Protein neddylation in lung tumorigenesis: Target validation and targeted therapy

Abstract

Much akin to ubiquitylation, neddylation is catalyzed by a cascade of three enzymes: E1 NEDD8-activating enzyme, E2 NEDD8-conjugating enzyme (UBE2M or UBE2F), and E3 NEDD8 ligases. The best-known neddylation substrates are the members of cullin family, leading to the activation of Cullin-RING ligases, which regulate a variety of downstream biological processes largely via promoting ubiquitylation and subsequent proteasomal degradation of many key signaling proteins. Notably, neddylation enzymes and components of the Cullin-RING ligases are frequently altered in many human cancers and have been validated as promising cancer targets. As such, drug discovery efforts are underway to target neddylation-Cullin-RING ligases with a few selective small molecule inhibitors being advanced into various phases of clinical trials. This review firstly provides a brief introduction to neddylation, then focuses on lung cancer, and summarizes a wealth of current data showing how neddylation-Cullin-RING ligases are altered and affect the growth and survival of lung cancer cells, lung tumorigenesis, lung tumor microenvironment, and inflammatory response. A few reported small molecule inhibitors of neddylation enzymes as well as their activity against lung cancer cells are also summarized, and future perspectives with an ultimate goal of discovering effective treatment of lung cancer via targeting neddylation-Cullin-RING ligases are proposed.

1. Introduction

Protein neddylation, a ubiquitin-like post-translational modification (PTM), is a process of attaching a ubiquitin-like protein NEDD8 (the neural precursor cell expressed, developmentally downregulated protein 8) to a substrate protein by an isopeptide bond between the NEDD8 C-terminal glycine76 (Gly76) and a lysine residue on the target proteins [1], not for substrate degradation, but for functional modulation of the substrates. Like ubiquitylation, neddylation is mediated by a cascade of three enzymes, an NEDD8-activating enzyme (NAE) E1, an NEDD8-conjugating enzyme E2, and an NEDD8 ligase E3 [2]. The cascade starts with the NEDD8 precursor maturation, a proteolytic process to expose the C-terminal Gly76 by NEDD8 protease 1 (NEDP1, also referred to as deneddylase 1, DEN1) [3]. The matured NEDD8 is activated by the E1, a heterodimer complex consisting of NAE1/APPBP1 and UBA3/NAEβ, in an ATP-dependent reaction. The activated NEDD8 is transmitted to the E2, UBE2M/UBC12 or UBE2F, which accepts and shuttles it to the E3 ligase. The substrate-specific E3 ligase ultimately conjugates NEDD8 to a target substrate protein [2]. Unlike ubiquitin E3 ligases which are anticipated to have over 600 members in the human genome, there are only a few members of NEDD8 E3 ligases. Most of them contain really interesting new gene (RING) finger domain, which includes RING-box protein 1 (RBX1, also known as ROC1) and RING-box protein 2 (RBX2, also known as ROC2/SAG) [4,5], along with a few other E3s, including DCN1-LIKE proteins (DCNL1-5), MDM2, SCF^FBXO11, c-CBL, TRIM40, IAPs, RNF111, and TFB3 [6]. Notably, all known neddylation E3s also serve as E3s for ubiquitination.

The most well-known substrates for neddylation are the cullin family of proteins, consisting of 8 members, including Cullin-1 (CUL-1), Cullin-2 (CUL-2), Cullin-3 (CUL-3), Cullin-4A (CUL-4A) and 4B (CUL-4B), Cullin-5 (CUL-5), Cullin-7 (CUL-7) and Cullin-9 (CUL-9) with CULs 1-5 being well-studied [7]. It is known that the UBE2M-DCN1-RBX1 E2-E3 pair catalyzes CULs 1-4 neddylation, while the UBE2F-SAG pair promotes the neddylation of CUL-5 [8,9] (Fig. 1). The cullin neddylation via multimodal means [10] activates Cullin-RING ligases (CRLs), known as the largest E3 ubiquitin ligase subfamily, comprising a scaffold cullin, a substrate-receptor protein, an adaptor protein, and an E2 binding RING protein [7]. CRLs are necessary for the ubiquitination of ∼20% of cellular proteins for proteasome degradation [11], thereby regulating many important biological processes, such as cellular responses to hypoxia and oxidative stress, immune response, cell cycle progression, DNA replication and repair, gene transcription, virus infection, apoptosis, and tumorigenesis, among others [2,6,[12], [13], [14]. Other non-cullin substrates were also subjected to neddylation [6]. However, none of them has been fully characterized as physiological substrates, with only the neddylated form being detected in conventional western blotting. This review will only summarize the enzymes that promote cullin neddylation and the cullin substrates.

Fig. 1.

A schematic illustration of the neddylation cascade, enzymes and substrates. Neddylation is a process that tags the ubiquitin-like small molecule NEDD8 (N8) onto its substrate through an enzymatic cascade involving the NEDD8-activating enzyme E1, the NEDD8-conjugating enzyme E2 and substrate specific NEDD8 E3 ligases. Cullin family members are the best-known physiological substrates of neddylation, and cullin neddylation activates CRLs.

Lung cancer has been the leading cause of cancer deaths worldwide for the last several decades, accounting for 12%-13% of new cancer cases and 21% of new cancer deaths in 2022 [15]. The 5-year survival rates for lung cancer patients diagnosed between 2010 and 2014 are only 10%-20% in most countries [16]. Thus, understanding the fundamental molecular mechanism that drives lung cancer occurrence, progression, and metastasis, identifying novel therapeutic targets, and discovering potent growth-suppressing molecular agents against lung cancer cells are crucial for guiding the treatment of lung cancer. In the past decade, numerous studies were conducted to understand the critical roles of neddylation and the underlying mechanism of neddylation as a regulator in lung tumorigenesis and altered tumor microenvironment (TME). This review summarizes the alterations of the neddylation pathway and its regulation in lung cancer cell survival and proliferation, in lung tumor formation and TME, as well as anti-lung cancer activities of small molecular inhibitors of enzymes for cullin neddylation.

