The Skp2 Pathway: A Critical Target for Cancer Therapy

https://doi.org/10.1016/j.semcancer.2020.01.013Get rights and content

Abstract

Strictly regulated protein degradation by ubiquitin-proteasome system (UPS) is essential for various cellular processes whose dysregulation is linked to serious diseases including cancer. Skp2, a well characterized component of Skp2-SCF E3 ligase complex, is able to conjugate both K48-linked ubiquitin chains and K63-linked ubiquitin chains on its diverse substrates, inducing proteasome mediated proteolysis or modulating the function of tagged substrates respectively. Overexpression of Skp2 is observed in various human cancers associated with poor survival and adverse therapeutic outcomes, which in turn suggests that Skp2 engages in tumorigenic activity. To that end, the oncogenic properties of Skp2 are demonstrated by various genetic mouse models, highlighting the potential of Skp2 as a target for tackling cancer. In this article, we will describe the downstream substrates of Skp2 as well as upstream regulators for Skp2-SCF complex activity. We will further summarize the comprehensive oncogenic functions of Skp2 while describing diverse strategies and therapeutic platforms currently available for developing Skp2 inhibitors.

Introduction

Protein degradation, as a necessary component of protein turnover, is essential for cells to rapidly recycle amino acids in response to various extracellular stimuli. UPS serves as the most critical posttranslational modification machinery that governs protein degradation [1]. Functional UPS mediated proteolysis is responsible for maintaining protein homeostasis by eliminating misfolded protein, which is important for regulating various cellular processes such as apoptosis [2] and cell cycle progression [3]. Moreover, recent studies have suggested that the non-proteasomal ubiquitin signals are also significantly involved in processes such as metabolic regulation [4], DNA repair [5], autophagy [6], signal transduction [7] and immune response [8]. Importantly, dysfunction of UPS may result in serious diseases such as cancer [9], Parkinson disease [10] and Alzheimer's disease [11].

In essence, protein degradation is tightly regulated by two sequential processes: the conjugation of ubiquitin moieties to targeted substrates, followed by proteolysis of ubiquitin tagged protein in the 26S proteasome. The covalent link of ubiquitin moieties to its targets requires several critical components, including ubiquitin, ATP, the ubiquitin-activating enzyme (E1), the ubiquitin-conjugating enzyme (E2) and the ubiquitin-protein ligase (E3) [12]. Mechanistically, three reaction steps are involved in the E1-E2-E3 catalyzing cascade. First, the cysteine residue on E1 forms a linkage with the carboxyl group of Glycine residue on ubiquitin through a thioester bond, thus activating ubiquitin in an ATP-consuming manner. Then, activated ubiquitin is transiently transferred to Cysteine residue of E2 through a thioester linkage. Finally, E3 covalently attaches ubiquitin to the amino group of Lysine residue on targeted substrates [13] (Fig. 1). Repetitions of these sequential reactions lead to the consecutive incorporation of ubiquitin moieties onto targeted protein, which is termed polyubiquitination.

Ubiquitin is a 76 amino-acid protein which is highly conserved among various species and ubiquitously expressed in all types of cells. Notably, seven Lysine residues (K6, K11, K27, K29, K33, K48 and K63) or Methionine residue (M1) on ubiquitin molecules could serve as a receptor to conjugate other ubiquitin resulting in diverse linkages of polyubiquitination to exert different cellular functions [14,15]. Generally, K48-linked polyubiquitin chains often induce 26S proteasome mediated proteolysis of tagged substrates [16]. However, rather than mediating protein degradation, K63-linked polyubiquitin chains regulate protein-protein interaction, protein activity or intracellular protein trafficking involved in numerous biological processes such as immune regulation [17], DNA repair [18], cell proliferation and survival [19,20], stress response [21] and autophagy [22]. In addition to K48- and K63-linked ubiquitin chains, which are referred to as canonical ubiquitin linkage types, other atypical linkage types have begun emerging in recent years [23]. Similar to K48-linked ubiquitination, K11-linked polyubiquitin chains are also recognized by proteasomal receptor for protein degradation, which plays a critical role in cell division [24]. Meanwhile, K27-linked polyubiquitination on histone 2A (H2A), representing the major ubiquitin linkage type on chromatin upon DNA damage, does not trigger protein degradation, but is crucial for the recruitment of DNA repair machinery [25]. Smurf1 mediated K29-linked polyubiquitination on Axin results in its dissociation with Wnt co-receptors LRP5/6, thus leading to the suppression of Wnt/β-Catenin signal [26]. Interestingly, M1-linked linear ubiquitination on NEMO modified by LUBAC is essential for the activation of IKK complex, which serves as a key regulator in NF-κB signal [27]. With the advancement of innovative technologies in proteomics and structural biology, the physiological relevance of these non-canonical ubiquitin linkages will be further delineated.

