FBXO45 is a potential therapeutic target for cancer therapy.

What are the physiological functions of FBXO45 in a variety of human malignancies? What are unknown targets of FBXO45 that are critically involved in tumorigenesis? How can a novel approach to identify new substrates of FBXO45 be established? What are the physiological functions of FBXO45 in a variety of human malignancies? What are unknown targets of FBXO45 that are critically involved in tumorigenesis? How can a novel approach to identify new substrates of FBXO45 be established?

Recently, FBXO45 has also been identified to have a pivotal role in tumorigenesis and progression. In the following paragraphs, we will describe the expression of FBXO45 in human tumour specimens and identify substrates of FBXO45 in cancer and its biological functions in the regulation of proliferation, apoptosis, cell cycle, motility and metastasis.

FBXO45 exerts its functions by targeting substrates for ubiquitination and degradation. FBXO45 has a pivotal role in carcinogenesis and progression. Targeting FBXO45 might be a novel strategy for cancer therapy. FBXO45 exerts its functions by targeting substrates for ubiquitination and degradation. FBXO45 has a pivotal role in carcinogenesis and progression. Targeting FBXO45 might be a novel strategy for cancer therapy.

A wealth of studies have uncovered that FBXO45 could have important roles in tumorigenesis and progression. Data from The Cancer Genome Atlas (TCGA) and GTEx, termed GEPIA, show that FBXO45 is highly expressed in a majority of human cancers (Supplementary Fig. 1 ). Compared with that in normal lung tissue, FBXO45 is highly expressed in squamous-cell lung carcinoma (SCLC) tissues according to TCGA and Gene Expression Omnibus data 21 . Compared with that in adjacent tissues, an increased level of FBXO45 was confirmed in SCLC tissues by reverse transcription polymerase chain reaction analysis. Furthermore, it was also illustrated that high expression of FBXO45 was correlated with poor survival in patients with SCLC by obtaining data from the Kaplan–Meier plotter website and the TCGA database 21 . Consistent with the results in SCLC, FBXO45 expression was higher in gastric cancer tissues than in normal gastric tissues 22 . However, gastric cancer patients with low FBXO45 expression exhibited poorer outcomes, such as worse survival, than those with high FBXO45 expression 22 . Consistently, data from TCGA show that high expression of FBXO45 is correlated with shortened overall survival in multiple types of human cancers (Supplementary Fig. 2 ). Moreover, Dahlem et al. 23 proved that overexpression of insulin-like growth factor 2 (IGF2) mRNA-binding protein IMP2 (IGF2BP2) existed and was associated with poor outcomes in pancreatic cancer patients by investigating publicly available datasets FBXO45 is positively associated with IMP2 expression, indicating that FBXO45 might play a potential oncogenic role in pancreatic cancer progression 23 .

A study showed that miR-27a could inhibit the expression of FBXO45, leading to accumulation of the downstream substrates of FBXO45 and modulation of cancer initiation and progression, indicating that FBXO45 is a direct target of miR-27a 30 . One group revealed that Hey1 could promote FBXO45 translocation from the cytoplasm into the nucleus, indicating that Hey1-mediated translocation of FBXO45 could be a useful way to degrade the nuclear substrates of FBXO45 31 . One elegant study revealed that lncRNA RP11-138 J23.1 (RP11) was overexpressed in colorectal cancer compared with matched normal colorectal tissues by microarray analysis 32 . Furthermore, they found that the expression of RP11 was associated with the progression of colorectal cancer stage. Further investigations showed that m 6 A upregulated the lncRNA RP11 via promotion of its nuclear accumulation and subsequently enhanced the mRNA degradation of FBXO45, eventually preventing the ZEB1 degradation, inducing EMT and increasing migration and invasion in colorectal cancer 32 . An amino-terminal of PAF could competitively bind to FBXO45, resulting in an increase in cell apoptosis owing to Par-4 accumulation. This PAF induced apoptosis and suppressed tumour growth in drug-resistant cells, suggesting that it could overcome drug resistance in cancer therapy 26 . Therefore, modulation of miR-27a, Hey1, m 6 A and RP11 could affect the FBXO45 expression level as a potential approach to treat cancer patients with high FBXO45 expression (Fig. 1 ).