2. Alterations of neddylation enzymes and cullin substrates in lung cancer

The protein abundance of NEDD8 and all three neddylation enzymes, including two subunits of NAE E1 (NAE1/APPBP1 and UBA3/NAEβ), two E2s UBE2M and UBE2F, and two E3s (RBX1/ROC1 and RBX2/SAG) for cullin neddylation are relatively higher in lung tumor tissues, compared with adjacent normal tissues, and higher levels of neddylation enzymes are related to low survival rates of lung cancer patients in most cases [17], [18], [19], [20], [21], [22]. Specifically, NEDD8 and global protein neddylation were relatively higher in the mRNA and protein levels in large cell lung cancer (LCLC), adenocarcinoma, and squamous carcinoma. Furthermore, upregulation of NEDD8 expression was found to result in poorer overall survival in patients with squamous carcinoma and adenocarcinoma [19]. The mRNA level of neddylation E2 UBE2M/UBC12 in lung cancer tissues was also much higher than in normal lung tissues and positively correlated with NEDD8 expression and global protein neddylation. Again, the patients with high UBC12 or NEDD8 mRNA levels showed poorer overall survival [21]. Zhou et al. found that UBE2F was overexpressed in non-small cell lung cancer (NSCLC) cells, and higher UBE2F expression was associated with poor patient survival [18]. Neddylation E3 ROC1 was also found to be upregulated in various human cancer tissues and cell lines, including lung cancer [17,22]. Moreover, our large-scale analyses of neddylation E3 SAG/RBX2 mRNA expression levels and prognosis data from 442 lung adenocarcinoma patients demonstrated that high SAG mRNA level was significantly related to increased tumor size and poorly differentiated tumors and patients with high SAG expression had lower overall survival [20].

Consistently, the expressions of neddylation substrates cullins are also upregulated in lung tumor tissues and cell lines [23], [24], [25], [26], [27], [28]. Specifically, Salon et al. analyzed CUL-1 expression in 128 human lung cancer clinical samples. They found that CUL-1 was overexpressed in 40% of all lung tumors, including 34% of NSCLCs, 75% of carcinoids, and 30% of high-grade neuroendocrine lung tumors [26]. Li et al. also found that, in lung adenocarcinoma tissues, the neddylated to un-neddylated CUL-1 ratio was almost three times higher than that in adjacent normal tissues [24]. High levels of CUL-4A and CUL-4B expression in human NSCLCs and a large cohort of lung cancer patients were also observed, which was significantly correlated with the reduced overall survival of lung cancer patients [23,27]. Moreover, Men et al. compared the CUL-7 expression profile in normal versus lung cancer human tissues and observed overexpression of CUL-7 in most lung cancer specimens [25]. In contrast, Zhou et al. reported that CUL-3 overexpression was associated with better overall survival in lung adenocarcinoma patients [28]. Taken together, cullin family members in most cases, are overexpressed in lung cancer settings, which is positively correlated with poor patient survival (Table 1).

Table 1.

Overexpression of neddylation enzymes and cullin substrates in lung cancer patients.

NEDD8 Enzymes	Cullin Substrates	Lung Cancer Types	Patient Survival	Refs
NEDD8	–	Large cell lung cancer, non-small cell lung cancer	Poor	[19]
NEDD8, UBE2M	–	Large cell lung cancer, non-small cell lung cancer	Poor	[21]
UBE2F	–	Non-small cell lung cancer	Poor	[18]
ROC1	–	Non-small cell lung cancer	Poor	[17]
SAG	–	Non-small cell lung cancer	Poor	[20]
–	CUL-1	Neuroendocrine lung tumor, non-small cell lung cancer	Poor	[26]
–	CUL-1	Non-small cell lung cancer	Poor	[24]
–	CUL-3	Non-small cell lung cancer	Favorable	[28]
–	CUL-4	Non-small cell lung cancer	Poor	[23,27]
–	CUL-7	Non-small cell lung cancer	Poor	[25]

3. Neddylation regulates lung cancer cell survival and proliferation as well as lung tumor formation

Given that most neddylation enzymes are overexpressed in lung tumors, and associated with poor survival rates, many follow-up preclinical studies were conducted to determine whether this overexpression is causally related to accelerated growth of lung cancer cells or lung tumorigenesis, using both genetic (siRNA knockdown or genetically modified mouse models) and pharmacological (MLN4924) approaches (Fig. 2; Table 2).

Fig. 2.

Neddylation modulates lung cancer cell growth and inflammatory responses in lung cancer tissue. Inhibition of neddylation by MLN4924 or knockdown of various NEDD8 enzymes suppresses the growth and survival of lung cancer cells in vitro and lung tumorigenesis in vivo (left) as well as regulates inflammatory responses in lung cancer tissues (right).

Table 2.

Neddylation regulates lung cancer cell proliferation and survival, lung tumorigenesis, and lung tumor microenvironment and inflammatory responses.

