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. 2024 Apr 17;15(4):274.
doi: 10.1038/s41419-024-06647-0.

ELK3 destabilization by speckle-type POZ protein suppresses prostate cancer progression and docetaxel resistance

Cheol-Jung Lee  1   2 , Heejung Lee  3 , Seo Ree Kim  4 , Soo-Bin Nam  1   2 , Ga-Eun Lee  1 , Kyeong Eun Yang  2 , Guk Jin Lee  4 , Sang Hoon Chun  4 , Han Chang Kang  1   5 , Joo Young Lee  1   5 , Hye Suk Lee  1   5 , Sung-Jun Cho  6 , Yong-Yeon Cho  7   8
Affiliations

ELK3 destabilization by speckle-type POZ protein suppresses prostate cancer progression and docetaxel resistance

Cheol-Jung Lee et al. Cell Death Dis. .

Abstract

Accumulating evidence demonstrates that the activity regulation of ELK3, a member of the E26 transformation-specific oncogene family, is critical to regulating cell proliferation, migration, and survival in human cancers. However, the molecular mechanisms of how ELK3 induces chemoresistance in prostate cancer (PCa) have not been elucidated. In this study, we found that SPOP and ELK3 are an interacting partner. The interaction between SPOP and ELK3 resulted in increased ELK3 ubiquitination and destruction, assisted by checkpoint kinase-mediated ELK3 phosphorylation. Notably, the modulation of SPOP-mediated ELK3 protein stability affected the c-Fos-induced cell proliferation and invasion of PCa cells. The clinical involvement of the SPOP-ELK3 axis in PCa development was confirmed by an immunohistochemical assay on 123 PCa tissues, with an inverse correlation between increased ELK3 and decreased SPOP being present in ~80% of the specimens. This observation was supported by immunohistochemistry analysis using a SPOP-mutant PCa specimen. Finally, docetaxel treatment induced cell death by activating checkpoint kinase- and SPOP-mediated ELK3 degradation, while SPOP-depleted or SPOP-mutated PCa cells showed cell death resistance. Notably, this observation was correlated with the protein levels of ELK3. Taken together, our study reveals the precise mechanism of SPOP-mediated degradation of ELK3 and provides evidence that SPOP mutations contribute to docetaxel resistance in PCa.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. ELK3 stability was regulated by Cul3-mediated proteasome pathway.
a–c Determination of ELK3 degradation pathways. The cell lysates extracted from HeLa cells treated with MG132 (a), chloroquine (b), or MLN4924 (c) were used to visualize ELK3 by WB. d ELK3 interaction with Cullin3. The cell lysates extracted from HEK293T cells transfected with indicated plasmids and treated with MG132 (10 μM) for 4 h were used to evaluate the interaction between ELK3 and each of Cullins by IP and WB. e Cullin3 decreases ELK3 half-life. The cell lysates extracted from HEK293T cells transfected with indicated plasmids and treated with cycloheximide (CHX, 10 μg/ml) were used to evaluate the half-life of ELK3 by WB. f Knockdown of Cullin3 increases ELK3. The cell lysates extracted from HeLa cells stably expressing sh-mock or sh-CUL3 were used to evaluate the ELK3 protein levels by WB. g Cullin3 knockdown inhibits ELK3 destabilization. The cell lysates extracted from PC-3 cells stably expressing sh-mock or sh-Cul3 and treated with CHX (10 μg/ml) were used to evaluate the ELK3 half-life by WB. h Cullin3 induces ELK3 ubiquitination. The cell lysates extracted from HEK293T cells transfected with indicated plasmids and treated with MG132 (10 μM) for 4 h were used to evaluate the ELK3 ubiquitination by IP and WB. Graphs in e and g The band intensity ELK3 bands measured using NIH image J computer program and normalized by β-actin intensity were plotted. Data, three independent experiments; Significance, *p < 0.05 obtained by Student t test.
Fig. 2
Fig. 2. SPOP interacts with ELK3 for its ubiquitination and degradation.