In summary, FBXO45 is critically involved in carcinogenesis and cancer progression by targeting its downstream substrates for ubiquitination and degradation (Table 1 ). Targeting FBXO45 with inhibitors might be a novel strategy for achieving a therapy benefit in cancer patients. One alternative approach is to target the upstream regulators of FBXO45, leading to regulation of the FBXO45 expression level. It is worth noting that several critical questions need to be addressed to fully understand the role of FBXO45 in carcinogenesis and cancer progression. For instance, what are the biological functions of FBXO45 in other types of human malignancies that have not been reported? To answer this, it is better to use conditional transgenic knock-in or knockout mouse models to determine the role of FBXO45 in specific tissues. In addition to the several substrates of FBXO45 that are mentioned above, what are other targets of FBXO45 in human cancer cells? How can a novel approach for identifying new substrates of FBXO45 be established? It is also necessary to explore the upstream factors controlling FBXO45 expression, which will help us to fully dissect the crosstalk between FBXO45 and other signalling pathways. Because the implications for the therapeutic targeting of FBXO45 are not obvious, in-depth exploration is required to uncover the functions of FBXO45 and the underlying molecular mechanisms to develop effective inhibitors of FBXO45 for the treatment of human diseases. Table 1 FBXO45 targets substrates for degradation in human diseases. Substrates Cell lines Functions Reference ERα HeLa, MCF-7 Involves in tissue-selective oestrogen complex-mediated endometriosis therapy 10 Munc13-1 293T, hippocampal neuron cells, COS. Controls synaptic activity, neuropathic allodynia 12 , 13 NMNAT2 293T Regulates axon degeneration 17 , 18 p73 BT-20, 293T, HeLa Reduction of cell death 24 Par-4 HeLa, 293T Reduction of cell apoptosis, promotes cell survival 25 FBXW7 U2OS, HeLa Governs cell fate decision, chemotherapy resistance 27 ZEB1/2, Snail1/2, Twist1 293T, HeLa, U2OS, MCF-7, MDA-MB-231, PANC-1 Regulates EMT 29 , 30 FBXO45 targets substrates for degradation in human diseases.