Neddylation Inhibition	Effect of Neddylation Inhibition in Lung Cancer	Refs
Genetic approaches
NEDD8 knockout	Induces G2/M arrest, and subsequent apoptosis or senescence through accumulation of tumor suppressors p21, p27 and Wee1	[19]
NEDD8, NAE knockdown	Inhibits metastasis by suppressing CCL2 transactivation and reducing macrophage population	[47]
UBE2M knockdown	Inactivates CRLs 1-4 and induces G2/M arrest through accumulation of tumor suppressors p21, p27 and Wee1	[21]
UBE2F knockdown	Inactivates CRL5 and induces apoptosis through accumulation of proapoptotic protein NOXA	[18]
ROC1 knockdown	Inhibits mTOR activity through accumulation of DEPTOR and independently induces autophagy and p21-depedent senescence	[17,22]
Sag conditional knockout	Inhibits NF-κbB and mTor signaling through accumulation of tumor suppressors Iκbα, Deptor, p21, p27, Noxa and Bim, and suppresses proliferation	[20]
CUL-4A knockdown	Inhibits AKT signaling through decreased EGFR transcription and phosphorylation and suppresses cell proliferation and promotes apoptosis; increases sensitivity to chemotherapy drugs through accumulation of tumor suppressors TIEG1, TGFBI and p33ING1b	[27,[30], [31], [32]
CUL-5 knockdown	Triggers apoptosis through accumulation of proapoptotic protein NOXA and re-sensitization to CDK9 inhibitor which targets antiapoptotic protein MCL1	[33]
CUL-7 knockdown	Activates DNA damage response pathway and inhibits cell proliferation through accumulation of tumor suppressors p53, p21 and p27	[25]
Pharmacological approaches
MLN4924	Inactivates CRL1 and CRL4 and leads to accumulation of CDT1, which triggers DNA re-replication and induces apoptosis	[11]
MLN4924	Blocks degradation of HIF-1α and c-JUN to upregulate UBE2M which couples with CRL3 and Parkin to promote degradation of UBE2F, resulting in accumulation of CRL5 substrate NOXA to induce apoptosis	[18,34]
MLN4924	Induces G2/M arrest and apoptosis through accumulation of proapoptotic protein NOXA	[24]
MLN4924	Blocks NF-κB and mTOR signals and causes accumulation of p21, p27, NOXA and BIM to inhibit proliferation	[20]
MLN4924	Sensitizes lung cancer cells to chemotherapeutic drugs and promotes DNA damage and apoptosis	[35,36]
MLN4924	Triggers p21-dependent senescence through accumulation of p21, p27 and Wee1, and DNA damage response through accumulation of CDT1 and ORC1	[37,39]
MLN4924	Induces autophagy by DEPTOR accumulation to inhibit mTOR	[39]
MLN4924	Inhibits mTOR signal and induces autophagy by accumulated HIF-1α, followed by activation of the HIF1-REDD1-TSC1 axis	[40]
MLN4924	Blocks NF-κB transcriptional activity by IκBα accumulation to suppress CCL2 transactivation, resulting in decreased infiltration of MDSCs and TAMs in lung tumor sites	[45,47]
TAS4464	Inhibits cullin neddylation and induces CDT1, p27 accumulation and IκBα phosphorylation, which shows greater inhibitory effects than MLN4924	[59]

3.1. Genetic approaches

NEDD8 appears to be growth essential for lung cancer cells. Jiang et al. reported that NEDD8 depletion significantly suppressed the growth of A549 lung cancer cells and tumor formation in a xenograft model by inducing accumulation of tumor suppressor (including p21, p27, and Wee1) to trigger G₂-phase cell cycle arrest and senescence via inactivation of CRLs [19].

Li et al. reported that the knockdown of UBE2M/UBC12 inhibited neddylation of CULs 1-4, but not CUL-5, inactivated CRLs 1-4 to cause accumulating tumor suppressor proteins (p21, p27, and Wee1), leading to suppression of cellular proliferation by inducing G₂/M arrest in lung cancer cells [21]. Similarly, Zhou et al. reported that UBE2F E2 and SAG/RBX2 E3 cooperatively promote CUL-5 neddylation to activate CRL5 and subsequently ubiquitylate and degrade NOXA in lung cancer cells. As such, UBE2F knockdown by siRNA caused the inactivation of CRL5, thereby leading to accumulation of NOXA, and selectively inhibited proliferation and survival of lung cancer cells by inducing apoptosis both in cell culture and xenograft tumor models. Notably, the depletion of NOXA in part rescues the phenotype triggered by knockdown of UBE2F, indicating a causal role of NOXA in this experimental setting [18].

Jia et al. reported earlier that siRNA-based knockdown of RBX1/ROC1 E3 inhibited the growth of multiple human cancer cell lines, including lung cancer cell line H1299 in part via induction of apoptosis and cellular senescence. The senescence induction that occurred in H1299 cells by ROC1/RBX1 silencing was likely coupled with DNA damage in p16/pRB- and p53/p21-independent manner [17]. Furthermore, Yang et al. explored whether ROC1/RBX1 knockdown triggers autophagy in several cancer cell lines, including H1299, and found that ROC1 knockdown significantly inhibited cancer cell proliferation in vitro and in vivo by sequentially and independently inducing autophagy and p21-dependent cellular senescence. Mechanistically, ROC1 knockdown-induced autophagy was triggered by inhibition of mammalian target of rapamycin (mTOR) kinase activity in part due to the accumulation of DEP domain containing mTOR interacting protein (DEPTOR), an endogenous inhibitor of mTOR complexes (mTORCs), and knockdown of endogenous DEPTOR blocked the autophagy pathway following ROC1 knockdown. Unlike apoptotic response, which was significantly enhanced upon ROC1 silencing, the autophagy pathway inhibition did not obviously affect senescence induction [22].

To define the role of Sag/Rbx2 E3 in lung tumor formation, Li et al. generated Sag^fl; Kras^G12D compound mice, and found that knockout of Sag significantly inhibited lung tumorigenesis induced by Kras^G12D and extended mouse life-span. Mechanistically, Sag depletion caused accumulation of tumor suppressor proteins, including IκBα, Deptor, Noxa, Bim, p21, and p27, leading to inactivation of nuclear factor kappa B (NF-κB) and mTOR signals and suppression of proliferation [20]. Similarly, Rbx1 E3 deletion in the same Kras^G12D mouse lung model also suppressed tumor formation and extended mouse life-span via causing accumulation of tumor suppressor p21 and Foxo 1, and inactivating the pErk and mTorc1 signals [29].

