The ubiquitin ligase adaptor SPOP in cancer

Abstract

The dysregulation of ubiquitin-mediated proteasomal degradation has emerged as an important mechanism of pathogenesis in several cancers. The speckle-type POZ protein (SPOP) functions as a substrate adaptor for the cullin3-RING ubiquitin ligase and controls the cellular persistence of a diverse array of protein substrates in hormone signalling, epigenetic control and cell cycle regulation, to name a few. Mutations in SPOP and the resulting dysregulation of this proteostatic pathway play causative roles in the pathogenesis of prostate and endometrial cancers, whereas overexpression and mislocalization are associated with kidney cancer. Understanding the molecular mechanism of the normal function of SPOP as well as the cause of SPOP-mediated oncogenesis is thus critical for eventual therapeutic targeting of SPOP and other related pathways. Here, we will review SPOP structure, function and the molecular mechanism of how this function is achieved. We will then review how mutations and protein mislocalization contribute to cancer pathogenesis and will provide a perspective on how SPOP may be targeted therapeutically.

Keywords: SPOP; endometrial cancer; intrinsically disordered proteins; liquid-liquid phase separation; prostate cancer; ubiquitin ligase; ubiquitination.

Figure 1:. SPOP domain and complex structure.

(A) Schematic of SPOP domain structure. (B) Crystal structures of the SPOP MATH domain (green ribbon representation) with substrate peptides reveal an extended substrate binding site. Superposition of the MATH/Puc peptide (yellow peptide, PDB code 3HQL) and the MATH/human PDX (orange peptide, PDB code 6F8F) complexes reveals similar interactions [5, 22]. (C) Crystal structure of the SPOP BTB domain dimer. The two-fold symmetric dimer (represented with one monomer in red and the other gray) is the Cul3 interaction site. (D) Crystal structure of the C-terminal SPOP BACK domain dimer (represented with one monomer in blue and the other gray, PDB code 4HS2) [28]. (E) A schematic diagram of the domain structure of SPOP monomer (left) and SPOP oligomer (far right). The linear SPOP oligomer schematic is colored based on panel A. The concentration dependent association of SPOP dimers through BACK domain interactions indicates the possibility for indefinite self-association [8]. (F) Model of SPOP oligomer created through superposition of known crystal structures: SPOP, 3HQI [5] and 4HS2 [28]. (G) Model of SPOP/Cul3 oligomer created through superposition of the SPOP/cullin-3 (PDB code 4EOZ [26]) and cullin-1/Rbx1/UbcH5 (PDB code 1LDK [59]) crystal structures with corresponding domains from the SPOP oligomer shown in panel F. The central SPOP octamer is colored as in panel A and the cullin component of the complex is colored in orange.

Figure 2:. Functional implications of SPOP oligomerization.

(A) Higher-order oligomeric SPOP ubiquitinates substrates more effectively than SPOP dimers or monomers. In vitro ubiquitination assays with CRL3^SPOP and a fragment of Gli3 as a substrate (residues 1–455). Comparison of WT SPOP and self-association defective mutants mutBTB (mutations L186D, L190D, L193D, I217K), mutBACK (mutation Y353E) and a combination of both, mutBTB/BACK. All SPOP versions comprise residues 28–359. (Panel reprinted with permission from [20]). (B) Model of the role of multivalent interactions between oligomeric SPOP and multiple SB motifs in a single substrate molecule. Dimeric SPOP recruits substrates with low affinity and is shown to miss suitable steric access to lysine acceptor sites on the substrate or on ubiquitin. Oligomeric CRL3^SPOP binds substrates with enhanced affinity via avidity effects and mediates effective polyubiquitination through multiple catalytic centers in the oligomeric CRL3. A SPOP tetramer is shown for clarity. SB motifs are depicted as pink bars, and the color saturation decreases for weaker motifs. (Adopted from [23] with permission.) (C) Phase separation via multivalent interactions. SPOP and DAXX undergo phase separation in vitro. Fluorescence microscopy images of a mixture of SPOP (green fluorescent, residues 28–359) and cDAXX (red fluorescent, residues 495–740). (D) The SPOP prostate cancer mutant W131G is defective for co-localization with DAXX in HeLa cells; WT SPOP colocalizes with DAXX in nuclear bodies that are distinct from nuclear speckles. SC-35 (magenta) marks nuclear speckles. (Adopted from [8] with permission.) (E) Schematic illustration of the role of phase separation in SPOP-mediated substrate turnover. SPOP phase separates with multivalent substrates and is able to target and ubiquitinate substrates localized to membrane-less organelles. SPOP cancer mutants are defective at phase separation and therefore co-localization and ubiquitination.

Figure 3:. Distinct sets of SPOP missense mutations in different cancer types.

(A) Lollipop plots with mutation site and number in SPOP in prostate cancer (top) and endometrial cancer (bottom). Data from cBio portal [41]. Green, red and black lollipops indicate missense mutations, short in-frame insertions/deletions and truncations, respectively. (B) Ribbon diagram of SPOP MATH domain with recurrent missense mutations in prostate cancer (left) and endometrial cancer (right).

Figure 4:. Possibilities for therapeutic interventions in SPOP-related cancers.

SPOP dysfunction plays key roles in the cancer pathogenesis of subsets of patients with prostate, endometrial, breast cancer and ccRC. In prostate cancer, loss-of-function SPOP mutations lead to accumulation of substrates [6, 10, 31], which could be targeted via PROTACs. These mutants also favor the use of non-homologous end joining (NHEJ) instead of homologous recombination (HR) as the DNA damage response, potentially rendering combinations with PARP inhibitors useful [42]. In endometrial cancer, gain-of-function SPOP mutations lead to enhanced ubiquitination and turnover of BET proteins BRD2, BRD3 and BRD4, rendering cells sensitive to BET inhibitors [11]. In breast cancer, the often observed SPOP amplification could make a SPOP inhibitor useful [52]. In ccRC, hypoxia leads to HIF-mediated SPOP induction and mislocalization to the cytoplasm, where CRL3^SPOP mediates ubiquitination and subsequent degradation of tumor suppressors PTEN, DUSP6 and DUSP7 [44]. A SPOP inhibitor could prevent this turnover [52]. Lastly, SPOP mutations may perturb the driving force for phase separation of SPOP with substrates [8], and small molecules could be used to readjust it.