Several substrates of FBXO45 have been identified in recent years. For example, FBXO45 targets p73, which belongs to the p53 family, for ubiquitylation and degradation, leading to a reduction in cell death 24 . Consistently, deletion of FBXO45 led to an accumulation of p73 and subsequently triggered cell death in cells 24 . Another study validated that prostate apoptosis response protein 4 (Par-4), an anticancer protein that induces apoptosis, was a downstream substrate of FBXO45 in cancer cells 25 . Specifically, FBXO45 mediated the ubiquitylation and proteolysis of Par-4, leading to a reduction in cell apoptosis. In line with this finding, depletion of FBXO45 led to elevated apoptosis due to upregulation of Par-4. This study indicated that FBXO45 could promote cell survival in human cancer cells 25 . Keeping abreast with this, other studies further demonstrated that the VASA segment is a crucial portion of Par-4 that can bind with FBXO45 and subsequently lead to Par-4 degradation. In contrast, a Par-4 amino-terminal fragment (PAF), generated by therapy-sensitive cancer cells and containing this VASA segment, recovered Par-4-mediated apoptosis by competitively binding FBXO45 26 . Richter et al. 27 identified FBXW7 as a new substrate of FBXO45 and dissected the mechanism of cell fate decisions in cancer cells. FBXW7 has been well characterised as a tumour suppressor in carcinogenesis and progression 28 . It has been accepted that FBXW7 exerts its tumour-suppressive function by targeting its substrates for ubiquitination and degradation, including Notch, c-Jun, cyclin E, c-Myc and Mcl-1. FBXW7 is potentially involved in the regulation of multiple cellular processes including cell proliferation, apoptosis, migration, invasion and metastasis. Accumulated evidence has also revealed that FBXW7 governs the epithelial-to-mesenchymal transition (EMT), stem cell differentiation and drug resistance in tumour cells 28 . Numerous upstream regulators of FBXW7 have also been discovered such as p53, Numb, microRNAs and Pin-1 28 . In one study, FBXO45 was validated as an F-box protein targeting another F-box protein, FBXW7, for proteolysis 27 . First, the authors measured the protein levels of FBXW7 in cells during prolonged mitotic arrest and found that FBXW7 expression was reduced in this process. Second, coimmunoprecipitation analysis showed that the N-terminal domain of FBXW7 interacted with FBXO45 and the MYCBP2 complex. Third, the results from immunoblotting and in vivo ubiquitylation demonstrated that FBXO45-MYCBP2 triggered FBXW7 ubiquitination and proteolysis during mitotic arrest. Fourth, using live-cell imaging analysis, the FBXO45-MYCBP2 complex was shown to decrease the cellular sensitivity to spindle poisons, including nocodazole, Taxol and vincristine 27 . Moreover, this complex inhibited mitotic cell deaths and promoted mitotic slippage mainly by targeting FBXW7 27 . Therefore, this study revealed that blockade of FBXO45-mediated FBXW7 degradation might be useful for enhancing mitotic cell death to overcome drug resistance during chemotherapeutic treatment 27 . However, other reports indicate that FBXO45 might have an inverse role in specific types of human malignancies. For example, ZEB1, a well-known marker related to EMT, was degraded via the caspase-8-associated protein 2 (CASP8AP2 or FLASH)-dependent SIAH1 E3 ubiquitin ligase and the FBXO45 atypical E3 ligase 29 . Silencing SIAH1 or FBXO45 restored the expression of ZEB1 protein 29 . Likewise, FBXO45 inhibited EMT by targeting EMT-inducing transcription factors, including Zeb1/2, Snai1/2 and Twist1, for ubiquitination and degradation in cancer cells 30 . FBXO45 was related to several biological processes, including cell adhesion, immune response, cell signal transduction and angiogenesis, in GO and KEGG pathways analyses 21 . Interestingly, a study in SCLC further revealed that diminished expression of FBXO45 blocked the colony forming ability of H520 cells, but markedly augmented migration and prompted EMT 21 . Although these studies explored the molecular mechanisms of FBXO45-related tumorigenesis and progression (Fig. 1 ), the underlying and comprehensive mechanisms have not been fully elucidated, which is required for in-depth exploration. Fig. 1 Molecular mechanism of FBXO45 in controlling cellular functions via targeting multiple substrates. FBXO45 targets numerous substrates for ubiquitination and degradation, including FBXW7, p73, Par-4 and ZEB1/2. Hey1, miR-27a and m 6 A regulate the expression of FBXO45. FBXO45 has a critical role in cell death, apoptosis, EMT and drug resistance. FBXO45 targets numerous substrates for ubiquitination and degradation, including FBXW7, p73, Par-4 and ZEB1/2. Hey1, miR-27a and m 6 A regulate the expression of FBXO45. FBXO45 has a critical role in cell death, apoptosis, EMT and drug resistance.

The ubiquitin proteasome system (UPS), which applies posttranslational modifications (PTMs) to proteins, is a vital pathway that drives protein degradation in cells 1 . It is responsible for ~80% of intracellular protein degradation and subsequently modulates a series of biological procedures, such as transcription, mitosis, cell cycle, proliferation, apoptosis, genomic stability and signalling pathways 2 , 3 . Two well-defined steps are implicated in UPS-mediated protein degradation 4 , 5 . Primarily, the substrate protein is labelled by ubiquitination (monoubiquitination or polyubiquitination) by three-step enzymatic reactions involving an E1 ubiquitin activating enzyme, an E2 ubiquitin conjugating enzyme, and an E3 ubiquitin ligase. Subsequently, the ubiquitinated substrate is degraded by the 26S ribosomal proteasome complex. Mechanistically, the E3 ubiquitin ligase specifically recognises numerous substrates for ubiquitination and degradation 6 . The Cullin-RING ligase complex family, which contains Skp1-Cullin1-F-box protein (SCF)-type ligases composed of Skp1, Cullin1 (Cul1), Rbx1 and an F-box protein, is one of the large E3 enzyme families 4 . Known to be subunits of the SCF E3 ligase complex, F-box proteins are generally categorised into three subfamilies, including FBXW (F-box with WD 40 amino-acid repeats), FBXL (F-box with leucine-rich amino-acid repeats) and FBXO (F-box only with uncharacterised domains) 7 . F-box proteins have been reported to participate in the development of many diseases, including cancer.

Supplementary figure 1 Supplementary figure 2 Supplementary Figure legend