Among E1-E2-E3 catalyzing cascades, only two E1s (UBA1 and UBA6 [28]) have been well defined so far. However, around 40 E2s and more than 600 E3s encoded by the human genome [29] are identified. Compared with E1 and E2 enzymes, E3 ligases receive greater attention, since the high variability of E3 ligases plays a critical role in determining the specificity of substrates and targeting E3 could enhance drug efficacy by minimizing the off-target effects.

Based on the characteristic domain as well as the mechanism of how ubiquitin is transferred to substrate protein, E3 ligases can be classified into three major families: HECT (homologous to E6-associated protein C terminus) E3s, RBR (RING-in-between-RING) E3s and RING (Really Interesting New Gene) E3s [30]. A summary of the differences among these three families is illustrated in Fig. 1. In mammals, around 30 HECT E3 ligases have been identified. HECT E3 family members share a conserved HECT domain at C terminus, which is featured by a bi-lobar structure: N-terminal lobe, responsible for ubiquitin-charged E2 binding and substrate recognition and C-terminal lobe with the catalytic Cysteine for receiving and transferring ubiquitin. Two lobes are tethered by a flexible hinge, which ensures the free rotation of these two lobes during ubiquitin transfer [31]. Two sequential steps are required for HECT E3s to catalyze substrate ubiquitination: ubiquitin is first transferred from E2s to the catalytic Cysteine of C-lobe and then it is passed from E3 to the Lysine residue of the substrate whose specificity is determined by N-lobe [32]. Intramolecular interactions critically regulate the catalytic activity of HECT E3s by autoinhibition, which is released upon various stimuli. Three subfamilies of HECT E3s have been further categorized based on the characteristic domain on N-terminal: (1) Nedd4 subfamily including 9 members such as Nedd4, Itch and Smurf1, which contain two to four Tryptophan-Tryptophan (WW) domains, (2) HERC (HECT and RCC1-like domain) subfamily including 6 members as HERC1∼6, which contain one or more chromosome condensation 1 (RCC1)-like domains (RLDs), (3) Other HECT E3s such as E6AP, which contain diverse domains [33].

In recent years, RBR E3s have been gradually identified. This enigmatic subfamily is composed of 14 members, including PARKIN, whose dysregulation is related to the pathogenesis of early-onset Parkinson's disease [10]. As indicated by its name, RBR E3s harbor two RING domains which are separated by an in-between-RING domain (IBR). In general, RING1 is responsible for ubiquitin charged E2 recruitment, while RING2 with a catalytic cysteine residue is involved in receiving ubiquitin from RING1 through a trans-thioesterification reaction [34]. Similar to HECT E3s, RBR E3s catalyze substrate ubiquitination in a two-step manner: ubiquitin is first transferred to the Cysteine residue of E3 from E2 and then passed to a substrate. Other than RBR domains, these E3s also contain additional functional domains, based on which, several subfamilies are further classified: the Ariadne family members harbor featured Ariadne domains including TRIAD1, PARC and others, while the rest contain various other domains including PARKIN, HOIP and others [35].

The remaining large numbers of E3s (more than 600) belong to the RING family. They are characterized by the existence of a zinc-binding RING domain or a U-box domain, which shares a similar RING fold albeit without zinc-binding [36]. Notably, unlike HECT E3s and RBR E3s, RING E3s catalyze a direct transfer of ubiquitin from E2 to targeted substrates [29]. Both RING domain and U-box domain can work as monomers (c-CBL, E4B), homodimers (RNF4, Prp19), or heterodimers (BRCA1-BARD1). Other RING E3s consist of multiple subunits, such as the Cullin-RING ligases (CRLs) and the APC (Anaphase promoting complex), which are well documented and characterized. Importantly, Cullin-RING E3s, representing the largest family of E3 ubiquitin ligases, account for the ubiquitination of approximately 20% of cellular proteins degraded through UPS [37]. These Cullin-RING ligases are featured by the common Cullin scaffold protein and can be further divided into several categories: Skp1/Cullin 1/F-box protein complex (SCF), Cullin 2-Elongin B/C-VHL or SOCS proteins (CRL2), Cullin 3-BTBs (CRL3s), Cullin 4-DDB1-DCAFs (CRL4), Cullin 5-ElonginB/C-SOCS proteins (CRL5) and Cullin 7/FBXW8 (CRL7) [37,38].

