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Research ArticleCell biology
Open Access | 
10.1172/JCI189048
1Department of Urology, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, China.
2State Key Laboratory of Genetic Engineering, MOE Engineering Research Center of Gene Technology, Shanghai Engineering Research Center of Industrial Microorganisms, School of Life Sciences, Fudan University, Shanghai, China.
Address correspondence to: Lei Li (last listed author) or Jian Ma, Department of Urology, The First Affiliated Hospital of Xi’an Jiaotong University, 710061, Xi’an, China. Phone: 86.8532.3661; Email: lilydr@163.com; majian0922@gmail.com. Or to: Chenji Wang, State Key Laboratory of Genetics and Development of Complex Phenotypes, MOE Engineering Research Center of Gene Technology, Shanghai Engineering Research Center of Industrial Microorganisms, School of Life Sciences, Fudan University, Shanghai 200438, P.R. China. Phone: 86.21.31246559; Email: chenjiwang@fudan.edu.cn.
Authorship note: ZW and Lei Li (second listed author) contributed equally to this work.
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1Department of Urology, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, China.
2State Key Laboratory of Genetic Engineering, MOE Engineering Research Center of Gene Technology, Shanghai Engineering Research Center of Industrial Microorganisms, School of Life Sciences, Fudan University, Shanghai, China.
Address correspondence to: Lei Li (last listed author) or Jian Ma, Department of Urology, The First Affiliated Hospital of Xi’an Jiaotong University, 710061, Xi’an, China. Phone: 86.8532.3661; Email: lilydr@163.com; majian0922@gmail.com. Or to: Chenji Wang, State Key Laboratory of Genetics and Development of Complex Phenotypes, MOE Engineering Research Center of Gene Technology, Shanghai Engineering Research Center of Industrial Microorganisms, School of Life Sciences, Fudan University, Shanghai 200438, P.R. China. Phone: 86.21.31246559; Email: chenjiwang@fudan.edu.cn.
Authorship note: ZW and Lei Li (second listed author) contributed equally to this work.
Find articles by Li, L. in: PubMed | Google Scholar
1Department of Urology, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, China.
2State Key Laboratory of Genetic Engineering, MOE Engineering Research Center of Gene Technology, Shanghai Engineering Research Center of Industrial Microorganisms, School of Life Sciences, Fudan University, Shanghai, China.
Address correspondence to: Lei Li (last listed author) or Jian Ma, Department of Urology, The First Affiliated Hospital of Xi’an Jiaotong University, 710061, Xi’an, China. Phone: 86.8532.3661; Email: lilydr@163.com; majian0922@gmail.com. Or to: Chenji Wang, State Key Laboratory of Genetics and Development of Complex Phenotypes, MOE Engineering Research Center of Gene Technology, Shanghai Engineering Research Center of Industrial Microorganisms, School of Life Sciences, Fudan University, Shanghai 200438, P.R. China. Phone: 86.21.31246559; Email: chenjiwang@fudan.edu.cn.
Authorship note: ZW and Lei Li (second listed author) contributed equally to this work.
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1Department of Urology, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, China.
2State Key Laboratory of Genetic Engineering, MOE Engineering Research Center of Gene Technology, Shanghai Engineering Research Center of Industrial Microorganisms, School of Life Sciences, Fudan University, Shanghai, China.
Address correspondence to: Lei Li (last listed author) or Jian Ma, Department of Urology, The First Affiliated Hospital of Xi’an Jiaotong University, 710061, Xi’an, China. Phone: 86.8532.3661; Email: lilydr@163.com; majian0922@gmail.com. Or to: Chenji Wang, State Key Laboratory of Genetics and Development of Complex Phenotypes, MOE Engineering Research Center of Gene Technology, Shanghai Engineering Research Center of Industrial Microorganisms, School of Life Sciences, Fudan University, Shanghai 200438, P.R. China. Phone: 86.21.31246559; Email: chenjiwang@fudan.edu.cn.
Authorship note: ZW and Lei Li (second listed author) contributed equally to this work.
Find articles by Lei, Y. in: PubMed | Google Scholar
1Department of Urology, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, China.
2State Key Laboratory of Genetic Engineering, MOE Engineering Research Center of Gene Technology, Shanghai Engineering Research Center of Industrial Microorganisms, School of Life Sciences, Fudan University, Shanghai, China.
Address correspondence to: Lei Li (last listed author) or Jian Ma, Department of Urology, The First Affiliated Hospital of Xi’an Jiaotong University, 710061, Xi’an, China. Phone: 86.8532.3661; Email: lilydr@163.com; majian0922@gmail.com. Or to: Chenji Wang, State Key Laboratory of Genetics and Development of Complex Phenotypes, MOE Engineering Research Center of Gene Technology, Shanghai Engineering Research Center of Industrial Microorganisms, School of Life Sciences, Fudan University, Shanghai 200438, P.R. China. Phone: 86.21.31246559; Email: chenjiwang@fudan.edu.cn.
