S-phase kinase-associated protein 2 (Skp2), a member of the F-box family of proteins, was discovered by Zhang et al. (1995). Skp2 functions as a substrate-specific adaptor in Skp1-CUL1-ROC1-F-box (Lyapina et al., 1998) E3 ubiquitin ligases, and many proteins playing antioncogenic roles are recognized by Skp2 and tagged with ubiquitin by SCF^Skp2(Lyapina et al., 1998; Tsvetkov et al., 1999). For example, Skp2 mediates the K48-linked ubiquitination of p27 and p21, both of which are tumor suppressors, resulting in the proteasomal degradation of these proteins and cell-cycle promotion (Yu et al., 1998; Tsvetkov et al., 1999). In addition, overexpression of Skp2 is observed in numerous human cancers (Gstaiger et al., 2001; Shigemasa et al., 2003). Accordingly, Skp2 is regarded as an oncogene and has remained a highly active topic in cancer-therapy research since its discovery.

Protein p27, cyclin dependent kinase inhibitor 1B, was previously found to bind and inhibit cyclin-CDK to arrest the cell cycle, and recently p27 was also reported to function as an oncogene to involve in tumor migration, invasion and regulation of gene expression in tumor (Razavipour et al., 2020). Skp2 was a rate-limiting factor of the degradation of p27 protein (Carrano et al., 1999; Tsvetkov et al., 1999), and the Skp2-p27 axis controlled S phase entry, tumor stemness as well (Wang J. et al., 2021). The upstream regulation factors of Skp2-p27 axis have also been reported, such as nuclear casein kinase and cyclin dependent kinase substrate 1(NUCKS1) (Hume et al., 2021), focal adhesion kinase 1 (Jeong et al., 2022), DnaJ heat shock protein family member C5(Li et al., 2021; Wang H. et al., 2021). Various Skp2 inhibiting compounds have been developed to inhibit the degradation of p27 mediated by Skp2 for cancer treatment, such as Skp2E3LIs (Wu L. et al., 2012), and the p27 protein level was taken as a point for selecting Skp2 inhibiting compound, such as when screening SMIP004 (Rico-Bautista et al., 2010). We referred to these compounds in this manuscript.

Since been reported to regulate cell cycle, promoting cell proliferation via p27 (Carrano et al., 1999), Skp2 was discovered to have various other functions in tumor pathological processes, including apoptosis (Nakayama et al., 2000), DNA repair (Wu J. et al., 2012), tumor metastasis (Li et al., 2004), and senescence (Lin et al., 2010; Wang et al., 2012).

Regarding the apoptosis regulation, Skp2 regulates various signaling pathways via binding and ubiquitination of its substrates to affect cellular apoptosis. For example, programmed cell death protein 4 is ubiquitinated and degraded by SCF^Skp2, and its degradation inhibits translation of p53 directly, leading to cell apoptosis (Li et al., 2019). In another case, the activation of TNF-related apoptosis-inducing ligand receptor 1/2(TRAIL-R1/R2 (DR4/DR5) death receptors induce forming of the death-inducing signaling complex (DISC) and lead to the cell apoptosis. Overexpression of Cullin-1/Skp2 enhances the ubiquitination of CASP8 and FADD like apoptosis regulator (FLIP), a DISC member, and this ubiquitination promotes turnover of FLIP between long form and short form, resulting modulation of TRAIL-R2-mediated apoptosis (Roberts et al., 2020). In Rb1-deficient tumors, Skp2 regulates apoptosis by limiting E2F transcription factor 1(E2F1) activity via a pRb-Skp2-p27-cyclin A-E2F1 pathway(Lu et al., 2014).

Skp2 affects cellular DNA repair in both homologous recombination (HR) and Non-homologous end joining (NHEJ) manners. In response to DNA double-strand breaks, Skp2 promotes K63-linked ubiquitination of Nijmegen breakage syndrome protein 1(NBS1), a vital event for facilitating ataxia telangiectasia mutated (ATM) protein recruitment to the DNA foci and activation (Wu J. et al., 2012). The knocked down of Skp2 also decreases the protein level of Ku70, which contributes to the NHEJ pathway (Jia et al., 2020).