In addition to NEDD8 enzymes, a few studies were conducted to investigate how the expression of cullins, the best-known physiological substrates of neddylation, affects lung cancer cell proliferation and survival or lung tumorigenesis. Among these studies, CUL-4A was found to be overexpressed and acted as an oncogene in several human cancers, including lung cancer [27,[30], [31], [32]. CUL-4A knockdown in NSCLC cells suppressed cell proliferation and promoted apoptosis, thereby suppressing the growth of lung cancer cell lines and inhibiting lung tumorigenesis in the lung cancer xenograft model [27]. At the mechanistic level, CUL-4A silencing dramatically decreased the level of EGFR transcripts and EGFR phosphorylation, leading to inhibition of AKT signaling, which was causally related to CUL-4A-induced cell proliferation [27]. Similarly, CUL-4A knockdown inhibited lung cancer cell proliferation and sensitized lung cancer cells to chemotherapy drugs, gemcitabine, and paclitaxel, via causing accumulation of tumor suppressors TIEG1, TGFBI, and p33ING1b, respectively [30], [31], [32]. Kabir et al. found that CUL-5 knockdown in lung cancer cells caused the accumulation of pro-apoptotic protein NOXA, consequently rejuvenating the responsiveness of lung cancer cells to CDK9 inhibitors, which indirectly targeted anti-apoptotic protein MCL1 to trigger apoptosis [33]. Moreover, Men et al. have unveiled the impact of CUL-7 knockdown on lung cancer progression. Knockdown of CUL-7 markedly suppressed lung cancer cell proliferation and survival in part by accumulating tumor suppressors p53 and p21/p27 and activating DNA damage response pathway. The p53 knockdown significantly rescued the phenotype triggered by CUL-7 knockdown [25]. In contrast, Zhou et al. found that lentivirus-mediated CUL-3 overexpression in NSCLCs acted as a tumor suppressor in a manner dependent on the CUL3‑NRF2‑RHOA axis [28]. Mechanistically, in H358 and H1299 lung cancer cells, CUL-3 overexpression significantly promoted the degradation of antioxidant transcription factor NRF2, and consequently reduced the levels of RHOA (a RAS homologous protein of GTPases), thus inhibiting cell proliferation and invasion [28]. Therefore, although knockdown of most cullins indeed suppressed the proliferation of lung cancer cells, exception does exist in the case of CUL-3, which can be a topic worthy of further in-depth investigation.

3.2. Pharmacological approaches

MLN4924, a selective inhibitor of UBA3 (neddylation E1 subunit) [11], has been widely used in preclinical studies to demonstrate that neddylation pathway is required for lung cancer cell proliferation, survival, and lung tumorigenesis, thus is a valid lung cancer target. As a neddylation E1 inhibitor, MLN4924 effectively blocks neddylation of all cullin family members, thus inactivating the entire family of CRL E3 ligases, resulting in subsequently accumulating diversity of CRL substrates. Many preclinical models, both in in vitro cell culture and in in vivo xenograft lung tumor models, have shown that MLN4924 inhibited the proliferation and survival of lung cancer cells through multiple mechanisms (Fig. 2; Table 2).

3.2.1. MLN4924 induces growth arrest and apoptosis

MLN4924 was first reported by Soucy et al. in 2009 as a potent anti-cancer agent by suppressing the growth of various human cancer cell lines [11], assessed in both in vitro cell culture and in vivo xenograft models. Specifically, MLN4924 effectively inhibits NEDD8 E1 NAE and inactivates NEDD8 E3 ligases CRL1 and CRL4 in Calu-6 and H460 lung cancer cell lines, causing accumulation of chromatin licensing and DNA replication factor 1 (CDT1), which triggers the S-phase defects and DNA re-replication, and ultimate induction of apoptosis. MLN4924 suppressed growth of lung tumor xenografts was also observed in mice bearing H522 or Calu-6 [11] as well as H358 [18] lung tumor xenografts.

Our group found that MLN4924 induced accumulation of hypoxia-inducible factor (HIF)-1 and Jun proto-oncogene (JUN) to increase UBE2M levels, which couples with CRL3 and Parkin ubiquitin E3 to promote UBE2F ubiquitylation and degradation, resulting in accumulation of NOXA, a CRL5 substrate, to induce apoptosis in lung cancer cells [18,34]. Li et al. found that MLN4924 induced G₂/M arrest and apoptosis in human lung cancer H460 cells and murine Lewis lung carcinoma (LLC) cells, and the apoptosis induction in H460 cells was causally related to NOXA accumulation, since it was blocked by NOXA knockdown [24]. The authors also found that MLN4924 significantly suppressed tumor formation and metastasis using in vivo LLC lung metastasis model [24].

Our group also found that MLN4924 inhibits lung tumorigenesis in vivo and lung cancer cell proliferation in vitro by blocking downstream mTOR and NF-κB signals and causing the accumulation of p21/p27, NOXA, and BIM [20]. Specifically, the LSL-Kras^G12D-driven mouse lung cancer model was used to determine the effect of MLN4924 in lung tumor formation. The LSL-Kras^G12D mice at 6–8 weeks were intra-nasally administrated with Ad-Cre to remove the STOP fragment, thus activating Kras^G12D. After 12 weeks when tumors had already formed, mice were treated with MLN4924 using a nontoxic dose (s.c., 60 mg/kg, once a day, 5 days per week for 4 weeks). Significant inhibition of lung tumor formation was observed. Similarly, in cell culture setting, MLN4924 treatment significantly suppressed growth and survival of lung cancer cells [20].

MLN4924 was also shown to sensitize lung cancer cells to chemotherapeutic drugs. Xu et al. investigated the effects of MLN4924 on paclitaxel (PTX)‑resistant NSCLC cells and found that MLN4924 promoted apoptosis and DNA damage in PTX‑resistant A549 and H460 lung cancer cells [35]. In addition, MLN4924 mediated the accumulation of CDT1, which promotes DNA re-replication and ultimate induction of apoptosis in these cells. Guo et al. showed that MLN4924 also sensitized lung cancer cells to poly-ADP ribose polymerase (PARP) inhibitor Olaparib, by suppressing the breast cancer 1 (BRCA1) complex recruitment to DNA damage sites, thereby impairing the DNA repair process and suppressing the cell growth [36].