Authorship note: ZW and Lei Li (second listed author) contributed equally to this work.
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1Department of Urology, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, China.
2State Key Laboratory of Genetic Engineering, MOE Engineering Research Center of Gene Technology, Shanghai Engineering Research Center of Industrial Microorganisms, School of Life Sciences, Fudan University, Shanghai, China.
Address correspondence to: Lei Li (last listed author) or Jian Ma, Department of Urology, The First Affiliated Hospital of Xi’an Jiaotong University, 710061, Xi’an, China. Phone: 86.8532.3661; Email: lilydr@163.com; majian0922@gmail.com. Or to: Chenji Wang, State Key Laboratory of Genetics and Development of Complex Phenotypes, MOE Engineering Research Center of Gene Technology, Shanghai Engineering Research Center of Industrial Microorganisms, School of Life Sciences, Fudan University, Shanghai 200438, P.R. China. Phone: 86.21.31246559; Email: chenjiwang@fudan.edu.cn.
Authorship note: ZW and Lei Li (second listed author) contributed equally to this work.
Find articles by Ye, L. in: PubMed | Google Scholar
1Department of Urology, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, China.
2State Key Laboratory of Genetic Engineering, MOE Engineering Research Center of Gene Technology, Shanghai Engineering Research Center of Industrial Microorganisms, School of Life Sciences, Fudan University, Shanghai, China.
Address correspondence to: Lei Li (last listed author) or Jian Ma, Department of Urology, The First Affiliated Hospital of Xi’an Jiaotong University, 710061, Xi’an, China. Phone: 86.8532.3661; Email: lilydr@163.com; majian0922@gmail.com. Or to: Chenji Wang, State Key Laboratory of Genetics and Development of Complex Phenotypes, MOE Engineering Research Center of Gene Technology, Shanghai Engineering Research Center of Industrial Microorganisms, School of Life Sciences, Fudan University, Shanghai 200438, P.R. China. Phone: 86.21.31246559; Email: chenjiwang@fudan.edu.cn.
Authorship note: ZW and Lei Li (second listed author) contributed equally to this work.
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1Department of Urology, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, China.
2State Key Laboratory of Genetic Engineering, MOE Engineering Research Center of Gene Technology, Shanghai Engineering Research Center of Industrial Microorganisms, School of Life Sciences, Fudan University, Shanghai, China.
Address correspondence to: Lei Li (last listed author) or Jian Ma, Department of Urology, The First Affiliated Hospital of Xi’an Jiaotong University, 710061, Xi’an, China. Phone: 86.8532.3661; Email: lilydr@163.com; majian0922@gmail.com. Or to: Chenji Wang, State Key Laboratory of Genetics and Development of Complex Phenotypes, MOE Engineering Research Center of Gene Technology, Shanghai Engineering Research Center of Industrial Microorganisms, School of Life Sciences, Fudan University, Shanghai 200438, P.R. China. Phone: 86.21.31246559; Email: chenjiwang@fudan.edu.cn.
Authorship note: ZW and Lei Li (second listed author) contributed equally to this work.
Find articles by Huang, W. in: PubMed | Google Scholar
1Department of Urology, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, China.
2State Key Laboratory of Genetic Engineering, MOE Engineering Research Center of Gene Technology, Shanghai Engineering Research Center of Industrial Microorganisms, School of Life Sciences, Fudan University, Shanghai, China.
Address correspondence to: Lei Li (last listed author) or Jian Ma, Department of Urology, The First Affiliated Hospital of Xi’an Jiaotong University, 710061, Xi’an, China. Phone: 86.8532.3661; Email: lilydr@163.com; majian0922@gmail.com. Or to: Chenji Wang, State Key Laboratory of Genetics and Development of Complex Phenotypes, MOE Engineering Research Center of Gene Technology, Shanghai Engineering Research Center of Industrial Microorganisms, School of Life Sciences, Fudan University, Shanghai 200438, P.R. China. Phone: 86.21.31246559; Email: chenjiwang@fudan.edu.cn.
Authorship note: ZW and Lei Li (second listed author) contributed equally to this work.
Find articles by Xu, S. in: PubMed | Google Scholar
1Department of Urology, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, China.
2State Key Laboratory of Genetic Engineering, MOE Engineering Research Center of Gene Technology, Shanghai Engineering Research Center of Industrial Microorganisms, School of Life Sciences, Fudan University, Shanghai, China.
