Opposing effects of cancer-type-specific SPOP mutants on BET protein degradation and sensitivity to BET inhibitors

Abstract

It is generally assumed that recurrent mutations within a given cancer driver gene elicit similar drug responses. Cancer genome studies have identified recurrent but divergent missense mutations affecting the substrate-recognition domain of the ubiquitin ligase adaptor SPOP in endometrial and prostate cancers. The therapeutic implications of these mutations remain incompletely understood. Here we analyzed changes in the ubiquitin landscape induced by endometrial cancer-associated SPOP mutations and identified BRD2, BRD3 and BRD4 proteins (BETs) as SPOP-CUL3 substrates that are preferentially degraded by endometrial cancer-associated SPOP mutants. The resulting reduction of BET protein levels sensitized cancer cells to BET inhibitors. Conversely, prostate cancer-specific SPOP mutations resulted in impaired degradation of BETs, promoting their resistance to pharmacologic inhibition. These results uncover an oncogenomics paradox, whereby mutations mapping to the same domain evoke opposing drug susceptibilities. Specifically, we provide a molecular rationale for the use of BET inhibitors to treat patients with endometrial but not prostate cancer who harbor SPOP mutations.

Figure 1. Endometrial and prostate cancer SPOP mutants induce opposing effects on BET protein levels.

(a) Outer surface of the SPOP substrate recognition domain with recurrently mutated amino acid residues highlighted in blue for prostate cancer and red for endometrial cancer, respectively. Substrate in green in the substrate binding cleft. (b) Scatter plot of protein expression changes of SPOP mutants (MTs) vs. SPOP wild type (WT) in Ishikawa endometrial cancer cells, dotted red line = 2 s.d. (c) Representative Western blot (WB) validation for indicated proteins in Ishikawa cells stably expressing vector control, SPOP-WT, or endometrial cancer SPOP-MTs (n=5). (d) Representative WB for indicated proteins in Ishikawa endometrial cancer cells expressing prostate cancer SPOP-MTs (n=3). (e) Representative WB for indicated proteins in 22Rv1 prostate cancer cells stably expressing prostate cancer SPOP-MTs (n=3). (f) Representative images of primary human endometrial cancer tissues stained for BRD2, BRD3 and BRD4 with corresponding expression analysis on primary tumors stratified accordingly SPOP mutation status. Scale bars, 20µm. (g) BRD2, BRD3 and BRD4 expression analysis of primary human prostate cancer tissues stratified accordingly SPOP mutation status (R correlation coefficient and p values are derived from Kendall’s tau-b). N indicates the number of independent experiments performed.

Figure 2. BET proteins are bona fide substrates of wild type SPOP.

(a) Schema of BET proteins with bromodomain 1 and 2 (BD1 and BD2), extraterminal (ET) domain, and C-terminal domain (CTD), ubiquitylated lysines (K-ɛ-GG) detected by mass-spectrometry, and SPOP degron motif. (b) Effect of transient SPOP-WT overexpression on protein levels of HA-BRD3-WT and HA-BRD3-Degron-MT assessed by WB in Ishikawa cells (n=3). (c) Interaction between SPOP-WT and BRD3-WT or HA-BRD3-Degron-MT. HA-immunoprecipitation (IP) and whole cell extract (WCE) of transiently transfected 293T cells (n=3). (d) In vivo ubiquitylation of HA-BRD3-WT and HA-BRD3-Degron-MT by SPOP-WT. 293T cells transfected with 8xHis-Ubiquitin (Ub) and indicated constructs followed by MG132 treatment. 8xHis-Ub pull down using nickel beads on lysed cells (n=3). (e) HA-BRD3 protein level by WB in Ishikawa cells transiently expressing SPOP-WT and HA-BRD3 with or without MG132 treatment (n=3). (f) Representative WB for indicated proteins upon knockdown of SPOP with shRNA (left) or siRNA (right) in Ishikawa cells (n=3). Representative WBs are shown. N indicates the number of independent experiments performed.

Figure 3. BET proteins are differentially ubiquitylated and degraded by endometrial and prostate SPOP mutants.

(a) Representative WB (n=4) of BET proteins and SPOP in Ishikawa and EN human endometrial cell lines. Statistical significance was determined by unpaired, two-tailed Student’s t-test (n.s., non-signicant). (b) Representative WB of indicated proteins in Ishikawa and EN cells with or without MG132 treatment (n=3). (c) Representative WB of indicated proteins after cycloheximide (CHX) treatment in Ishikawa and EN cells (n=3). (d) Interaction between HA-BRD3 and SPOP-WT, endometrial cancer mutants (SPOP-E50K, -R121Q), and one prostate cancer mutant (SPOP-W131G). HA-IP and WCE of transiently transfected 293T cells overexpressing HA-BRD3 and indicated SPOP constructs (n=3). (e) Effects of SPOP-WT and SPOP mutants on in vivo ubiquitylation of HA-BRD3 (n=3). (f) In vivo ubiquitylation of HA-BRD3-WT or HA-BRD3-Degron-MT by SPOP-E50K (n=3). Representative WBs are shown. N indicates the number of independent experiments performed.