As far as tumor metastasis and senescence is concerned, dysregulation of Skp2 level reduces osteosarcoma cell invasion and lung metastasis both in vitro and vivo (Ding et al., 2017; Zhang et al., 2018). Knockdown of Skp2 also attenuates the migration of colon carcinoma and gastric cancer cells (Wei et al., 2013; Chen et al., 2014). Skp2 orchestrates with Myc to recruit p300 to the promoter of zinc finger E-box binding homeobox 1 (Zeb1), which leads to induction of Zeb1 transcription, finally promoting tumor migration and invasion(Wang et al., 2022). Skp2 also mediates the ubiquitination and degradation of Amino-terminal enhancer of split (AES), which is a tumor and metastasis suppressor, and enhances tumor metastasis (Wang Z. et al., 2021). Downregulation of Skp2 also induces senescence in glioma (Wu et al., 2020). The ERK/SKP2/p27 pathway played important roles in cellular senescence induced by 14-3-3β depletion in glioblastoma cells (Seo et al., 2018). In addition to degradation of p21 and p27, Skp2 also regulates cell senescence by cascade. Skp2 decreased the K63-linked ubiquitination of histone modification enzyme Jumonji/ARID Domain-Containing Protein 1B (JARID1B) by E3 ubiquitin ligase TRAF6, and inactivation of Skp2 induces senescence through JARID1B accumulation in cells (Lu et al., 2015).

The AMPK-SKP2-CARM1 pathway is involved in the autophagy regulation after nutrient starvation, and targeting Skp2 leads to activation of autophagy (Shin et al., 2016). Skp2 mediated the ubiquitination and degradation of autophagy regulator Beclin1 (BECN1) to decrease autophagy, and this inhibitory effect of Skp2 on autophagy was regulated by a phosphorylation cascade involving HSP90 cochaperone FK506 binding protein (FKBP)51, PH domain leucine-rich repeat protein phosphatase (PHLPP) and AKT1 (Gassen et al., 2019).

Emerging evidence showed that Skp2 highly correlated with drug resistance and poor prognosis, rendering Skp2 as a therapeutic target (Wu et al., 2021). Skp2 contributes to drug resistance by disturbing various cellular procedures, such as cell proliferation and apoptosis, cell cycle, transcription, et al. Skp2 has been reported to involve in the resistance of various drugs, such as paclitaxel, cisplatin, tamoxifen, and mTOR inhibitor. Family with sequence similarity 60A (FAM60A) mediates the cisplatin-resistant in human lung adenocarcinoma A549 cells, and it exerts its function via upregulating expression of Skp2 and the following inhibition of cell death as well (Hou et al., 2020). POU Domain Transcription Factor OCT4 mediates the tamoxifen resistance in breast cancer cells, and its expression is repressed by NK3 Homeobox 1(Nkx3-1), which is a substrate protein of Skp2 (Bhatt et al., 2016).

In addition, Skp2 is also involves in metabolism regulation (Chan et al., 2012). Activation of Epidermal Growth Factor (EGF) signaling pathway induces AKT localize to mitochondrion to interact with Hexokinases (HK)2, and promote HK2 mitochondrial localization consequently. As a substrate protein of Skp2, the K63-linked ubiquitination of AKT mediated by SCF^Skp2 is required in this signaling pathway (Yu et al., 2019). The decreased Skp2 protein level reprogrammes cellular aerobic glycolysis. Protein Isocitrate Dehydrogenase (NADP(+)) (IDH) 1/2, a substrate protein of Skp2, promotes the cell metabolic fluctuation between glycolysis and tricarboxylic acid (TCA) cycle, by its Skp2-dependent protein fluctuation in cell cycle. Skp2 inhibition leads to IDH1/2 accumulation and promotes cell metabolism shift from glycolysis to TCA cycle. This Skp2–IDH1 signaling axis is also involved in the cancer “Warburg” metabolic phenotype (Liu J. et al., 2021).