3.2.2. MLN4924 induces senescence

In addition to apoptosis induction, Jia et al. have shown that MLN4924 also induced irreversible senescence in various cancer cell lines including lung cancer cells through which MLN4924 treatment stabilized p21, p27, and Wee1 tumor suppressive CRL substrates [37]. Specifically, MLN4924-triggered senescence in lung cancer line H1299 was an irreversible process and dependent on p21, a mediator of senescence, likely associated with cellular response to DNA damage [37]. It appeared that CDT1 and origin recognition complex subunit 1 (ORC1), both of which are critically involved in DNA re-replication, were accumulated after MLN4924 treatment in H1299 to trigger DNA damage response. Notably, p21 accumulation induced by MLN4924 treatment was sustained even after drug removal in H1299 cells, so were the DNA damage response proteins pCHK1 and pH2AX, which reflects the notion that the DNA damage response likely triggers cellular senescence in this experimental setting. The senescence occurred in H1299 cells independently of pRB/p16 and p53 [37], consistent with our earlier findings that senescence induction in H1299 cells by ROC1/RBX1 silencing was likely coupled with DNA damage in p16/pRB- and p53-independent manner [17].

3.2.3. MLN4924 induces autophagy

In response to therapeutic stresses, autophagy either acts as a pro-survival signal against unfavorable conditions or pro-death signal that causes cell death [38]. Many studies showed that besides apoptosis and senescence induction, MLN4924 also effectively induced autophagy in lung cancer cells and other multiple human cancer cell lines [39,40]. Luo et al. reported that MLN4924-induced autophagy exerted a pro-survival role in liver cancer cells as well as H1299, HeLa, and U87 glioblastoma cells, in response to CRLs inactivation [39]. Inhibition of autophagy by siRNA-mediated knockdown of autophagy-related genes enhanced MLN4924-induced apoptosis in vitro and in vivo. They showed that MLN4924-induced autophagy was partially attributed to the inhibition of mTOR kinase activity triggered by accumulation of DEPTOR [41]. In addition to DEPTOR, Zhao et al. found that accumulation of HIF-1α, followed by activation of the HIF1-REDD1-TSC1 signaling pathway, was also involved in mediating mTOR inhibition and autophagy induction by MLN4924 treatment in multiple human cancer cell lines [40]. Similarly, induction of autophagy in this study was a pro-survival signal for cancer cells and combination of blockage of the autophagy pathway with MLN4924 treatment remarkably enhanced MLN4924-induced apoptosis.

Taken together, in these preclinical studies, MLN4924 acts as a potent inhibitor of lung cancer cell proliferation and survival, alone or in combination with other anti-cancer agents via multiple approaches. These studies provide proof-of-concept evidence of targeting neddylation as an effective anti-lung cancer therapy. However, it remains to be determined, mechanistically, as to under what physiological or stressed conditions with accumulation of what type of substrates, along with other possible off-target effects [42], [43], [44] would determine the cell fate after MLN4924 treatment described above, although it appears to be, in general, cell-type dependent.

4. Neddylation regulates tumor microenvironment and inflammatory responses in lung cancer

In addition to regulating cell growth, survival, and tumorigenesis, neddylation pathway was also found to modulate tumor microenvironment (TME) (Fig. 2; Table 2), which consists of a variety of cells, including tumor and immune cells, cancer-associated fibroblasts, and endothelial cells, and soluble factors [45].

4.1. Genetic approaches

The macrophages in tumor tissues are tumor-associated macrophages (TAMs), which are the major type of immune cells to infiltrate and trigger the TME for cancer progression and metastasis [46]. One study showed that neddylation inactivation significantly inhibited TAM infiltration, thereby suppressing lung cancer metastasis [47]. Mechanistically, neddylation inactivation by knockdown of NEDD8 or NAE in H1299 lung cancer cells suppressed transactivation of chemotactic cytokine ligand 2 (CCL2), leading to reduced chemotaxis of monocytes. Furthermore, Nedd8 knockout significantly decreased the population of both M1 and M2 macrophages in lung tumor tissues, suggesting that inhibiting neddylation likely regulates the chemotaxis of TAMs [47]. Xiong et al. generated LysM-Cre/Sag^fl/fl mice in which Sag is selectively deleted in myeloid cell subsets, namely macrophages and neutrophils. Sag deletion impaired lipopolysaccharides (LPS)-induced degradation of IκBα, thereby resulting in suppressing NF-κB activation in macrophages and decreased levels of proinflammatory cytokines IL-6 and TNF-α [48]. Interestingly, LysM-Cre/Sag^fl/fl mice showed increased mortality in response to LPS likely due to the enhanced release of proinflammatory cytokines in neutrophils [48].

In addition to macrophages, Mathewson et al. previously found that knockdown of SAG or β-TrCP (an F-box protein of CRL1) in dendritic cells (DCs) suppressed the release of proinflammatory cytokine TNF-α [49]. Sag genetic knockout in T cells also significantly decreased the activation and proliferation of T cells in response to stimulation [50]. Mechanistic studies showed that, in contrast to macrophages and DCs where the inhibition of NF-κB translocation to the nucleus was responsible for decreased functions [48,49,51], Sag T cell knockout had minimal effect on NF-κB translocation, but caused accumulation of suppressor of cytokine signaling (SOCS) proteins SOCS 1 and 3 to negatively regulate the T cell function [50]. Furthermore, our recent study of conditional knockout of two neddylation E2 (Ube2m and Ube2f) and two E3 (Rbx1 and Sag), individually in regulatory T (Treg) cells, showed that while Treg knockout of Ube2f or Sag has no phenotype, Treg knockout of Rbx1 developed an early-onset fatal inflammatory disorder with disrupted Treg homeostasis and functions [52]. Rbx1 substrate Bim, a pro-apoptotic protein, accumulated in Rbx1-deficient Treg cells, and Bim deletion in Rbx1-deficient Treg cells attenuated the inflammatory phenotype but failed to rescue early lethality, indicating Bim plays a moderate role, if any, in the process [52]. Similar but less severe phenotypes were observed in Treg Ube2m knockout mice. Collectively, the Ube2m-Rbx1, but not the Ube2f-Sag, axis was found to be essential for the maintenance of the Treg cell fitness [52].

While these studies clearly demonstrated that neddylation indeed modulated the functions of these immune cells, how these modulations actually affect the establishment and development of TME in the lung is a subject of future investigation.

