Targeting E3 ubiquitin ligases to sensitize cancer radiation therapy

Radiotherapy is an effective treatment for many cancer patients to eliminate malignant cells and increase survival rate. However, cancer cells can develop resistance in response to radiation through activation of signaling pathways that promote cell cycle progression, DNA damage response, cell survival, and inflammation. Various combination therapies are developed to sensitize radiotherapy by targeting key signaling proteins involved in radioresponse. The past decade has seen significant advances in the knowledge of ubiquitin signaling in cancer biology. Developing E3 ubiquitin ligase‐related molecules as novel strategies to cure cancer has become an emerging field. This review briefly discusses the potential of targeting diverse E3 ubiquitin ligases as promising strategies for radiosensitization in cancer.


INTRODUCTION
Cancer radiotherapy is widely used as a standard first-line treatment for numerous types of cancer. 1 The purpose of radiotherapy is to kill cancer cells or inhibit their growth through high-energy radiation. Radiation generates ions that pass through cells, damaging the genomic DNA of cells, and thus blocking their ability to proliferate and causing cell death. 2 Although radiation could impair both normal and cancer cells, normal cells usually more readily recover than cancer cells, because cancer cells are defective in DNA repair machinery. 2,3 Radiotherapy, combined with surgery and chemotherapy, has significantly improved the survival rates of many cancer patients. 4,5 However, the therapeutic effect of radiation still remains unsatisfactory in many cases where tumor cells develop radioresistance and normal tissues suffer from severe adverse reactions to irradiation. 3 processes. 3,6 In the present review, we briefly summarize the ubiquitin E3 ligases that are potential targets to sensitize cancer radiation therapy.
The ubiquitination system involves E1-activating enzymes, E2 conjugation enzymes, and E3 ligases working in unison to covalently attach ubiquitin to the lysines of substrate proteins. 7 The ubiquitinproteasome system is the major protein degradation machinery in the cell to define the proteome at the post-translational level, which is dependent on Lysine-48 (K48) polyubiquitin chains that mark substrate proteins for proteasomal degradation. [7][8][9] The indication of targeting ubiquitin signaling to sensitize cancer radiation therapy is supported by the fact that bortezomib, a clinically approved proteasome inhibitor to treat several cancers, has been shown to be a tumor radiosensitizer. [10][11][12][13] However, such proteasome inhibitors are problematic, in that they non-selectively block protein degradation, thus impacting too many pathways and leading to severe toxicity. The new direction of therapeutic development is focused on targeting individual or subtypes of over 600 ubiquitin E3 ligases, which confer specificity of substrates. 7,14 Other than ubiquitin-proteasome system, there are various other types of polyubiquitination or monoubiquitination that serve as non-proteolytic signals in DNA repair, subcellular localization, and other signal transduction pathways. 14 There are three types of E3 ligases, including RING E3s, HECT E3s, and RING-between-RING (RBR) E3s. 7,[14][15][16] In the present review, we discuss the indication of members in these three major E3 families as targets for radiosensitization. to facilitate ubiquitin transfer. [19][20][21][22][23] There are six major cullins (CUL1, CUL2, CUL3, CUL4A, CUL4B, and CUL5), each assembling with a distinct set of >200 substrate receptor modules. 18 CRLs and related enzymes constitute a very dynamic system. Catalyzed by CAND1/2, CRLs undergo active exchange of substrate receptors in response to particular cellular events to turn over specific substrates. [24][25][26][27][28] Regulation of CRL enzymatic activity also involves dynamic activation/deactivation cycles: conjugation of NEDD8 (neddylation) to cullin activates CRLs, [29][30][31] and the COP9 signalosome complex (CSN), which deneddylates cullin, puts CRLs into an inactive state. 32 Such detailed illustration of the regulation of the CRL system enabled discoveries of several targeted therapies for different CRL components, which have been tested for radiosensitization.