The serine/threonine kinase AKT, one upstream regulator of Skp2, increases the expression of Skp2 mRNA, and it also modified by Skp2 post-translationally. The phosphoinositide-3-kinase (PI3K)/AKT signaling pathway promotes E2F1 bind to the promoter of SKP2 gene (Reichert et al., 2007), relying on maintaining the c-myc expression in a glycogen synthase kinase-3 (GSK3)-dependent fashion (Schild et al., 2009), thus increases the expression levels both of Skp2 mRNA and protein. AKT also binds and phosphorylates Skp2, triggering SCF^Skp2 E3 ligase formation. This phosphorylation of Skp2 also promotes Skp2 localize to the cytosol (Lin et al., 2009), preventing ubiquitination and degradation of Skp2 (Gao et al., 2009). Meanwhile, the Skp2 mediates the K63-linked ubiquitination of AKT (Gao et al.), which is required for epidermal growth factor receptor (ErbB receptor)-mediated AKT activation (Chan et al., 2012). This ubiquitination of AKT associates closely with functions of AKT protein in tumor development, such as cell membrane recruitment (Wang G. et al., 2019), mitochondrial localization (Yu et al., 2019), especially increasing the Skp2 level by this AKT-Skp2 positive feedback loop.

In addition to AKT-Skp2, there are other feedback loops reported involve Skp2. The Skp2 autoinduction loop, i.e., a self-amplifying feedback loop, increases Skp2 mRNA and protein level in G1 phase, which contains Skp2, p27, retinoblastoma 1 (RB) and E2F (Yung et al., 2007). During the process of human papillomavirus (HPV) infection, Skp2 was involved in a negative feedback loop of E2-Skp2. The HPV-18 E2 protein inhibits function of anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase, leading to Skp2 accumulation. E2 itself is a substrate of Skp2 and ubiquitinated by the increased Skp2 protein finally, resulting in the expression of E6 and E7(Bellanger et al., 2010).

Skp2 itself is also degraded by ubiquitin-proteasome system. In cell cycle, ubiquitin ligase anaphase-promoting complex/cyclosome and its activator Cdh1(Cdh1/APC) has been reported to ubiquitinate Skp2 and promote its degradation in G1 phase (Bashir et al., 2004). This degradation is regulated by the phosphorylation of Skp2 via cyclin dependent kinase 2 (CDK2) and cell division cycle 14B (Cdc14B) (Rodier et al., 2008). Upon TGFβ stimulation, Skp2 translocates from cellular cytosol to nucleus, and then is ubiquitinated by Cdh1/APC, which is critical for TGFβ cytostatic effect (Hu et al., 2011). The tumor suppressor F-Box and WD-40 domain-containing protein 2 (FBXW2) was also proved to ubiquitinate Skp2 and promote its degradation. The axis β-TrCP-FBXW2-SKP2 are related to cancer cell growth regulation (Xu et al., 2017).

The stability of Skp2 protein is also regulated by deubiquitylases (DUBs). USP10(Liao et al., 2019), USP13(Chen et al., 2011), USP2(Zhang et al., 2021), and USP14(Wu et al., 2022) was reported to deubiquitylate Skp2. The binding between USP2 and Skp2 disrupts the Skp2-substrate binding, and stabilizes both Skp2 and substrates (Zhang et al., 2021). Various transcription factors have been reported to regulate Skp2 expression transcriptionally, such as selenocysteine tRNA gene transcription-activating factor, Forkhead box protein O3 (FOXO3a) (Wu et al., 2013; Shin et al., 2016), NUCKS1(Hume et al., 2021).

Skp2 relates to several important cellular signaling pathways, such as Hippo, WEE1(Pan et al., 2021), autophagy and so on. The Hippo pathway effector yes-associated protein (YAP) induces the acetylation of Skp2 to suppress cell ploidy and tumorigenesis (Zhang et al., 2017), while YAP is also ubiquitinated by Skp2 to induce its nuclear localization and transcriptional activity (Yao et al., 2018). Skp2 also promotes the ubiquitination and mitochondrial localization of AKT, to regulate cell glycolysis and proliferation (Yu et al., 2019). In autophagy pathway, Skp2 regulates the level of co-activator-associated arginine methyltransferase 1 in the AMPK-SKP2-CARM1 signaling cascade to regulate cell autophagy (Chen et al., 2008; Shin et al., 2016).