Address correspondence to: Lei Li (last listed author) or Jian Ma, Department of Urology, The First Affiliated Hospital of Xi’an Jiaotong University, 710061, Xi’an, China. Phone: 86.8532.3661; Email: lilydr@163.com; majian0922@gmail.com. Or to: Chenji Wang, State Key Laboratory of Genetics and Development of Complex Phenotypes, MOE Engineering Research Center of Gene Technology, Shanghai Engineering Research Center of Industrial Microorganisms, School of Life Sciences, Fudan University, Shanghai 200438, P.R. China. Phone: 86.21.31246559; Email: chenjiwang@fudan.edu.cn.
Authorship note: ZW and Lei Li (second listed author) contributed equally to this work.
Find articles by Wang, K. in: PubMed | Google Scholar
1Department of Urology, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, China.
2State Key Laboratory of Genetic Engineering, MOE Engineering Research Center of Gene Technology, Shanghai Engineering Research Center of Industrial Microorganisms, School of Life Sciences, Fudan University, Shanghai, China.
Address correspondence to: Lei Li (last listed author) or Jian Ma, Department of Urology, The First Affiliated Hospital of Xi’an Jiaotong University, 710061, Xi’an, China. Phone: 86.8532.3661; Email: lilydr@163.com; majian0922@gmail.com. Or to: Chenji Wang, State Key Laboratory of Genetics and Development of Complex Phenotypes, MOE Engineering Research Center of Gene Technology, Shanghai Engineering Research Center of Industrial Microorganisms, School of Life Sciences, Fudan University, Shanghai 200438, P.R. China. Phone: 86.21.31246559; Email: chenjiwang@fudan.edu.cn.
Authorship note: ZW and Lei Li (second listed author) contributed equally to this work.
Find articles by Liu, J. in: PubMed | Google Scholar
1Department of Urology, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, China.
2State Key Laboratory of Genetic Engineering, MOE Engineering Research Center of Gene Technology, Shanghai Engineering Research Center of Industrial Microorganisms, School of Life Sciences, Fudan University, Shanghai, China.
Address correspondence to: Lei Li (last listed author) or Jian Ma, Department of Urology, The First Affiliated Hospital of Xi’an Jiaotong University, 710061, Xi’an, China. Phone: 86.8532.3661; Email: lilydr@163.com; majian0922@gmail.com. Or to: Chenji Wang, State Key Laboratory of Genetics and Development of Complex Phenotypes, MOE Engineering Research Center of Gene Technology, Shanghai Engineering Research Center of Industrial Microorganisms, School of Life Sciences, Fudan University, Shanghai 200438, P.R. China. Phone: 86.21.31246559; Email: chenjiwang@fudan.edu.cn.
Authorship note: ZW and Lei Li (second listed author) contributed equally to this work.
Find articles by Gao, Y. in: PubMed | Google Scholar
1Department of Urology, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, China.
2State Key Laboratory of Genetic Engineering, MOE Engineering Research Center of Gene Technology, Shanghai Engineering Research Center of Industrial Microorganisms, School of Life Sciences, Fudan University, Shanghai, China.
Address correspondence to: Lei Li (last listed author) or Jian Ma, Department of Urology, The First Affiliated Hospital of Xi’an Jiaotong University, 710061, Xi’an, China. Phone: 86.8532.3661; Email: lilydr@163.com; majian0922@gmail.com. Or to: Chenji Wang, State Key Laboratory of Genetics and Development of Complex Phenotypes, MOE Engineering Research Center of Gene Technology, Shanghai Engineering Research Center of Industrial Microorganisms, School of Life Sciences, Fudan University, Shanghai 200438, P.R. China. Phone: 86.21.31246559; Email: chenjiwang@fudan.edu.cn.
Authorship note: ZW and Lei Li (second listed author) contributed equally to this work.
Find articles by Wang, C. in: PubMed | Google Scholar
1Department of Urology, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, China.
2State Key Laboratory of Genetic Engineering, MOE Engineering Research Center of Gene Technology, Shanghai Engineering Research Center of Industrial Microorganisms, School of Life Sciences, Fudan University, Shanghai, China.
Address correspondence to: Lei Li (last listed author) or Jian Ma, Department of Urology, The First Affiliated Hospital of Xi’an Jiaotong University, 710061, Xi’an, China. Phone: 86.8532.3661; Email: lilydr@163.com; majian0922@gmail.com. Or to: Chenji Wang, State Key Laboratory of Genetics and Development of Complex Phenotypes, MOE Engineering Research Center of Gene Technology, Shanghai Engineering Research Center of Industrial Microorganisms, School of Life Sciences, Fudan University, Shanghai 200438, P.R. China. Phone: 86.21.31246559; Email: chenjiwang@fudan.edu.cn.
Authorship note: ZW and Lei Li (second listed author) contributed equally to this work.
Find articles by Ma, J. in: PubMed | Google Scholar
1Department of Urology, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, China.