RING E3 LIGASES
Early studies using RNAi to silence RBX1/2, the catalytic subunit of CRLs, confirmed that crude inhibition of CRL activity could sensitize cancer cells to radiation. 33,34 The role of NEDD8 conjugation on cullins as an essential activating modification 29 makes the neddylation enzymes promising targets to modulate CRL activities and enhance radiation therapeutic efficacy. This is supported by Wang et. al. that silencing of UBC12, the NEDD8 E2 enzyme, sensitizes prostate cancer cells to radiation. 35 In 2009, a small-molecule inhibitor called MLN4924 was developed to specifically target the neddylation E1 enzyme, NAE, potently blocking cullin neddylation and activation. 36 MLN4924 treatment leads to deneddylation within 1 h, followed by accumulation of numerous CRL substrates impacting multiple facets of biology. [36][37][38] Indeed, MLN4924 has been shown to exert a radiosensitization effect in many cancer types, including pancreatic, lung, breast, neck-and-oral, prostate, and blood tumors. 35,[39][40][41][42][43][44] In these studies, it is well established that MLN4924 sensitizes cancer radiotherapy through inducing G2/M cell cycle arrest and DNA damage response. 39,40,42 The underlying mechanism is that MLN4924 treatment results in an accumulation of CRL substrates, such as Wee1, p21, p27, and Cdt1, regulators of cell cycle and DNA damage response. 39,40,42 Knocking down p21, Wee1, or Cdt1 could attenuate the radiosensitizing effect of MLN4924. 39,40 Although most studies attribute the mechanism of MLN4924 to the proteins and pathways mentioned above, it is proposed that MLN4924 mediates radiosensitization in different cancer types through different mechanisms. 45 Considering the diversity of the CRL pool and their substrates, 27,38 it is plausible that the repertoire of active CRLs in different cancer types could be variable, and they might respond differently to MLN4924. It is also a major concern that MLN4924 as a non-selective inhibitor of CRL system could cause toxicity in normal tissues. 46  These studies showed the radiosensitization effect of targeting these RING E3s, and there are probably many more RING E3 ligases involved in radioresponse signaling. Developing specific inhibitors for these RING E3s is a challenging, but exciting, direction in the future.

HECT AND RBR ELIGASES
As mentioned above, HECT and RBR E3 ligases contain a catalytic cysteine that receives ubiquitin from E2 7 , forming an E3∼Ub thioester, and then transfers the ubiquitin to substrate proteins. There are much fewer members in the HECT and RBR families, and studies directly investigating the radiosensitizing effect of them are generally lacking.
However, recent progress in the mechanistic insights into the autoregulatory mechanisms of these enzymes has led to the discovery of novel inhibitors, which showed promising antitumor activity. Considering the vital roles these E3s are playing in controlling numerous cellular functions in cancer cells, 62,63 it is conceivable that therapies targeting some members in the HECT and RBR families will greatly improve radiotherapy.
There are 32 HECT E3s in the human genome, which are featured by comprising a HECT catalytic domain. 62  Deletion of HUWE1 leads to p53 protein accumulation, growth arrest, and apoptosis in MYC-driven B-cell lymphomas. 71 Knocking down WWP1 leads to p53 accumulation and HCC cell apoptosis. 72 Interestingly, instead of degrading p53, WWP1 stabilizes p53, leading to its cytoplasmic accumulation and reduction of transcriptional activity. 73 Constitutively active EGFR is overexpressed in many cancer types and is associated with poor prognosis. Ionizing radiation can activate EGFR and its downstream signaling pathway, and EGFR-targeted therapy has been approved by the FDA for the treatment of headand-neck cancer in combination with radiation. 6  Indole-3-carbinol is a natural compound found in vegetables, such as broccoli, cauliflower, and cabbage, which exerts the ability to inhibit NEDD4-1 and WWP1, and thus activates PTEN in tumor cells. 78,89 Given the fact that p53 and PTEN are two important players in regulating radiosensitivity and radioresistance, their ubiquitin E3s inhibitors could be potential radiosensitizers, whereas such investiga- tions are yet to be carried out.
RBR E3s contain two RING domains, one of which docks E2∼Ub, and the other harboring a catalytic cysteine receiving and transferring ubiquitin. 16 There are 12 major RBR E3s. 16 Parkin, an RBR E3 ligase, has been extensively studied in Parkinson's disease. 90

CONCLUSION AND FUTURE PERSPECTIVES
Targeting ubiquitin E3 ligases has proved to be a promising direction to improve the efficacy of cancer radiotherapy. In the past decades, numerous seminal breakthroughs have been made in basic science to understand the structure, biochemistry and regulation of various E3 ligases, as well as in translational research, which unraveled the roles of different E3s in cancer, and targeted compounds for them, showing therapeutic potentials. Accumulating evidence from pre-clinical studies using RNAi justified that many E3 ligases are candidate targets for radiosensitizers. However, there is still little known about most members of the large E3 ligase family. Current advanced high-throughput technologies, such as RNA sequencing, CRISPR screening, and proteomics, should be carried out to uncover new E3 ligases with critical functions in cancer and radioresponse. Furthermore, there are dozens of new inhibitors for the ubiquitin system that remain to be tested for radiosensitization, such as DCN1 inhibitors, CSN5i-3, and Heclin. With the enthusiasm of both academia and industry in developing therapies targeting E3s nowadays, it is likely that more and more new compounds will be made available, and it will be exciting to explore their radiosensitizing effect in the future.

CONFLICT OF INTEREST
The authors declare that they had read the article and there are no competing interests.