The structure of Skp2 has been described. It has an F-box domain, a typical structural feature of F-Box family proteins. This 40-amino-acid-residue F-box motif is located next to the N-terminal region, which comprises ∼100 amino acids. Three non-canonical leucine-rich repeats (LRRs) act as “linkers” connecting the F-box to seven other canonical LRRs, which act as protein-protein interaction modules, directly binding to substrates. Finally, the C-terminal tail is located next to the LRR structure (Schulman et al., 2000; Zheng et al., 2002).

The SCF^Skp2 complex is a RING E3 ubiquitin ligase characterized by the existence of a zinc-binding RING domain. In this complex, a cullin protein acts as a scaffold, binding the RING-box protein Rbx at its N terminus and the adaptor Skp1 and substrate receptor Skp2 at its C terminus (Zheng et al., 2002; Chan et al., 2013).

Before a protein is ubiquitinated, the ubiquitin molecule is activated upon forming a highly reactive thioester bond between its C terminus and the active site Cys of the ubiquitin-activating enzyme (E1). This process, which forms an E1–Ub complex, is catalyzed by E1 and is ATP-dependent. Then, the activated ubiquitin is transferred onto the conserved catalytic Cys residue of ubiquitin-conjugating enzyme (E2) by a transthiolation reaction, generating an E2-Ub complex. The E2 interacts with SCF^Skp2 E3 ligase, in which Skp2 recognizes and binds with the substrate protein. Finally, the substrate protein is positioned into the gap between Skp2 and E2, indicating that the SCF^Skp2 E3 ligase acts as scaffold to orient the E2–ubiquitin complex close to the substrate protein, facilitating the transfer of the ubiquitin from the E2 to the substrate directly and efficiently. The Skp2 protein determines the substrate protein and the specificity of this ubiquitination (Scheffner et al., 1995; Morreale and Walden, 2016; Asmamaw et al., 2020).

Until recently, Skp2 has shown the ability to conjugate both K48-linked and K63-linked ubiquitin chains on its substrates, such as C/EBPα (Thacker et al., 2020) and NBS1 (Wu J. et al., 2012). The K48-linked ubiquitination promotes the degradation of substrates, while the K63-linked ubiquitination enhances the stability and modulates the function of the substrate (Deng et al., 2000; Cai et al., 2020).

Skp2 was reported to be overexpressed in various aggressive cancers both at mRNA level and protein level since its discovery, and the protein level of Skp2 was reported to associate with the prognosis in various cancer patients. Due to its close relation with cancers, Skp2 was regarded as an attractive target for cancer therapy. However, besides cancers, Skp2 was also reported to associate with other non-cancer diseases, such as chronic kidney disease (Suzuki et al., 2011), pulmonary Fibrosis(Li et al., 2016; Mikamo et al., 2018), systemic dysregulation of COVID-19 patients(Gassen et al., 2021), and asthma(Park et al., 2016; Liu B. et al., 2021) as well. In our manuscript, we would like to focus on the Skp2-inhibiting compounds that was used to treat tumors experimentally.

In the last 20 years, numerous attempts have been made to develop antitumor drugs targeting Skp2 proteins, and various Skp2 inhibitory compounds have been developed. However, none has been proved suitable for clinical use. In this review, we introduce the structural features of Skp2, discuss the ubiquitination functionality of SCF^Skp2, and summarize the diverse range of natural and synthetic Skp2 inhibitors or inhibitory compounds reported to date.

The synthetic small-molecule Skp2-inhibiting compounds were separated into two groups, i.e., “structure-based” and “non-structure-based”. In the “structure-based” group, all the reported compounds were selected by structure-based approached, in which the compound binds with Skp2 and disrupts the protein interaction, thus inhibiting Skp2’s functions directly. In the ‘non-structure-based’ group, the compounds were identified by various methods, leading to different mechanisms when related to how compounds worked.

