracellular stresses it has created. Neddylation is a protein post-translational modification by which the ubiquitin-like protein NEDD8 conjugates to protein targets via an E1-E2-E3 multi-enzymatic cascade. As a vital survival mechanism in maintaining cell and tissue homeostasis in all eukaryotes, neddylation appears to be upregulated in multiple types of cancers, therefore targeting neddylation has become a promising anticancer strategy [1]. To this end, a small molecule, MLN4924 (Pevonedistat) has been developed to specifically inhibit NEDD8 activating enzyme (NAE) (E1), the apex of NEDD8 E1-E2-E3 cascade [2]. NAE is the only NEDD8 E1 identified and a heterodimer of the regulatory subunit NAE1 and the catalytic subunit UBA3. MLN4924 forms a tight, irreversible NEDD8-MLN4924 covalent adduct that resembles NEDD8-AMP and prevents the docking of NEDD8-AMP to the catalytic pocket in UBA3, a process that is required for NEDD8 activation, thereby effectively blocking neddylation [3]. Increasing evidence from pre-clinical studies and clinical trials have consistently demonstrated the effectiveness and potency of MLN4924 in inhibition of tumourigenesis and metastasis in diverse cancers ranging from hematological malignancies to solid tumours [4], highlighting its therapeutic potential. However, similar to other target-based chemotherapies, mutations on UBA3 that diminish the binding affinity of NEDD8-MLN4924 to UBA3 render tumour cells resistant to MLN4924 treatment and may limit its clinical application [5]. Therefore, there is an emergent need to identify additional drug targets in the neddylation pathway in order to develop the next generation of neddylation inhibitors.

In an article in EBioMedicine, Li and colleagues explored the possibility of targeting the E2 neddylation conjugation enzyme to suppress tumour growth [6]. In mammalian cells, two NEDD8 E2s have been identified, the well-characterized UBC12 (also known as UBE2M) and the less-studied UBE2F. These two E2s appear to show distinct function: UBC12 interacts with RBX1 to mediate neddylation of Cullin 1–4, whereas UBE2F pairs with SAG/RBX2 to facilitate neddylation of Cullin 5. While UBE2F was recently shown to promote the survival of lung cancer cells by sustaining CRL5 to degrade NOXA [7], little is known about the role of UBC12 in lung cancers.

In this study, Li and colleagues first analyzed two published Affymetrix microarray datasets which revealed that UBC12, but not NAE1 and UBA3, is overexpressed in multiple lung cancers and predicts poor survival rate. Targeted deletion of UBC12 in two lung cancer cell lines led to inhibition of Cullin neddylation, accumulation of multiple cell-cycle inhibitors that are known to be degraded by Cullin-based RING ubiquitin ligases (CRLs), and cell cycle arrest. In vivo, deficiency of UBC12 also greatly suppressed tumour growth and metastasis. Together, these results confirm a critical role of UBC12 in controlling neddylation and are reminiscent of those observed by others in MLN4924-treated tumour cells, thereby identifying UBC12 as an attractive alternative therapeutic target in cancer treatment.

Further, the authors addressed whether targeting UBC12 could be a potential strategy to resolve MLN4924-induced drug resistance. In an MLN4924-resistant cancer cell line, whose sensitivity to MLN4924 has dropped ~20-fold compared to its parent wild-type cell line, knockdown of UBC12 was able to effectively inhibit Cullin neddylation and thus repress cell growth, suggesting that inhibition of UBC12 could be effective to suppress the growth of tumours resistant to MLN4924. Future studies are warranted to investigate whether deletion of UBC12 has any impact on tumour growth in mice transplanted with MLN4924-resistant tumours, and to develop a pharmacological compound that specifically inhibits UBC12.

While aberrant neddylation has been repeatedly reported to associate with tumour malignancy, very little is known about upstream mechanisms regulating neddylation. UBC12 is a stress-responsive protein and is transcriptionally controlled by HIF1α and AP-1 signaling [8]. Thus, it is conceivable that the upregulation of UBC12 in lung cancer cells, as reported in this study, could be consequent to transactivation of HIF1α in the hypoxic tumour microenvironment. Nevertheless, it remains to be determined whether its protein levels are consequently increased and whether such changes are sufficient to enhance the abundance of neddylated proteins in cancer cells.

To pinpoint the underlying mechanism by which UBC12 controls tumourigenesis, Li and colleges performed quantitative proteomic analysis. The results revealed an accumulation of more than 500 proteins in cancer cells lacking UBC12 [6] and coincide with the loss of function of CRLs. Yet, UBC12 deficiency also caused significant downregulation of ~500 proteins in cancer cells, indicating that neddylation is necessary for other cellular processes beyond Cullin-mediated proteolysis. To date, only a dozen of non-Cullin NEDD8 substrates have been identified, which has prevented us from fully understanding the cellular functions of neddylation in not only cancer cells, but also in other tissues. Identification of novel NEDD8 targets is challenging because of the low abundance of neddylated proteins, the transient and reversible nature of the modification, and concerns over the fidelity of targets identified from cells in which NEDD8 is overexpressed. Supported by this study, ectopic expression of UBC12 could a feasible approach to upregulate neddylated proteins, thereby facilitating the search for bona fide NEDD8 targets.

Besides cancers, dysregulation of neddylation is implicated in various other diseases. In particular, specific inhibition of neddylation in neurons and cardiomyocytes has been shown to cause defects in synapse formation [9] and cardiac maturation [10], respectively. An important yet unanswered question is whether normal cells/tissues, like cancer cells, could benefit from the global enhancement of neddylation under pathological conditions. Thus far, a valid tool to enhance neddylation in vivo is still lacking. As seen in this study and in cases of other ubiquitin-like proteins such as SUMO, overexpression of their E2 enzymes is able to stimulate the respective protein modification. Thus, this strategy could be employed to promote neddylation in vivo, ideally in a cell type-specific manner, and to ultimately address this question.

Declaration of Competing Interest

The authors declare no conflicts of interest.