a, b ELK3 interacts with SPOP. The cell lysates extracted from HEK293T cells transiently transfected with indicated plasmids and treated with MG132 (10 μM) for 4 h were used to determine ELK3 interaction to E3 ligases by IP and WB. c Evaluation of endogenous interaction between ELK3 and SPOP. The cell lysates extracted from HeLa cells treated with MG132 (10 μM) for 8 h were used to evaluate the ELK3 and SPOP interaction by IP and WB. d Confirmation of ELK3 and SPOP interaction. The cell lysates extracted from HEK293T cells transiently transfected with His-ELK3 plasmid were used to confirm the ELK3 and partially purified bacterial His-SPOP by pulldown assay (PD) and WB. e SPOP induces ELK3 destabilization. The cell lysates extracted from HEK293T cells transiently transfected with His-ELK3 and dose-increasing Myc-SPOP plasmids were used to evaluate ELK3 protein level by WB. GFP was used as an internal control for equal transfection. f SPOP accelerates ELK3 destabilization. The cell lysates extracted from HEK293T cells transiently transfected with indicated plasmids and treated with CHX (10 μg/ml) were used to evaluate the ELK3 half-life by WB. g SPOP knockdown increases ELK3 protein levels. The cell lysates extracted from HeLa cells stably expressing sh-mock or sh-SPOP were used to evaluate the ELK3 protein levels by WB. h Depletion of SPOP did not affect ELK3 RNA expression. The levels of ELK3 mRNA extracted from HeLa cells stably expressing sh-mock or sh-SPOP were assessed by quantitative real-time RT-PCR using TaqMan RNA-to-CT 1-step kit. i SPOP knockdown prolongs ELK3 half-life. The cell lysates extracted from PC-3 cells stably expressing sh-mock or sh-SPOP and treated with CHX (10 μg/ml) were used to evaluate ELK3 half-life by WB. j, k SPOP induces ELK3 ubiquitination in cell and in vitro ubiquitination assay system. The cell lysates extracted from HEK293T cells transiently transfected with indicated plasmids and treated with MG132 (10 μM) for 4 h were used to evaluate ELK3 ubiquitination by IP and WB (j). In vitro ubiquitination assay was conducted using commercially available E1, E2, and ubiquitin (Ubi) proteins, along with His-tagged ELK3 purified from E. coli and Myc-tagged SPOP isolated from HEK293T cells overexpressing SPOP (k). Graphs in f and i The band intensity of ELK3 quantified by NIH image J computer program normalized by β-actin intensity was plotted. Data, three independent experiments; Significance, *p < 0.05 obtained by Student t test.
Fig. 3
Fig. 3. Cancer-derived SPOP mutations fail to promote ELK3 degradation.
a ELK3 interacted with MATH domain of SPOP. Upper panel Schematic diagram of SPOP domains. The numbers at the MATH domain indicates SPOP mutations observed in PCa patients. Bottom panels The cell lysates of HEK293T cells transiently transfected with indicated plasmids and treated MG132 (10 μM) for 4 h were used to determine the SPOP’s binding domain by IP and WB. b MATH domain deletion of SPOP suppressed ELK3 ubiquitination. The cell lysates of HEK293T cells transiently transfected with indicated plasmids and treated MG132 (10 μM) for 4 h were used to evaluate the SPOP-dependent ELK3 ubiquitination by IP and WB. c The deletion of the MATH or BTB domain of SPOP abolishes ELK3 destabilization. The cell lysates of HEK293T cells transiently transfected with indicated plasmids were used to evaluate ELK3 protein levels by WB. GFP was used as an internal control for equal transfection. d MATH domain mutation of SPOP abrogates interaction with ELK3. The cell lysates of HEK293T cells transiently transfected with the indicated plasmids and treated with MG132 (10 μM) for 4 h were used to evaluate the interaction between SPOP and ELK3 by IP and WB. e MATH-F133V mutation of SPOP critically affects ELK3 stability. The cell lysates of HEK293T cells transiently transfected with indicated plasmids and treated with CHX (10 μg/ml) for indicated period were used to evaluate ELK3 protein levels by WB. The band intensity of ELK3 quantified by NIH image J computer program normalized by β-actin intensity was plotted. GFP was used an internal control for equal transfection. Data, three independent experiments; Significance, *p < 0.05 obtained by Student t test. f SPOP mutations at MATH domain inhibits endogenous ELK3 destabilization. The cell lysates of 22Rv1 cells stably expressing SPOP-wt or each of SPOP mutants (Y87C, W131G, and F133V) were used to evaluate ELK3 protein levels by WB. g SPOP mutations at MATH domain abolishes ELK3 ubiquitination. The cell lysates of HEK293T cells transiently transfected with indicated plasmids and treated with MG132 (10 μM) for 4 h were used to evaluate ELK3 ubiquitination by IP and WB.
Fig. 4
Fig. 4. Determination of ELK3 degron motif for SPOP.