As an inhibitor should be able to bind to the target protein directly, in this manuscript, the Skp2-inhibiting compound which binds Skp2 directly and disrupts the binding between Skp2 and other proteins is called Skp2 inhibitor, while the compound only inhibits the expression or activity of Skp2 is called Skp2 inhibitory compound.

The IC₅₀ data of the Skp2 inhibitors or inhibiting compounds in various kinds of tumors at cellular level were collected in Table 2. In prostate cancer cell lines PC3 and LNCaP, the IC₅₀ values of Skp2 inhibitor SZL-P1-41 (i.e., #25) was 5.61 μM and 1.22 μM respectively (Chan et al., 2013), much lower than the natural Skp2-inhibiting compounds Gartanin (13.56 μM, 8.32 μM) (Pham et al., 2020) and Safranal (0.512 mM, 0.234 mM)(Jiang et al., 2020), and also lower than SMIP004(40 μM in LNCaP)(Rico-Bautista et al., 2010), a non-structure-based compounds. However, the treatment time course was quite different between these compounds, with 4 days for SZL-P1-41, 48 h for Safranal, and 72 h for Gartanin, which might lead to a bias of the IC₅₀ in this comparation.

This phenomenon that structure-based inhibitor of Skp2 was more efficient than non-structure-based compounds, was also observed in lung cancer cell line H520, H69, H196 and primary mouse small cell lung cancer (SCLC) cell, in which IC₅₀ value of Skp2 inhibitor SKPin C1 was lower than MLN4924 and Flavokawain A (Zhao et al., 2020). H69 and H196 are both SCLC cell lines, so we investigated the data of non-small cell lung cancer (NSCLC) cell lines. In H460 and A549 cell lines, the Skp2 inhibitors did not show strong inhibitor effects. The MLN4924 had the lowest IC₅₀ values in H460(0.43 μM), lower than Skp2 inhibitor SZL-P1-41(5.15 μM) and SKPin C1(33 μM). SMIP004 also exerted stronger inhibitor effects than SZL-P1-41, with IC₅₀ value of 500 nM at 24 h (Zhao et al., 2020). Flavokawain A and SMIP004 was Skp2-inhibiting compound, while MLN4924 is an SCF^Skp2 complex neddylation inhibitor which might not bind with Skp2. In the above comparisons, the SZL-P1-41 was also with the longest treatment time course of 4 days, however, it’s not the potent inhibitor with IC₅₀ value higher than other inhibitor or compounds, so we ignore the bias of the IC₅₀ lead by the differences of treatment time courses, and concentrate on the potent compounds with much lower IC₅₀ values and shorter treatment time. The different performances between Skp2 inhibitor and Skp2-inhibiting compounds in SCLC cell lines and NSCLC cells lines (Figure 1), might provide a clue that Skp2 played more important roles in SCLC than in NSCLC cells from the point of tumor pathological mechanisms. Furthermore, when treating lung cancers, different Skp2 targeting compound with respective effects should be selected according to the type, stage, pathological mechanisms of lung cancers. However, the wild-type RB1 existence should also be taken into consideration, for instance, the IC₅₀ value of SKPin C1 in lung squamous cell carcinoma cell line H520 with wild-type RB1 was higher than that in SCLC cell line H69 with deficient RB1 (Zhao et al., 2020). As lung cancer cells were quite heterogenous, we should take these data seriously.

