JackedCarpenter33
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Abstract
Perturbations of the circadian clock are linked to multiple diseases, including cancers. Pharmacological activation of REV-ERB nuclear receptors, the core components of the circadian clock, has antitumor effects on various malignancies, while the impact of SR9009 on prostate cancer (PCa) remains unknown. Here, we found that SR9009 was specifically lethal to PCa cell lines but had no cytotoxic effect on prostate cells. SR9009 significantly inhibited colony formation, the cell cycle, and cell migration and promoted apoptosis in PCa cells. SR9009 treatment markedly inhibited prostate cancer subtype 1 (PCS1), the most lethal and aggressive PCa subtype, through FOXM1 pathway blockade, while it had no impacts on PCS2 and PCS3. Seven representative genes, including FOXM1, CENPA, CENPF, CDK1, CCNB1, CCNB2, and BIRC5, were identified as the shared genes involved in the FOXM1 pathway and PCS1. All of these genes were upregulated in PCa tissues, associated with worse clinicopathological outcomes and downregulated after SR9009 treatment. Nevertheless, knockdown or knockout of REV-ERB could not rescue the anticancer effect of SR9009 in PCa. Further analysis confirmed that it was LXRα rather than REV-ERBs which has been activated by SR9009. The expression levels of these seven genes were changed correspondingly after LXRα knockdown and SR9009 treatment. An in vivo study validated that SR9009 restrained tumor growth in 22RV1 xenograft models and inhibited FOXM1 and its targeted gene expression. In summary, SR9009 can serve as an effective treatment option for highly aggressive and lethal PCS1 tumors through mediating the LXRα/FOXM1 pathway independently of REV-ERBs.Results
SR9009 was specifically lethal to PCa cell lines
We assessed the impact of SR9009 on cell viability in three PCa cell lines and 1 normal prostate cell line. SR9009 markedly inhibited 22RV1, PC3 and DU145 cell viability in a dose-dependent manner but had no impact on RWPE-1 cells (Fig. 1A, Fig. S1A–D). We further showed that SR9009 significantly inhibited colony formation (Fig. 1B, C) and induced cell cycle arrest in PCa cells (Fig. 1D, E). SR9009 also promoted apoptosis in PCa cells compared with controls (Fig. 1F, G). Moreover, SR9009 impaired PCa cell migration in vitro in scratch and Transwell assays (Fig. 1H, I, Fig. S1D–E). These results indicated that SR9009 could inhibit PCa cell growth and migration but had no influence on normal prostate cells in vitro.Fig. 1: SR9009 is selectively lethal in PCa cells.
A CCK-8 assay of RWPE-1, PC3, 22RV1, and DU145 cell lines treated with different concentrations of SR9009 for 48 h; means ± SDs; unpaired t test. B, C Clone formation assay of PC3, 22RV1, and DU145 cell lines treated with DMSO or SR9009 (20 μM) for 7–14 days; n = 3; means ± SDs; unpaired t test. D, E Cell cycle tests of PC3, 22RV1, and DU145 cells treated with DMSO or SR9009 (20 μM) for 48 h; n = 3; means ± SDs; ANOVA. F, G Cell apoptosis tests of PC3, 22RV1, and DU145 cells treated with DMSO or SR9009 (20 μM) for 48 h; n = 3; means ± SDs; unpaired t test. H, I Transwell migration assays of PC3, 22RV1, and DU145 cells treated with DMSO or SR9009 (20 μM) for 48 h; n = 3; means ± SDs; unpaired t test. ns, not significant; p < 0.05; *p < 0.01; p < 0.001; *p < 0.0001.
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SR9009 inhibited the cell cycle pathway and suppressed lethal PCS1
A total of 559 upregulated and 553 downregulated genes were identified through RNA-seq (Fig. 2A, |Log2FC|> 2, adjusted Pvalue < 0.05). KEGG pathway enrichment analysis revealed that SR9009 significantly promoted ferroptosis and glycine, serine and threonine metabolism (Fig. 2B) and inhibited the cell cycle pathway (Fig. 2C). GSEA indicated that SR9009 mainly functioned by regulating the PCa cell cycle (Fig. 2D, E). A recent analysis classified PCa into three distinct subtypes, named the “PCS classification” [16], and this classification performed better in distinguishing luminal and basal PCa than the PAM50 classification [19]. Our subsequent GSEA found that SR9009 markedly inhibited PCS1, the most aggressive and lethal PCa subtype (enrichment score [ES] = −0.95, P < 0.0001), whereas it had no influence on PCS2 (ES = −0.30, P = 0.25) and PCS3 (ES = 0.30, P = 0.09) when compared with the control groups (Fig. 2F–H, Fig. S2A, B). The results indicated that SR9009 significantly reduced 71 of 82 genes in PCS1 (Fig. 2H). The sequencing results were validated at the mRNA and protein levels (Fig. 2I–K).Fig. 2: SR9009 downregulated the cell cycle pathway and suppressed lethal prostate cancer subtype 1 (PCS1).
A Volcano plot of differentially expressed genes between the SR9009 group (n = 3) and DMSO group (n = 3) after RNA sequencing. B, C Kyoto Encyclopedia of Genes and Genome (KEGG) pathway analysis of up- (B) and downregulated (C) pathways after SR9009 or DMSO was added; D, E Gene Set Enrichment Analysis (GSEA) analysis of the top 5 upregulated (D) and downregulated (E) pathways applying “c2.cp.v7.2.symbols.gmt [Curated]” from the MSigDB database. F GSEA of PCS1 after SR9009 or DMSO administration. G Cluster analysis of genes in PCS1, PCS2, and PCS3 between the SR9009 and DMSO groups. H Heatmap of genes in PCS1 after SR9009 or DMSO administration. I, J qPCR validations of genes downregulated in PCS1 in 22RV1 (I) and PC3 (J); n = 3; means ± SDs; ANOVA. K Western blot validation of SR9009 treatment on the expression of FOXM1 and RRM2 in PC3 and 22RV1 cells. ns, not significant; p < 0.05; *p < 0.01; p < 0.001; *p < 0.0001.
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