2State Key Laboratory of Genetic Engineering, MOE Engineering Research Center of Gene Technology, Shanghai Engineering Research Center of Industrial Microorganisms, School of Life Sciences, Fudan University, Shanghai, China.
Address correspondence to: Lei Li (last listed author) or Jian Ma, Department of Urology, The First Affiliated Hospital of Xi’an Jiaotong University, 710061, Xi’an, China. Phone: 86.8532.3661; Email: lilydr@163.com; majian0922@gmail.com. Or to: Chenji Wang, State Key Laboratory of Genetics and Development of Complex Phenotypes, MOE Engineering Research Center of Gene Technology, Shanghai Engineering Research Center of Industrial Microorganisms, School of Life Sciences, Fudan University, Shanghai 200438, P.R. China. Phone: 86.21.31246559; Email: chenjiwang@fudan.edu.cn.
Authorship note: ZW and Lei Li (second listed author) contributed equally to this work.
Find articles by Li, L. in: PubMed | Google Scholar
Authorship note: ZW and Lei Li (second listed author) contributed equally to this work.
Published July 15, 2025 - More info
Published in Volume 135, Issue 14 on July 15, 2025
AbstractNuclear size is crucial for cellular functions and often increases with malignancy. Irregular nuclei are linked to aggressive tumors, driven by genetic and epigenetic changes. However, the precise mechanisms controlling nuclear size are still not fully understood. In this study, we demonstrated that cancer-associated speckle-type POZ protein (SPOP) mutations enlarged nuclear size by reducing the protein level of lamin B2 (LMNB2), a key nuclear integrity protein. Mechanistically, SPOP bound to LMNB2 and promoted its mono-ubiquitination at lysine-484, which protected it from degradation by the E3 ubiquitin ligase WD repeat domain 26. SPOP mutations disrupted this process, leading to reduced LMNB2 levels and impaired nuclear envelope (NE) integrity. This compromised NE was more vulnerable to damage from farnesyltransferase inhibitors (FTIs), causing nuclear rupture in SPOP-mutant tumor cells. This study identified SPOP as a positive regulator of nuclear size; the findings suggest tumors with SPOP mutations may be vulnerable to FTI-based therapies.
Graphical Abstract
Introduction
Nuclear size is a key feature of cellular architecture, reflecting the intricate balance of cellular processes that govern growth, division, and function. In healthy cells, nuclear size is tightly regulated to maintain proper gene expression, chromatin organization, and cellular homeostasis (1, 2). However, in cancer, this regulation often becomes disrupted, leading to alterations in nuclear size—a hallmark of malignancy (3). Enlarged and irregularly shaped nuclei are frequently observed in various aggressive cancers, including those of the breast, prostate, colon, and pancreas (2, 4–6). Regulation of nuclear size—via nuclear envelope (NE) integrity (7–9), nuclear lamins (10, 11), and nucleocytoplasmic transport (12–16)—is essential for cellular order. Disruptions in these processes can lead to NE rupture, chromosomal instability, and increased metastatic potential (17–19). Understanding this interplay could pave the way for new targeted therapies, improving treatment options for aggressive cancers.
Nuclear lamins play a central role in controlling nuclear size by forming the nuclear lamina, a dense fibrous network that lines the inner surface of the NE in eukaryotic cells (6, 10). This network provides essential mechanical support, maintaining the nucleus’s shape, integrity, and organization (20). Nuclear lamins are divided into A type and B type. A-type lamins, such as lamin A and lamin C, contribute to nuclear stability and chromatin organization (21–25), whereas B-type lamins, including lamin B1(LMNB1) and LMNB2, are key to NE formation and maintenance (4, 26, 27). LMNB1 is degraded through the lysosome (28), but the regulation of LMNB2 in cancer is less well understood (29).
The SPOP gene encodes a substrate-binding adaptor subunit of the CULLIN3 (CUL3)-RING box 1 E3 ubiquitin ligase complex (30–33). SPOP is linked to oncogenesis through frequent mutations in cancers such as prostate and endometrial cancers (34–37). It targets several cancer-related proteins for degradation, including androgen receptor (AR) (38–40), BRD4 (41), SRC3 (42), and ATF2 (43). Additionally, SPOP mediates nondegradative ubiquitination of substrates like geminin (44), SQSTM1 (45, 46), and 53BP1 (47). Our previous research showed that SPOP regulates protein recruitment in the ER (48). Given the continuity between nuclear and ER membranes, changes in ER morphology could influence nuclear size (9, 12, 49). However, it remains unclear whether SPOP plays a role in nuclear size control.
In the present study, we showed that cancer-associated SPOP mutations increased nuclear size by decreasing LMNB2 protein levels. Normally, SPOP bound to LMNB2 and facilitated its mono-ubiquitination, which prevented degradation by WDR26. However, SPOP mutations led to enhanced LMNB2 degradation and disrupted NE integrity, causing nuclear rupture and making cancer cells more sensitive to farnesyltransferase inhibitor–based (FTI-based) therapies.