In addition, in T cell leukemia cell line TAIL7, two Skp2 inhibitors, SZL-P1-41(30 μM) and SKPin C1(2.4 μM) exhibited significantly different performances (Rodriguez et al., 2020). SZL-P1-41 binds to the Trp97 and Asp98 residues of Skp2, preventing Skp2–Skp1 interaction, while SKPin C1 targets the Skp2-p27 interaction interface to block p27 degradation. These data imply that in IL-7-dependent T-ALL cell line TAIL7, preventing a specific substrate(p27) degradation might exert stronger inhibitor effects than inhibition of Skp2 function by disrupting the SCF^Skp2 complex. However, this phenomenon was not observed in other tumor cell lines listed in Table 2. The IL-7 signaling pathway was proved to play important roles in Notch/Skp2/p27^Kip1 pathway to promote Skp2 induction in T-ALL development, and which might attribute to this observation that SKPin C1 exerted more inhibitory effects on TAIL7 than SZL-P1-41. This result indicated that one should choose Skp2 inhibitor compound according to the mechanisms of various cancers, and specific target should be selected according to the pathological mechanisms of specific cancer when developing drugs. Somehow, it might have stronger inhibitor effects when only block the binding between Skp2 and one specific substrate, other than inhibiting the function of the SCF^Skp2 complex in some situation.

To the contrary, in NSCLC cell line H460, SZL-P1-41(5.15 μM) exerted stronger inhibitory effects than SKPin C1(33 μM) (Zhao et al., 2020), indicating that in lung cancer with same mechanisms of H460, disrupting the SCF^Skp2 complex prevented tumor development more significantly than only preventing p27 degradation. Other Skp2 substrate proteins might also play important roles in lung cancer other than p27, implying the multiple roles of Skp2 in cancer development.

Natural Skp2 inhibitor should be considered additionally. A natural Skp2 inhibitor with high affinity and specificity, which could disrupt the binding between Skp2 and other proteins, might be a potential effective Skp2 inhibitor in tumor treatment, or used as template for developing more potent and safer drugs. For example, natural Skp2 inhibitor Betulinic acid performed so well to be used in clinical trials (He et al., 2021). According to the IC₅₀ data in Table 2, in several cancer cell lines, the Skp2 inhibitor exerted stronger inhibitory effects than the Skp2-inhibiting compounds, especially the natural Skp2 inhibitor gentian violet, which disrupted the binding between Skp2 and p27, with a IC₅₀ of 0.4 μM in human cervical cancer cell HeLa, and 0.6 μM in murine breast cancer cell tsFT210, comparing to the 5.3 μM in normal NIH3T3 cells (Ooi et al., 2013). However, the decreased IC₅₀ values of many natural inhibitors should be considered carefully. For instance, the gentian violet also has other cellular toxic functions in addition to Skp2 inhibitor, such as the genetic toxicity, uncoupling effect on mitochondrial oxidative phosphorylation, photodynamic action (Moreno et al., 1988). The low IC₅₀ value of gentian violet might be attributed to high permeability or non-specific off target toxicity, other than the direct effects on Skp2. There is another situation, many natural drugs always have multiple targets or multiple pharmacologic effects by affecting several signaling pathways. Take curcumin, for example, the observed tumor inhibition effects of curcumin might attribute to several inhibitor mechanisms including inhibition of Skp2. The really inhibitory effect of natural Skp2 inhibitor should be considered carefully. In conclusion, more confirmative tests should be carried out to evaluate the potence of natural inhibitors.

There are also two interesting findings. The first one, when treating primary mouse SCLC lung cell and primary mouse SCLC liver metastatic cell, the IC₅₀ value of SKPin C1 was almost the same, however, the IC₅₀ value of MLN4924 decreased from 1.8 μM in primary mouse SCLC lung cell to 0.18 μM in primary mouse SCLC liver metastatic cell (Zhao et al., 2020). MLN4924 is an inhibitor of NEDD8-activating enzyme (NAE), which inhibits the function of SCF^Skp2 complex by inactivation of cullin-RING ligases (Soucy et al., 2009). However, it also has some neddylation-independent activities, such as activating EGFR, promoting glycolysis, inhibiting IFN-β production, and activating the JNK signaling pathway (Jin et al., 2013). The different performances of MLN4924 in primary mouse SCLC lung cell and primary mouse SCLC liver metastatic cell, implied that some important mechanisms might change during SCLC cell metastasis, and this mechanism changes might relate to neddylation-dependent activities of MLN4924, indicating that SCF^Skp2 or other E3 neddylation might promote the SCLC cell metastasis, other than only Skp2 protein or the ubiquitination mediate by Skp2. In addition, this mechanism changes might also relate to neddylation-independent activities of MLN4924, indicating other signaling pathways might play important roles in SCLC cell metastasis process, which could be used as target for drug development or therapy potentially.