4.2. Pharmacological approaches

Few studies have shown that MLN4924 treatment altered inflammatory/immune-related signaling pathways in lung cancer cells [45,47]. To establish the link between neddylation pathway and myeloid-derived suppressor cell (MDSC) activation that contributes to an immunosuppressive microenvironment, a list of 22 MDSC-related genes was curated by literature search. Analysis of these genes with MLN4924 treatment in lung cancer cells demonstrated that most genes were significantly downregulated upon treatment, including CCL2 and chemokine (C-X-C motif) ligand 1 (CXCL1) critical chemokines for MDSCs and TAMs recruitment in tumor sites [45]. Functional study showed that using a lung metastasis mouse model, MLN4924 treatment decreased infiltration of MDSCs in lung tumor sites [45]. Similarly, MLN4924 treatment also significantly inhibited the infiltration of TAMs in lung tumor sites by suppressing the transactivation of Ccl2, leading to suppression of metastasis of murine Lewis lung cancer cells after tail-vein injection [47]. Mechanistically, MLN4924 inactivates CRL1 to cause accumulation of IκBα which blocks p65 nuclear translocation, thus inactivating NF-κB, a key transcription factor responsible for the expression of many inflammatory cytokines, such as CCL2, in innate and adapter immune responses [53]. Similarly, although it was not conducted in a lung cancer model, few studies showed that MLN4924 also repressed LPS-induced up-regulation of proinflammatory cytokines IL-6 and TNF-α in macrophage cells through inactivation of NF-κB [48,51,54]. Furthermore, our previous study also showed that MLN4924 treatment of DCs caused the accumulation of IκBα to inactivate NF-κB, resulting in suppressing the LPS-induced proinflammatory cytokine release and allogeneic T cell stimulating capacity in vitro and in vivo [49].

Cancer-associated fibroblasts (CAFs) and cancer-associated endothelial cells (CAEs) are important components of TME that contribute considerably to tumor angiogenesis and metastasis [55,56]. While there have been rare studies to investigate how neddylation pathway modulates CAFs in lung cancer, Zhou et al. indeed found that MLN4924 treatment or siRNA-based knockdown of NEDD8 or NAE1 reduced secretion of inflammatory chemokines, including CCL2, and suppressed proliferation, activation, and migration of CAFs isolated from hepatocellular carcinoma tissues [45]. Tan et al. found that Sag deletion or MLN4924 treatment of primary endothelial cells (ECs) isolated from lung tissues inhibited proliferation, migration, and tube formation, accompanied by p27 accumulation that is required for this effect [57]. Furthermore, although it was not conducted in a lung cancer model, Sag deletion or MLN4924 treatment significantly inhibited tumor angiogenesis and angiogenesis-associated tumor formation in mouse MS-1 ECs and a B16F10 melanoma model, respectively [57]. Similarly, Yao et al. reported that MLN4924 treatment or ROC1/RBX1 knockdown inhibited angiogenesis in human umbilical vein endothelial cells (HUVECs) and in various in vivo models of pancreatic cancer, leading to the suppression of tumor formation and metastasis in pancreatic cancer [58]. Mechanistically, the effect of MLN4924 against vascular ECs appeared to be stage-dependent. At the early stages of MLN4924 treatment, accumulation of RhoA inhibited cell migration and capillary tube formation. Prolonged MLN4924 exposure accumulated cell cycle-related CRLs substrates such as Wee1, p21, and p27, DNA replication licensing proteins, CDT1 and ORC1, and pro-apoptotic proteins such as NOXA, which in turn triggered cell cycle arrest, DNA damage response and apoptosis of endothelial cells, respectively [58].

Collectively, neddylation indeed modulates the functions of immune cells (e.g., MDSCs, TAMs) or their inflammatory responses, as well as of CAFs or ECs, although less studied in the sites of lung tumor, which would affect establishment and development of lung TME to accelerate the process of lung tumorigenesis. While targeting neddylation appears to be a sound approach to modulate TME, the future investigation in this area of study is needed to fully elucidate underlying mechanisms before it becomes practical.

5. Development of neddylation enzyme inhibitors

The studies discussed above in lung cancer cells as well as many other studies on a variety of human cancer cells have validated that neddylation pathway is an attractive anti-cancer target [2,6]. Currently, several small molecule inhibitors have been discovered to target all three neddylation enzymes, respectively [59], [60], [61], [62] (Fig. 3; Table 3).

Fig. 3.

Neddylation inhibitors for anti-lung cancer therapy[65]. MLN4924, TAS4464 and HA-1141 are NAE inhibitors. DI-591, 404, 1548, 1859, DC-2, DN-2, SK-464, Compound 27 & 40, NAcMs, and WS-383 are DCN1 inhibitors that target the interaction between DCN1 and UBE2M proteins. HA-9104 inhibits the UBE2F-CRL5 signaling axis. Gossypol inhibits Cul-5 and Cul-1 neddylation by directly binding to SAG-CUL5 and RBX1-CUL1 complexes, respectively.

Table 3.

Small molecule inhibitors targeting all three neddylation enzymes in lung cancer.

Inhibitors	Target Enzymes	Molecular Mechanism	Lung Cancer Models	FDA Approval	Clinical Trial	Refs
MLN4924	NAE	Inactivates NAE thus blocking cullin neddylation and inactivating CRLs, leading to accumulation of CRL substrates	Calu-6, H460, xenograft tumor model	Yes1, but for limited indications	Phase I-III2	[11]
TAS4464	NAE	Inhibits cullin neddylation and causes accumulation of CDT1, p27 and IκBα	NCI-H211	No	Phase I3	[59]
HA-1141	NAE	Inhibits neddylation of CULs 1-5 and triggers non-canonical ER stress, eventually leading to autophagy induction	H358, H1299, H2170, xenograft tumor model	No	N/A	[64]
HA-9104	UBE2F	Inhibits CUL-5 neddylation and inactivates CRL5, causing accumulation of NOXA to induce apoptosis, DNA damage and G2/M arrest	H2170, H1650, H358, xenograft tumor model	No	N/A	[62]
Gossypol	RBX1, SAG	Directly binds to RBX1-CUL1 or SAG-CUL5 complex, leading to accumulation of anti-apoptotic MCL1 and pro-apoptotic NOXA, respectively	H1299, H358, H2170	No	Phase I-IV4 (not for E3 inhibitor)	[60]
DCN1 inhibitors	DCN1	Disrupts UBE2M-DCN1 binding to block neddylation of cullins, particularly CUL-3, leading to accumulation of an antioxidant transcription factor NRF2	SK-MES-1, H2170, HC995, PC9, H1795	No	N/A	[61,[70], [71], [72], [73], [74], [75], [76], [77], [78], [79]