ResultsSPOP mutation increases cell nuclear volume. Histopathology images from The Cancer Genome Atlas (TCGA) prostate cancer data set (37), uterine corpus endometrial cancer data set (50), and our cohort revealed that SPOP-mutant tumors had larger nuclei compared with SPOP WT tumors (Figure 1, A–D). To investigate this further, we examined the impact of 2 common SPOP mutations, F102C and F133V, on nuclear size. Stable expression of these mutants increased nuclear volume in PC-3 and HeLa cells compared with the SPOP WT group (Figure 1, E and F, and Supplemental Figure 1, A–D; supplemental material available online with this article; https://doi.org/10.1172/JCI189048DS1). Similar results were observed in SPOP knockout cells (Figure 1, G and H, and Supplemental Figure 1, E–H). Given the central role of lamin proteins in nuclear size control (21, 22, 27, 51), we explored whether lamin involvement was linked to the enlarged nuclei in SPOP-associated cases. Co-immunoprecipitation (co-IP) assays showed that both ectopically expressed and endogenous SPOP interacted with LMNB2, but not with lamin A/C (LMNA) or LMNB1(Figure 1, I and J, and Supplemental Figure 1, I–K). This interaction was further confirmed in vitro, where LMNB2 directly bound to GST-SPOP purified from E. coli (Supplemental Figure 1L). Proteins targeted by SPOP typically contain a SPOP-binding consensus sequence (SBC) (Φ-π-S-S/T-S/T where Φ is a nonpolar residue and π is a polar residue) (52). Notably, LMNB2 contains 2 SBC motifs (SBC1: 417ATSSS421; SBC2: 610PRTTS614), which are absent in LMNA and LMNB1. Despite the high sequence similarity among LMNA, LMNB1, and LMNB2 (53), these SBC sites are unique to LMNB2 and conserved across species (Figure 1K and Supplemental Figure 1M). We further demonstrated that deletion of the 5 amino acids or mutation of 418TSS420 to 418TAA420 in SBC1 completely abolished SPOP’s interaction with LMNB2 in HEK293T cells (Figure 1L and Supplemental Figure 1N), confirming 417ATSSS421 as a functional SBC motif in LMNB2. Collectively, our data showed that cancer-associated SPOP mutation increased nuclear volume, likely through direct binding with LMNB2.
Figure 1SPOP mutation increases cell nuclear volume. (A and B) Quantification of nuclear area from diagnostic slides from patients in the SPOP WT (n = 354) and SPOT mutant (MUT) (n = 40) groups from the TCGA prostate adenocarcinoma data set (37) (A) and patients in the SPOP WT (n = 404) and SPOP MUT (n = 51) groups from the TCGA uterine corpus endometrial carcinoma data set (50) (B). (C and D) Representative H&E staining (C) images of prostate cancer specimens from the SPOP WT (n = 80) and SPOP MUT (n = 20) groups and quantification data (D). Scale bars: 200 μm in ×10 fields; 40 μm in ×40 fields. (E and F) PC-3 cells infected with lentivirus expressing WT, F102C, or F133V SPOP were analyzed using 2D and 3D IF (E) and quantified (F). Scale bar: 50 μm. (G and H) Control or SPOP knockout PC-3 cells were analyzed using 2D and 3D IF (G) and quantification (H). Scale bar: 50 μm. Data are shown as the mean ± SD of 3 biological replicates (n > 200). (I) Co-IP analysis of indicated proteins in 293T cells transiently transfected with Flag-LMNA, LMNB1, or LMNB2. (J) Co-IP analysis of endogenous proteins in 293T cells using indicated antibodies. (K) A LMNB2 structure diagram showing 2 putative evolutionally conserved SBC motifs (SBC1 and SBC2) located at the C-terminal of LMNB2. (L) Co-IP analysis of indicated proteins in 293T cells transiently transfected with Flag-LMNB2 WT, ΔSBC1, or ΔSBC2. *P < 0.05, ***P < 0.001 by Mann-Whitney test (A, B, D, and H) or 1-way ANOVA followed by Dunnett’s multiple comparisons test (F).