a Identification of ELK3 degron motifs for SPOP. ELK3 amino acid sequences were aligned to search putative degron motifs for SPOP. b Determination of ELK3 degron motif interacting to SPOP. The cell lysates of HEK293T cells transiently transfected with indicated plasmids and treated with MG132 (10 μM) for 4 h were used to evaluate the ELK3 degron motif by IP and WB. c Involvement of ELK3 Deg1 in its stabilization. The cell lysates of HEK293T cells transiently transfected with indicated plasmids were used to determine the ELK3 degron motif by WB. GFP was used an internal control for equal transfection. d ELK3 Deg1 deletion abrogates SPOP-mediated destabilization. Left panels The cell lysates of HEK293T cells transiently transfected with indicated plasmids and treated with CHX (10 μg/ml) for an indicated time were used to evaluate the ELK3 protein levels by WB. GFP was used as an internal control for equal transfection. Graph The band intensity of ELK3 quantified by NIH image J computer program normalized by β-actin intensity was plotted. GFP was used an internal control for equal transfection. Data, three independent experiments; Significance, *p < 0.05 obtained by Student t test. e ELK3 Deg1 deletion abolishes SPOP-mediated ubiquitination. The cell lysate of HEK293T cells transiently transfected with indicated plasmids and treated with MG132 (10 μM) for 4 h were used to evaluate SPOP-mediated ELK3 ubiquitination by IP and WB. f, g Protein–protein docking of ELK3 and SPOP. The SPOP structure (PDB ID: 3HQI) and ELK3 built by amino acid similarly modeling using Discovery Studio were used to predict SPOP and ELK3 interaction interface. Magenta, ELK3; Green, SPOP. The amino acids and interaction types involved in interaction between ELK3 and SPOP are provided in Supplementary Fig. S1.
Fig. 5
Fig. 5. CHK1/2-mediated ELK3 phosphorylation facilitates ELK3 destruction.
a Dephosphorylation of ELK3 prevent the interaction between ELK3 and SPOP. The cell lysates of HEK293T cells transiently transfected with indicated plasmids were treated with/without λ-phosphatase. The interaction between ELK3 and SPOP was evaluated by IP and WB. b CHK1/2 inhibition suppresses ELK3 destabilization. The cell lysates of HeLa cells treated with 5 μM of indicated inhibitor and CHX (10 μg/ml) for 12 h were used to evaluate the ELK3 protein levels by WB. c CHK1/2 inhibition abolishes ELK3 and SPOP interaction. The cell lysates of HEK293T cells transfected with indicated plasmids treated with 5 μM of indicated inhibitor for 12 h and MG132 (10 μM) for 4 h were used to evaluate the interaction between ELK3 and SPOP by IP and WB. d ELK3 interacts with CHK1 and CHK2. The cell lysates of HEK293T cells transiently transfected with indicated plasmids and treated with MG132 (10 μM) for 4 h were used to evaluate the interaction between ELK3 and CHK1 or CHK2 by IP and WB. e CHK2 facilitates ELK3 ubiquitination by SPOP. cell lysates of HEK293T cells transiently transfected with indicated plasmids and treated with MG132 (10 μM) for 4 h were used to evaluate the ELK3 ubiquitination by IP and WB. f CHK1/2 inhibition abrogates SPOP-mediated ELK3 ubiquitination. The cell lysates of HEK293T cells transiently transfected with indicated plasmids and treated with 5 μM of AZD7762 for 12 h and MG132 (10 μM) for 4 h were used to evaluate the SPOP-mediated ELK3 ubiquitination by IP and WB. g CHK1 knockdown increases ELK3 protein levels. The cell lysates of HeLa cells stably expressing sh-mock or sh-CHK1 or CHK2 were used to evaluate ELK3 protein levels by WB. h CHK1 and CHK2 phosphorylates ELK3. In vitro kinase assay using partially purified His-ELK3 and active CHK1 or CHK2 was conducted. ELK3 phosphorylation by CHK1 or CHK2 was evaluated RxxS/T antibody by WB. i ELK3 deg1 deletion abolishes CHK1- or CHK2-mediated phosphorylation. In vitro kinase assay using partial purified His-ELK3-wt or -∆Deg1 and active CHK1 or CHK2 was conducted. ELK3 phosphorylation by CHK1 or CHK2 was evaluated RxxS/T antibody by WB. j, k CHK2 phosphorylates ELK3 at Ser133 in cell system. The phosphorylation of ELK3 mediated by CHK2 in cell culture (j) and in vitro kinase assay system (k) was evaluated using phos-tag immunoblot analysis. Treatment with AZD7762 (j) and mutation of ELK3 Ser133 to Ala (l) abolished the phosphorylation of ELK3 induced by CHK2. l ELK3 phosphorylation at Ser133 is indispensable to interact with SPOP. ELK3 phosphorylation requirement for the interaction with SPOP was evaluated by IP and western blotting using cell lysates transiently expressing mock, His-ELK3-wt, His-ELK3-S133D, or His-ELK3-S133A.
Fig. 6
Fig. 6. SPOP-mediated ELK3 degradation suppresses c-fos expression.