¹https://www.takeda.com/newsroom/newsreleases/2020/takeda-announces-u.s.-fda-breakthrough-therapy-designation-granted-for-pevonedistat-for-the-treatment-of-patients-with-higher-risk-myelodysplastic-syndromes-hr-mds/.

²https://classic.clinicaltrials.gov/ct2/results?term=pevonedistat.

³https://classic.clinicaltrials.gov/ct2/show/NCT02978235?term=TAS4464&draw=2&rank=1.

⁴https://classic.clinicaltrials.gov/ct2/results?term=Gossypol.

5.1. Small molecule inhibitors targeting neddylation E1

1. MLN4924 (also known as pevonedistat)

Due to remarkable anti-cancer effects in many preclinical studies, MLN4924, as described for lung cancer cells above, was advanced to a variety of clinical trials. At present, there are 41 Phase I-III clinical trials for MLN4924, most in combination with chemotherapeutic drugs (https://clinicaltrials.gov/ct2/results?cond=&term=MLN4924&cntry=&state=&city=&dist=). On July 2020, the FDA approved its use for the treatment of high-risk myelodysplastic syndrome (HR-MDS) (https://www.takeda.com/newsroom/newsreleases/2020/takeda-announces-u.s.-fda-breakthrough-therapy-designation-granted-for-pevonedistat-for-the-treatment-of-patients-with-higher-risk-myelodysplastic-syndromes-hr-mds/).

2. TAS4464

In addition to MLN4924, TAS4464 was discovered in 2019 as yet another highly potent NAE inhibitor [59]. The compound effectively suppressed cullin neddylation, thereby resulting in accumulated CRL substrates such as p27, CDT1, and phosphorylated form of IκBα, in human cancer cell lines, including small-cell lung cancer (SCLC) NCI-H211. TAS4464 showed greater efficacy than MLN4924 with prolonged inhibitory effects in xenograft mouse models [59]. However, in Phase I clinical trial, TAS4464 showed liver toxicity and was terminated [63].

3. HA-1141

Through virtual screening and structural modification, our group recently reported the discovery of HA-1141. The compound was shown to directly bind to NAE in in vitro and in vivo assays and effectively inhibit neddylation of cullins 1–5 [64]. HA-1141 efficiently inhibited lung cancer cell proliferation and survival in culture cells and in vivo xenograft models. However, HA-1141 displays some off-target effect by inducing non-canonical endoplasmic reticulum (ER) stress, eliciting double‐stranded RNA‐dependent protein kinase (PKR)-mediated integrated stress response (ISR), and inactivating mTORC1 activity, eventually leading to autophagy induction [64].

4. Other neddylation E1 inhibitors

There is more than a dozen of neddylation E1 inhibitors reported so far, and some of them showed growth inhibitory activity against lung cancer cells, which have been summarized in a recently review [65], and will, therefore, not be further discussed here.

5.2. Small molecule inhibitors targeting neddylation E2s and E3s

1. HA-9104

Again, through virtual screening and structural modification, our group recently discovered a novel inhibitor, HA-9104, for the UBE2F-CRL5 axis. HA-9104 binds to UBE2F and reduces its protein levels, thereby inhibiting CUL-5 neddylation. HA-9104 treatment inactivated CRL5 E3 ligase activity thereby accumulating the CRL5 substrate NOXA to induce apoptosis. HA-9104 effectively suppressed lung cancer cell proliferation and survival as well as conferred radio-sensitization in culture cells and mouse xenograft assays [62].

2. Gossypol

Using an AlphaScreen based assay for SAG-CUL5 neddylation inhibitor screening, our group identified Gossypol, a cotton seed-derived natural compound, from a library consisting of 17,000 compounds [60]. Gossypol inhibited neddylation of cullin-5 and cullin-1 in lung and other cancer cell lines through direct binding to SAG-CUL5 or RBX1-CUL1 complex, and induced selective accumulation of pro-apoptotic protein NOXA and anti-apoptotic protein MCL1, the substrates of CUL-5 and CUL-1, respectively. Combination treatment with gossypol and MCL-1 inhibitor exhibited a synergistic effect in inhibiting proliferation of multiple cancer cell lines including H1299 lung cancer [60].

3. DCN1 inhibitors

DCN1 (defective in cullin neddylation 1), also referred to as DCNL1, DCUN1D1, or SCCRO (squamous cell carcinoma-related oncogene), is an evolutionarily conserved and frequently amplified gene in squamous cell carcinoma (SCC) with oncogenic potential. DCN1 amplification and overexpression in SCC correlated with poor clinical outcome [66]. The early studies showed that DCN1 acted as a scaffold-type E3 ligase and required for cullin neddylation [67,68]. In fact, DCN1 bound to UBE2M and collaborated with RBX1 to make efficient transfers of NEDD8 from UBE2M to CUL-1 [69]. Crystal structure analysis revealed that the N-terminus fragment of UBE2M inserts into a hydrophobic pocket of DCN1, which appeared to be amenable to the design of inhibitors [9,69]. Based upon this defined structure, several laboratories have moved on and discovered a battery of small molecules that disrupted UBE2M-DCN1 binding with potent activity to block neddylation of cullins, particularly CUL-3 [61,[70], [71], [72], [73], [74], [75], [76], [77], [78], [79]. Our study showed that DCN1 inhibitor DI-404 selectively inhibited CUL-3 neddylation in SCC lung cancer cell lines, including SK-MES-1 and H2170. However, the growth inhibitory effect was limited [61]. Likewise, two potent DCN1 inhibitors DI-1548 and DI-1859 effectively inhibited Cul-3 neddylation to accumulate an antioxidant transcription factor Nrf2 that acts as a ROS scavenger to protect acetaminophen-induced liver toxicity in mice [73]. However, it appears that DCN1 inhibitors may not be potent anti-cancer agents.