Another interesting finding is that when treating LNCaP cells with SMIP004, the IC₅₀ value decreased from 40 μM in LNCaP cells with normal Skp2 expression level (LNCaP-WT) to 1.09 μM in LNCaP cells with stable Skp2 overexpression (LNCaP-S14) (Rico-Bautista et al., 2010). In this study that SMIP004 was selected out, the increased Skp2 protein level was proved to be associated with the inhibitory effect of SMIP004. Recently, SMIP004 was reported to mediate a mitochondrial pathway (Rico-Bautista et al., 2013), leading to mitochondrial ROS formation, unfolded protein response activation and apoptosis finally. These results implied that Skp2 might also relate to the apoptosis induced by ROS and unfolded protein response, supplying a new clue for Skp2-targeting therapy. The previous reported findings were consistent with the roles of Skp2 in ROS generation and unfolded protein response. Skp2 has been reported to relate to the degradation of Myc which is necessary for the generation of ROS mediated by pre-existing Romo1 protein (Lee et al., 2011). The decreased Skp2 protein level was reported to relate with the unfolded protein response as well (Han et al., 2013).

Furthermore, the RNA expression of Skp2 showed low cancer specificity, and we collected the data of Skp2 RNA expression level in several of the cell lines in Table2 from the Human Protein Atlas database, with the data represented as nTPM (Figure 2A). The correlation between the RNA expression level and IC₅₀ value was analyzed in treatment groups of SZL-P1-41, MLN4924, Skpin C1, and Flavokawain A. No apparent significant correlation was observed (Figure 2B-E). However, the mRNA level might not always consistent with the Skp2 protein level, the correlation between protein level and IC₅₀ value should also be analyzed in the future when protein expression data is accessible. Meanwhile, one of the Skp2 key substrate protein, p27, was reported to relate with clinical stages and chemoresistance of various tumors. In hepatocellular carcinoma, the p27 protein level was significantly lower in advanced staged of disease (Tannapfel et al., 2000). In various other cancers, the protein level of p27 was also reported to be decreased in advanced tumor stage and relate to poor prognosis. In prostate cancer, for instance, the intensity of p27 expression was negatively associated with the tumor stage and Gleason scores, and the upregulation of p27 expression was observed in prostate adenocarcinoma cells after hormonotherapy (Nikoleishvili et al., 2007). In addition, p27 protein level was lower in breast cancer cell line MDA-MB-231 compared to BT474, and the MDA-MB-231 cells showed higher susceptibility to treatment of 5 gg, curcumin, and lycopene respectively, comparing to BT474. However, BT474 was more sensitive to quercetin (Huang et al., 2011). This observation implied that different mechanisms involve in the tumor development as MDA-MB-231 is ER/HER2-negative and BT474 was HER2-positive. Furthermore, in SCLC tissues, the observation that protein level of p27 was suppressed and protein level of Skp2 was increased was demonstrated by Immunohistochemistry assay (Zou et al., 2022). In NSCLC tissues, the total positive rate of p27 protein was significantly lower than in non-cancerous lung disease, meanwhile, the positive rate of p-AKT was significantly higher than these non-cancerous controls. Protein p-AKT was another important substrate of Skp2, which mediates the K63-linked ubiquitination of p-AKT rendering AKT more stable (Miao et al., 2006). The positive expression rate of AKT was also significantly higher in other cancer tissues, such as gastric cancer tissues compared to normal gastric mucosa (Gu et al., 2014). Skp2 has been reported to be the ubiquitin ligase not only for p27 but also other antioncogenic proteins, and it has other important oncogenic effects, suggesting the reduction of Skp2 level is a rational therapeutic method.