SPOP mutation destabilizes LMNB2 protein levels. Given the canonical role of SPOP as an E3 ubiquitin ligase, we investigated whether SPOP affects LMNB2 protein levels. Surprisingly, increased SPOP concentrations led to elevated LMNB2 protein levels (Figure 2A) and extended the half-life of endogenous LMNB2 in PC-3 cells (Figure 2, B and C). Consistently, SPOP mutations or knockout reduced LMNB2 protein levels in PC-3 and HeLa cells without affecting LMNB2 mRNA levels (Figure 2, D–G, and Supplemental Figure 2, A–D). The proteasome inhibitor MG132 treatment rescued LMNB2 protein level in both SPOP-mutant or knockout PC-3 and HeLa cells (Supplemental Figure 2, E and F). Immunofluorescence (IF) assays confirmed that SPOP mutation or knockout decreased LMNB2 signals compared with controls (Figure 2, H–K, Supplemental Figure 2, G and H, and Supplemental Figure 3, A and B). Moreover, LMNB2 overexpression successfully rescued the increased nuclear size induced by SPOP mutations and knockout in both PC-3 and HeLa cells (Supplemental Figure 3, C–J).
Figure 2SPOP mutation impairs LMNB2 protein levels. (A) IB analysis of whole-cell lysates (WCLs) derived from 293T cells with indicated plasmids. DMSO or 10 μM MG132 was added for 12 hours before harvest. (B and C) IB analysis (B) and quantification (C) of LMNB2 protein in WCLs from 293T cells transfected with or without SPOP after cycloheximide treatment. Data are shown as the mean ± SD of 3 biological replicates (n = 3). (D–G) IB analysis of WCL derived from PC-3 cells infected with lentivirus expressing WT, F102C or F133V SPOP (D) and PC-3 control and SPOP knockout cells (F). The LMNB2 RNA levels are shown in (E) and (G). Data are shown as the mean ± SD of 3 independent experiments (n = 3). (H and I) PC-3 cells infected with lentivirus expressing HA-WT, F102C, or F133V SPOP were subjected to IF. Representative images are shown in (H) and quantification in (I). Scale bar: 10 μm. Data are reported as the mean ± SD of 10 fields (>200 cells; n = 10) from 3 biological replicates. (J and K) PC-3 control and SPOP knockout cells were subjected to IF; representative images are shown in (J) and quantification in (K). Scale bar: 10 μm. Data are reported as the mean ± SD of 10 fields (>200 cells; n = 10) of 3 biological replicates. (L and M) Representative images of IHC staining (L) of LMNB2 antibodies on prostate cancer specimens from SPOP WT (n = 80) and SPOP MUT (n = 20) groups. The distribution of LMNB2 IHC levels is shown in (M). Scale bar: 200 μm in ×10 fields; 40 μm in ×40 fields. **P < 0.01 and ***P < 0.001 by 2-way ANOVA followed by Tukey’s multiple comparisons test (C) or 1-way ANOVA followed by Dunnett’s multiple comparisons test (E, G, and I) or 2-tailed unpaired Student’s t test (K) or Fisher’s exact test (M).
To further assess the impact of SPOP mutations on LMNB2 protein levels in patient specimens, we analyzed 100 primary prostate tumors from our cohort (Supplemental Table 1). Sanger sequencing identified 20 tumors with SPOP mutations. IHC revealed that 85% of SPOP-mutated tumors showed weak LMNB2 staining. In contrast, only 30% of SPOP-WT tumors had weak LMNB2 staining, with the majority (70%) exhibiting strong or intermediate staining (Figure 2, L and M). These findings suggested that LMNB2 protein levels were impaired in SPOP-mutated prostate cancer specimens.
SPOP maintains LMNB2 protein level by promoting its mono-ubiquitination at lysine-484. To further investigate how SPOP influences LMNB2 protein level, we examined whether LMNB2 is a ubiquitinating substrate of SPOP. Intriguingly, although SPOP knockout reduced total LMNB2 half-life (Supplemental Figure 4, A and B), we found that increased expression of SPOP did not induce the smeared pattern typical of polyubiquitination of LMNB2 but instead led to a prominent increase in a discreet band consistent with mono-ubiquitin addition (Figure 3A). Knockout of endogenous SPOP by CRISPR-Cas9 greatly attenuated both exogenous and endogenous LMNB2 mono-ubiquitination in HEK293T cells, and this effect was reversed by restored expression of SPOP (Figure 3B and Supplemental Figure 4C). Moreover, deletion of the SBC1 region of LMNB2 also inhibited its mono-ubiquitination, indicating that SPOP binding was necessary for LMNB2 mono-ubiquitination (Supplemental Figure 4D). To determine which lysine residues of LMNB2 are ubiquitinated by SPOP, we performed mass spectrometry on HEK293T cells transfected with Flag-tagged LMNB2, Myc-tagged SPOP, and HA-tagged ubiquitin plasmids. Ubiquitination at lysine residues 170, 484, and 549 in LMNB2 was detected by mass spectrometry (Figure 3, C–E). Mutagenesis analysis showed that mutation of Lys484 abolished SPOP-dependent LMNB2 mono-ubiquitination and reduced the half-life of LMNB2 in HEK293T cells (Figure 3, F–H). These findings indicated that SPOP maintained LMNB2 protein levels by promoting its mono-ubiquitination at Lys484.