6. Conclusion and future perspectives

Taken together, the findings presented here highlight the fact that protein neddylation modulates many important biological processes. Specifically, inhibition of neddylation pathway by small molecule inhibitors or siRNA-mediated knockdown inactivates the CRL E3 ligases to stabilize multiple tumor suppressor substrates, thereby suppressing lung cancer cell proliferation, cell survival, and lung tumorigenesis by inducing cell cycle arrest, cellular apoptosis and senescence, and autophagic response, along with modulation of TME and inflammatory/immune responses. These studies validated that neddylation is a promising oncogenic target for lung cancer. However, there are still quite few unsolved puzzles for future investigations (Fig. 4).

Fig. 4.

Outline of future perspectives. Four proposed future directions to advance the neddylation–lung cancer field: (1) the mechanisms of neddylation activation in lung cancer; (2) the mechanisms of cell fate determinants upon neddylation inactivation; (3) the mechanisms by which neddylation regulates lung tumor microenvironment; and (4) discovery of selective inhibitors for neddylation E2s or E3s.

1. What is the underlying mechanism by which NEDD8 and neddylation enzymes are overexpressed in lung cancer tissues? Is it derived from enhanced transcription or stabilization and/or reduced degradation in response to stresses triggered during lung tumorigenesis? Is the overactivation of neddylation pathway the consequence or the cause of lung tumorigenesis? Although our previous studies reported that SAG is transactivated by activator protein 1 (AP-1) [80] and HIF-1 [81], and plays a cooperative role in lung tumorigenesis induced by Kras^G12D [20], a systematic study on this aspect is currently lacking.

2. Inactivation of neddylation causes growth suppression via multiple mechanisms in a manner dependent of lung cancer cell lines. It is unclear what are the cell fate determinants, particularly under the in vivo setting.

3. How does neddylation coordinately regulate the function of a variety of cells in TME including lung cancer cells, endothelial cells and inflammatory cells? What is the exact role that neddylation played in the establishment and development of lung TME? Current studies, reporting that neddylation inhibition caused altered immune cell infiltration and altered secretion of few inflammatory cytokines, are rather descriptive in nature. The in-depth investigation in this area required well-defined models of TME in the lung with selectively targeting neddylation at the different types of TME cells.

4. Given associated cytotoxicity of normal cells by neddylation E1 inhibitors, targeted therapy efforts should be put more weight on the discovery of small molecule inhibitors specific to neddylation E2s or E3s. While these efforts are currently undergoing as described above, all studies are at the preclinical stage, and none of them have been advanced to clinical trials, which will be a future direction for drug discovery on targeting neddylation-CRLs for anticancer therapy.

CRediT authorship contribution statement

Yawen Zheng: Resources, Writing – original draft, Writing – review & editing, Visualization. Hiroyuki Inuzuka: Writing – review & editing. Wenyi Wei: Writing – review & editing. Yi Sun: Conceptualization, Writing – review & editing, Supervision, Funding acquisition.

Biographies

Yawen Zheng(BRID: 02582.00.53365) received her Ph.D. degree in biochemistry and molecular biology in June 2023 from the Institute of Translational Medicine, Zhejiang University, China. She was awarded a scholarship from China Scholarship Council and spent two years (2020–2022) as a visiting student in the Department of Radiation oncology, University of Michigan, USA. She currently works as a research associate in Dr. Yi Sun's laboratory. Her research interest is to elucidate the mechanistic roles of protein neddylation in regulation of lung inflammation, lung tumorigenesis, tumor microenvironment, and mitochondrial functions.

Yi Sun(BRID: 03506.00.23151) is a Qiushi Chair professor at Zhejiang University, China. He was endowed as Lawrence-Krause Research Professor in Radiation Oncology at the University of Michigan, USA. He served as a founding Dean at the Institute of Translational Medicine, Zhejiang University in 2014–2018, and has been a Fellow of the American Association for the Advancement of Science (AAAS) since 2012. The main research focus of Sun's laboratory is to define the role of Cullin-RING ligases (CRLs) and protein ubiquitylation/neddylation in regulation of tumorigenesis. His laboratory has validated CRLs and protein neddylation, upon abnormal activation in cancer, are promising cancer targets, and is actively engaging in the discovery of small molecule inhibitors of CRLs-neddylation for cancer therapy.

Appendix

Abbreviation list

CAEs	Cancer-associated endothelial cells
CAFs	Cancer-associated fibroblasts
CCL2	Chemotactic cytokine ligand 2
CRLs	Cullin-RING ligases
DCN1	Defective in cullin neddylation 1
ECs	Endothelial cells
HIF-1	Hypoxia-inducible factor 1
HUVECs	Human umbilical vein endothelial cells
LLC	Lewis lung carcinoma
MDSC	Myeloid-derived suppressor cell
mTOR	Mammalian target of rapamycin
NAE	NEDD8-activating enzyme
NEDD8	Neural precursor cell expressed, developmentally downregulated protein 8
NEDP1	NEDD8 protease 1
NF-κB	Nuclear factor kappa B
NSCLC	Non-small cell lung cancer
PARP	Poly-ADP ribose polymerase
PTM	Post-translational modification
RBX1/2	RING-box protein 1/2
RING	Really interesting new gene
SCC	Squamous cell carcinoma
SCLC	Small-cell lung cancer
SOCS	Suppressor of cytokine signaling
TAMs	Tumor-associated macrophages
TME	Tumor microenvironment
UBA3	Ubiquitin like modifier activating enzyme 3
UBE2M	Ubiquitin conjugating enzyme E2 M
UBE2F	Ubiquitin conjugating enzyme E2 F