a, b SPOP inhibits ELK3-mediated c-fos promoter activity. HEK293T cells transiently transfected with indicated plasmids were used to evaluate c-fos promoter activity by luciferase assay. c SPOP knockdown increases c-fos promoter activity. HEK293T cells stably expressing sh-SPOP were transfected with c-fos-luc reporter plasmid and evaluated c-fos promoter activity by luciferase assay. d Confirmation of increased c-Fos protein by SPOP knockdown. The cell lysates of 22Rv1 cells stably expressing sh-SPOP were used to evaluate the c-Fos protein level by WB. e Establishment of ELK3 and SPOP double knockdown cells. The cell lysates of 22Rv1 cells stably expressing sh-SPOP and/or sh-ELK3 as indicated were used to evaluate the SPOP and c-Fos protein levels by WB. f Increased cell proliferation by SPOP knockdown suppresses cell proliferation by ELK3. The 22Rv1 cells in e were used to evaluate the effects of cell proliferation by CCK-8 assay. g Increased cell invasion by SPOP knockdown abrogated by ELK3 knockdown. The 22Rv1 cells in e were used to evaluate the effects of cell invasion by cell invasion assay using Matrigel Invasion Chamber. a–c and f, g Data, three independent experiments; Significance, *p < 0.05 obtained by Student t-test.
Fig. 7
Fig. 7. SPOP-ELK3 axis regulates docetaxel-induced cell death.
a Inverse correlation of ELK3 and phospho-CHK1/2 protein levels under docetaxel treatment. The cell lysates of 22Rv1 cells treated with docetaxel (300 nM) for an indicated time were used to evaluate the indicated protein levels by WB. b Rescue of docetaxel-mediated decreased ELK3 protein level by MG132 treatment. The 22Rv1 cell lysates treated with docetaxel (300 nM) in the presence or absence of MG132 (10 μM) were used to evaluate ELK3 protein levels by WB. c Docetaxel-mediated decreased ELK3 protein is rescued by SPOP knockdown. The cell lysates of 22Rv1 cells stably expressing sh-SPOP and treated with docetaxel (300 nM) were used to evaluate ELK3 protein level by WB. d Docetaxel induces SPOP and ELK3 interaction. The cell lysates of HEK293T cells transfected with indicated plasmids and treated with docetaxel (300 nM) were used to evaluate the SPOP and ELK3 interaction by IP and WB. e Docetaxel induces ELK3 ubiquitination. The cell lysates of HEK293T cells transfected with indicated plasmids and treated with docetaxel (300 nM) were used to evaluate ELK3 ubiquitination by IP and WB. f Diminished interaction between SPOP and ELK3 increases PCa cell survivability. 22Rv1 cells stably expressing SPOP-WT or each of SPOP mutants treated with docetaxel (1 μM) for 48 h were used to cell survivability by CCK-8 assay. g SPOP and ELK3 double knockdown inhibits cell survivability by docetaxel in SPOP knockdown cells. 22Rv1 cells stably expressing sh-SPOP and/or sh-ELK3 treated with docetaxel (1 μM) for 48 h were used to evaluate cell survivability by CCK-8 assay. h SPOP and ELK3 double knockdown enhances apoptosis signaling by docetaxel. The cell lysates of 22Rv1 cells stably expressing sh-SPOP and/or sh-ELK3 treated with docetaxel (300 nM) were used to evaluate the protein levels of apoptosis-related signaling molecules by WB. i, j The SPOP-ELK3 axis plays a critical role in docetaxel-induced cell death. The cell lysates of 22Rv1 cells stably expressing sh-SPOP and/or SPOP were used to evaluate the protein levels of ELK3 and SPOP by WB (i), and to assess cell viability using the CCK-8 assay (j). k Increased ELK3 protein levels in PCa cancer tissues. IHC images of ELK3 in prostate cancer tissues (n = 117) stained by DAB staining were analyzed by TissueFAXS system. Data, DAB intensity in normal vs. cancer tissues; Significance was obtained by Anova t test. l Increased ELK3 protein levels in PCa patient harboring SPOP-F133V mutation. Representative IHC image of ELK3 protein levels by DAB staining in prostate cancer tissues harboring SPOP-WT or SPOP-F133V. f, g Data, three independent experiments; Significance, *p < 0.05 obtained by Student t test.
Fig. 8
Fig. 8. Graphical summary of the proposed mechanism.
A schematic diagram illustrates the working model in which SPOP mutants enhance ELK3 stabilization, leading to increased resistance to docetaxel-induced cell death. Consequently, the SPOP-ELK3 axis plays a pivotal role in the progression of prostate cancer (PCa).
See this image and copyright information in PMC

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