Figure 3SPOP maintains LMNB2 protein level by promoting its mono-ubiquitination at lysine-484. (A) Co-IP analysis of indicated proteins in 293T cells transfected with increased Myc-SPOP WT in combination with Flag-LMNB2 and HA-Ub. (B) Co-IP analysis of indicated proteins in control or SPOP knockout 293T cells transfected with the indicated plasmids. (C–E) Mass spectrometry analysis revealed LMNB2 ubiquitination at lysine residues 170(C), 484 (D), and 549 (E). (F) Co-IP analysis of indicated proteins in 293T cells transfected with Flag-WT or mutated LMNB2 in combination with other constructs. (G and H) IB analysis (G) and quantification (H) of Flag-LMNB2 protein in whole-cell lysates from 293T cells transfected with Flag-WT or K484R LMNB2 after cycloheximide treatment. Data are reported as the mean ± SD of 3 biological replicates (n = 3). (H) Statistical comparisons were performed using 2-way ANOVA followed by Tukey’s multiple comparisons test. *P < 0.05, **P < 0.01, and ***P < 0.001.
Lys484 mono-ubiquitination stabilizes LMNB2 by antagonizing WDR26-mediated degradation. Our finding that SPOP promoted mono-ubiquitination and prevented LMNB2 degradation (Figure 3) led us to hypothesize that SPOP stabilized LMNB2 by counteracting K48-linked, ubiquitination-dependent degradation mediated by another E3 ligase. Previous studies have identified WDR26 as a core subunit of the GID ubiquitin ligase complex that regulates the polyubiquitination and degradation of LMNB2 in zebrafish and mouse erythroblasts (29). We confirmed that WDR26-mediated LMNB2 degradation was blocked by the proteasome inhibitor MG132 in HEK293T cells (Figure 4A), suggesting that WDR26 facilitated proteasomal degradation of LMNB2. Notably, although the WT LMNB2 bound to WDR26, the K484R mutation, but not other mutations, enhanced both the exogenous and endogenous WDR26 binding (Figure 4B and Supplemental Figure 5A). Deletion of the Lamin-tail (LTD) domain, which includes Lys484, abolished LMNB2-WDR26 binding, indicating that mono-ubiquitination of LMNB2 at Lys484 blocked its interaction with WDR26 (Figure 4C). Indeed, increased SPOP expression reduced LMNB2 binding to WDR26 and decreased K48-linked polyubiquitination of LMNB2 (Figure 4, D and E). Conversely, SPOP knockout increased LMNB2-WDR26 binding and K48-linked polyubiquitination, which was reversed by the expression of WT SPOP but not the F133V mutant (Figure 4F and Supplemental Figure 5, B and C). We conducted in vitro ubiquitination assays using E. coli–purified proteins and confirmed that SPOP mediates LMNB2 mono-ubiquitination. Notably, although WDR26 facilitated LMNB2 polyubiquitination, the presence of SPOP-driven mono-ubiquitination inhibited this process (Figure 4G). Consistently, the K484R mutant LMNB2 failed to rescue the nuclear size increase caused by LMNB2 knockdown (Figure 4, H and I, and Supplemental Figure 5, D–G). These findings suggested that SPOP-mediated mono-ubiquitination of LMNB2 at Lys484 inhibited WDR26 binding and degradation of LMNB2, thereby stabilizing LMNB2 protein levels and maintaining nuclear size.
Figure 4Lys484 mono-ubiquitination stabilizes LMNB2 by antagonizing WDR26-mediated degradation. (A) IB analysis of whole-cell lysates (WCLs) derived from 293T cells with indicated plasmids. DMSO or 10 μM MG132 was added for 12 hours before harvest. (B) Co-IP analysis of indicated proteins in 293T cells transfected with Flag-WT or mutated LMNB2 in combination with other constructs. (C) Co-IP analysis of indicated proteins in 293T cells transiently transfected with Flag-LMNB2 WT or ΔLTD. (D and E) Co-IP analysis of indicated proteins (D) and K48 ubiquitination (E) in 293T cells transfected with increased Myc SPOP in combination with indicated constructs. (F) Co-IP analysis of indicated proteins in 293T control and SPOP knockout cells transfected with indicated plasmids. (G) Ubiquitination of LMNB2 using E. coli–purified proteins in vitro. (H and I) PC-3 control and LMNB2 knockdown cells transfected with Flag-WT or K484R LMNB2 were subjected to IF; representative images are shown in (H) and quantification in (I). Scale bar: 10 μm. Data are reported as the mean ± SD of 3 biological replicates (n = 100). (I) Statistical comparisons were performed using 1-way ANOVA followed by Dunnett’s multiple comparisons test. ***P < 0.001.