Figure 4. Cancer-type specific SPOP-mutants alter BET inhibitor sensitivity in an opposing manner.

(a) Response to JQ1 in Ishikawa cells stably over-expressing endometrial (E47K, E50K, E78K, S80R, M117V, R121Q, D140N) and prostate cancer (Y87C, F102C, W131G, F133L) SPOP mutants in 3D semi-solid cell culture condition (n=3). P values are indicated above the compared bars (two-way ANOVA with Dunnett’s post test, DF= 112 (degrees of freedom)). (b) Correlation of IC₅₀ (JQ1) shown in Supplementary Fig. 7c with BET protein levels quantified by mass-spectrometry in Ishikawa cells stably expressing recurrent endometrial SPOP-MTs (r- and p value Spearman rank correlation). (c) Response to JQ1 (250nM) of Ishikawa cells stably overexpressing SPOP-E50K and different BET protein degron mutant constructs (Degron MT) (n=3). (d) Effect of single shRNA-mediated depletion of BRD2, BRD3 and BRD4 on JQ1 (200nM) sensitivity in Ishikawa-SPOP-Y87C cells (n=3). (e) JQ1 sensitivity of SPOP-WT Ishikawa, SPOP-R121Q-mutant EN human endometrial cancer cell lines; SPOP-E47K-mutant NCI-H508 human large intestine cancer cell line and SPOP-E50K-mutant VM-CUB1 human urothelial cancer cell line in 3D semi-solid culture (n=4). P values are indicated above the compared bars (two-way ANOVA with Dunnett’s post test, DF= 30). N indicates the number of independent experiments performed. All error bars, mean ± SEM. Statistical significance was determined by unpaired, two-tailed Student’s t-test unless otherwise specified (*P < 0.05, **P < 0.01, ***P < 0.001).

Figure 5. Downregulation of FOSL1 sensitizes to JQ1 treatment.

(a) Venn diagram depicting the overlap of significantly differentially expressed genes in Ishikawa cells stably expressing either endometrial (E47K, E50K) or prostate (Y87C, W131G) cancer SPOP MTs without or with JQ1. Overlay is significant p value < 0.05 (Benjamini-Hochberg test). (b) Heat map showing the fold change of the 16 genes included in the intersection of panel a. (c) FOSL1 mRNA (normalized to Cyclophilin) and protein levels of Ishikawa cells stably expressing SPOP-WT and either endometrial or prostate cancer SPOP MTs (n=4). (d) FOSL1 mRNA expression in endometrial and prostate– cancer patient datasets stratified accordingly to SPOP status. P value was derived from an unpaired t-test with Welch’s correction. (e) Representative images of human primary endometrial cancer tissues stained for FOSL1 with corresponding expression analysis on human primary tumors stratified accordingly to SPOP mutation status (p value Kendall’s tau-b). Scale bars, 80µm. (f) FOSL1 mRNA and protein expression levels after JQ1 (500nM) treatment in Ishikawa cells stably expressing SPOP-WT and either two endometrial (E47K, E50K) or two prostate (Y87C, W131G) cancer SPOP MTs (n=3). (g) Dose-response curves to JQ1 of Ishikawa-SPOP-Y87C cells upon FOSL1 knockdown (n=3). P value is indicated below the dose-response curves by extra-sum of squares F test. Corresponding WB validation of FOSL1 knockdown. N indicates the number of independent experiments performed. All error bars, mean ± SEM. Statistical significance was determined by unpaired, two-tailed Student’s t-test unless otherwise specified (n.s., non-signicant, *P < 0.05, **P < 0.01, ***P < 0.001).

Figure 6. Endometrial SPOP mutants sensitize to JQ1 treatment in vivo.

(a) Tumor growth kinetics and graph showing the individual tumor weight with (n=9) or without (n=7) JQ1 in xenografts established from EN. (b) Tumor growth kinetics and graph showing the individual tumor weight with (n=7) or without (n=7) JQ1 in xenografts established from Ishikawa. (c) Representative histology and quantification of mitotic and apoptotic cells in EN and Ishikawa xenografts treated either with vehicle or JQ1. (d) Tumor growth kinetics and graph showing the individual tumor weight with (n=7) or without (n=6) JQ1 in xenografts established from Ishikawa stably over-expressing SPOP-E50K. (e) Tumor growth kinetics and graph showing the individual tumor weight with (n=6) or without (n=7) JQ1 treatment in xenografts established from Ishikawa stably over-expressing SPOP-S80R. Representative images of tumors for each xenograft group are shown. Mean tumor volume + SEM is shown. Statistical significance was determined by unpaired, two-tailed Student’s t-test (*P < 0.05, **P < 0.01, ***P < 0.001). (f) Model showing the differential effect of cancer-specific SPOP mutations on both BET protein levels and sensitivity to BET inhibitors.