The SCF complex consists of Skp1 (S-phase kinase-associated protein 1), Cullin 1, Rbx1 (Ring box protein 1)/Roc1 (Regulator of Cullin 1) and diverse F-box proteins [39]. Two key functional domains are presented in each of the identified F-box proteins: the C-terminal domain responsible for substrate recognition and the F-box motif connecting to other components of SCF complex through Skp1. In the human genome, 69 F-box proteins have been documented. These F-box proteins can be further divided into three categories based on the characterized domain beyond F-box: FBXWs with WD40 repeats, including 10 members such as β-TrCP, FBXLs with Leucine-rich repeats (LRR), including 21 members such as Skp2 and FBXOs with other various domains, including the remaining 38 members such as FBXO4.

Although most F-box proteins have been documented for many years, only a few of them have been well studied, among which Skp2 is the best characterized mammalian F-box protein with various proposed substrates. Because of its pro-oncogenic properties, which are involved in the pathogenesis of multiple cancers, Skp2 has become a promising target for cancer therapy [40]. Thus, in the following discussion, we will focus on the role of Skp2 in the development of cancer as well as the regulation of Skp2 activity during tumorigenesis, with a particular emphasis on its implication in cancer therapy and prevention.

Section snippets

Downstream targets of Skp2

As an F-box protein, Skp2 exerts its function mainly through its E3 ligase activity, although the involvement of Skp2 in transcriptional regulation is also defined, which is independent of its E3 ligase activity [41]. Two categories of substrates by Skp2 have been identified based on the ubiquitin linkage types, which are summarized in Fig. 2. Skp2 is initially found capable of inducing K48-linked ubiquitination, leading to proteasome mediated proteolysis of tagged substrates including p27 [42

Regulation of Skp2 expression and its E3 ligase activity

Aberrant Skp2 expression and dysregulation of its activity are associated with many human cancers [131]. The expression level of Skp2 as well as its E3 ligase activity are tightly regulated under multiple layers including transcription, protein stability and post-translational modifications, as illustrated in Fig. 3.

Evidence of tumor promoting function of Skp2

In 1995, Skp2, together with Skp1, was initially defined as a critical component of cyclin A-CDK2 kinase complex and deemed to be required for the S-phase entry in many transformed cells [167]. This was the first piece of evidence of the oncogenic property of Skp2. In the last 20 years, studies from diverse genetic mouse models, as well as human clinical tumor samples, have further confirmed the pro-tumor activity of Skp2.

Oncogenic property of Skp2

As a putative oncoprotein, Skp2 governs tumor formation and progression in many aspects such as promoting cell cycle, preventing cellular senescence and apoptosis, orchestrating cancer metabolism, maintaining cancer cell stemness, facilitating tumor metastasis, inducing drug resistance and other effects, as summarized in Fig. 4.

Targeting Skp2 for cancer therapy

Giving the critical involvement in governing cancer progression, Skp2 has emerged as an appealing pharmacological target for both cancer prevention and cancer treatment. In the last several decades, multiple strategies have been proposed to limit either Skp2 expression or Skp2-SCF complex activity for cancer therapy, as summarized in Fig. 5.

Conclusion and perspective

Skp2, as an E3 ligase coupling with Skp1, Cul1 and Rbx1/Roc1 to form Skp2-SCF complex, conjugates both K48-linked and K63-linked ubiquitin chains on its substrates, leading to proteasome mediated degradation as well as nonproteolytic functional regulation of the substrates respectively. The expression of Skp2 is under tight regulation during cell cycle progression in normal cells, while Skp2 overexpression is observed in multiple types of cancer such as breast, prostate, lung and others,

Declaration of Competing Interest

There are no conflicts of interest declared by the authors.

Acknowledgements

We would like to thank the members in Lin’s laboratory for the inputs and suggestions throughout the course of this study. This work was supported by start-up funds from Wake Forest School of Medicine and NIH grants (R01CA182424 and 1R01CA193813).

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