As a hot research spot for therapeutic target-drugs of cancers, inhibition of Skp2 by genetic deletion or pharmacological blockade showed little effect on normal tissues based on murine models, indicating that Skp2 inhibition may not induce serious adverse effect (Chan et al., 2010; Lin et al., 2010). Recently, Skp2 has been reported to be a therapeutic target in other non-cancer diseases, such as BM transplantation (Wang et al., 2011), depression (Wang D. et al., 2019). However, we should still consider the harmful aspect of Skp2 inhibition treatment seriously. For example, the Skp2 inhibition might affect the female patient’s fertile ability. The recurrent miscarriage patients were reported to have reduced expression in the decidual tissues compared to normal pregnant women, and Skp2-deficient mice showed impaired ovarian development and reduced fertility (Lv et al., 2021). The slowdown of cell cycle relates to cellular senescence closely, and the Skp2 inhibition induces the senescence of endothelial progenitor cell (EPC) (Wang H. H. et al., 2020), and inhibits angiogenic senescence, leading to aging-related vasculopathy, which finally affects atherosclerosis related disease or vascular event.

Until recently, there are emerging various Skp2-inhibiting compounds with different inhibitory mechanisms. Comprehensively considering the characteristics of Skp2 protein, such as the self-amplifying feedback loop of itself and potentially side effects of Skp2 inhibition (Yung et al., 2007), it could be suggested to adopt two or more Skp2-inhibiting compounds for Skp2 inhibitory treatment clinically, which would be a better way in tumor treatment than single Skp2 inhibitors. The choice of Skp2 inhibitor or inhibiting compound should be based on the disease pathological mechanisms. For example, the expression and stability of Skp2 protein was reported to relate to the vemurafenib-resistance in melanoma cells. Vemurafenib inhibits Skp2 on transcriptional level, and b-AP15 inhibits Skp2 on post-translational level. Combined use of both inhibitors is effective in treating vemurafenib-resistant melanoma (Wu et al., 2022). In addition, the combination of tubulin inhibitor VERU-111 and vemurafenib also inhibited vemurafenib-resistant melanoma effectively by inhibiting tubulin protein and Skp2 protein together (Cui et al., 2021). Another example, the SCF^Skp2 components related to BTZ resistance, and combined use of BTZ and Skp2 inhibitor inhibits BTZ-resistant multiple myeloma growth, rendering targeting SCF^Skp2as a strategy to overcome therapeutic resistance in multiple myeloma.

Skp2 is overexpressed in various human cancers, and as a component of SCF^Skp2 E3 ligase, it has been defined as an oncogene. Exploring the functions and pathological mechanisms of Skp2 in tumorigenesis and development could help us achieve a better understanding of tumors and provide new strategies for tumor treatment. The development of drugs that target Skp2 is a promising strategy for future cancer therapy.

In this review, we have summarized recently reported synthetic and natural Skp2 inhibitory compounds. These compounds inhibit Skp2 function or downregulate its expression by various mechanisms, and their effects have been evaluated in vivo by animal experiments. However, only the nature Skp2 inhibitor, Betulinic acid, was used in Phase 1 clinical trials to treat cutaneous metastatic melanoma in 2008. Two TKIs, Imatinib and GNF-5, which have been proved for clinical treatment, have been reported to suppress the expression of Skp2 indirectly via inhibiting its upstream factor. In addition, the BRAF^V600Einhibitor vemurafenib has been found to decrease Skp2 mRNA expression and has been used in several clinical trials for the treatment of cancers with BRAF mutation. These TKIs and vemurafenib was not the inhibitor of Skp2, however, these findings implied that targeting upstream factors of Skp2 might be a clue for Skp2 suppression in drug development.

In addition, not all the roles of Skp2 in cancer are fully understood. Additional functional and mechanistic studies of Skp2 are required, and more small molecules that target Skp2 are also needed, especially those that disrupt Skp2 stabilization and function without cytotoxic effects. However, due to the multiple functions of Skp2 in cancers, the cancer type, stage and pathological mechanisms should be taken into consideration when selecting Skp2-inhibiting compound for cancer treatment.