SPOP mutation increases NE rupture risk upon farnesyltransferase inhibition. SPOP mutations in prostate cancer predominantly occur within the MATH domain, which is essential for substrate binding (37, 54) (Figure 5A). Using co-IP assays, we found that the SPOP ΔMATH mutant loses its ability to bind and monoubiquitinate LMNB2. In contrast, the ΔBTB mutant, which lacks CUL3 binding and is unable to ubiquitinate substrates, retained its binding ability to LMNB2 but failed to promote its mono-ubiquitination (Figure 5, B and C, and Supplemental Figure 6A). We further generated 5 prostate cancer–associated SPOP mutants, and co-IP assays revealed that all 5 mutants showed impaired binding to LMNB2 compared with WT SPOP (Figure 5D). These mutations also reduced SPOP-mediated mono-ubiquitination of LMNB2 (Figure 5E). IF assays in PC3 and HeLa cell lines visually confirmed the colocalization of LMNB2 with WT SPOP, but not with the mutant forms (Figure 5F and Supplemental Figure 6B). Moreover, whereas LMNB2 knockdown resulted in an increase in nuclear size, the expression of either WT or mutant SPOP showed no significant effect on nuclear size control in both PC-3 and HeLa cells (Figure 5, G and H, and Supplemental Figure 6, C–F). These results indicated that pathophysiological mutations in SPOP compromised its ability to monoubiquitinate LMNB2 and regulate nuclear size.
Figure 5SPOP mutation increases NE rupture risk upon farnesyltransferase inhibition. (A) Schematic of domain organization of SPOP and major SPOP mutations in prostate cancers. (B and C) Co-IP analysis of indicated proteins (B) and ubiquitination (C) in 293T cells transfected with Myc-WT, ΔMATH, or ΔBTB SPOP in combination with other constructs. (D and E) Co-IP analysis of indicated proteins (D) and ubiquitination (E) in 293T cells transfected with Myc-WT or mutant SPOP in combination with other constructs. (F) Representative images of IF of Flag LMNB2 and HA SPOP from PC-3 cells. Scale bar: 10 μm. (G and H) PC-3 control and LMNB2 knockdown cells infected with lentivirus expressing WT or F133V SPOP were subjected to LMNB2 IF; representative images are shown in (G) and quantification in (H). Scale bar: 10 μm. Data are reported as the mean ± SD of 3 biological replicates (n = 100). (I and J) PC-3 cells infected with lentivirus expressing HA-WT, F102C, or F133V SPOP were subjected to mAb414 IF. DMSO, 10 μM tipifarnib, or 5 μM lonafarnib was added for 24 hours before harvest. Representative images are shown in (I), quantification of the cell rupture ratio is shown in (J). Scale bar: 5 μm. Data are reported as the mean ± SD of 3 biological replicates (n = 200). ***P < 0.001 by 1-way ANOVA followed by Dunnett’s multiple comparisons test (H) or Fisher’s exact test (J).
Given that LMNB2 is crucial for maintaining NE integrity, we investigated the effects of SPOP mutations on the NE. We observed that the NE rupture rate was slightly higher in SPOP-mutant cells compared with SPOP WT cells. FTIs such as tipifarnib and lonafarnib are known to disrupt normal lamin maturation and cause abnormal lamin localization, which can weaken NE stability (25, 55, 56). To determine whether farnesyltransferase inhibition further affects NE rupture in SPOP-mutant cells, we treated these cells with the FTIs. Although FTI treatment did not affect NE rupture in SPOP WT cells compared with the control, SPOP-mutant cells exhibited a substantial increase in NE rupture (Figure 5, I and J, and Supplemental Figure 6, G and H). These findings suggested that SPOP-mutant cells were more vulnerable to NE rupture when farnesyltransferase was inhibited.
SPOP-mutant cells are hypersensitive to farnesyltransferase inhibition. Because SPOP-mutant cells showed an increase in NE rupture upon farnesyltransferase inhibition (Figure 5, I and J, and Supplemental Figure 6, G and H), we hypothesized that these cells are hypersensitive to FTIs due to NE rupture. To test this, we evaluated the viability of SPOP mutant–expressing PC-3, C4-2, and HeLa cells treated with 2 FTIs, tipifarnib, and lonafarnib. A dose-response survival assay showed that the SPOP-mutant F133V resulted in a lower IC50 for both inhibitors compared with the empty vector control groups in PC-3, C4-2, and HeLa cells (Figure 6, A–D, and Supplemental Figure 7, A and B). FTI treatment also inhibited the growth of SPOP-mutant cells, leading to fewer and smaller colonies, whereas control cells had only a slight reduction in colony size and number (Figure 6, E–H, and Supplemental Figure 7, C and D). S
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