Inhibition of the Sterol Regulatory Element Binding Protein SREBF-1 Overcomes Docetaxel Resistance in Advanced Prostate Cancer

Abstract

Resistance to antiandrogens and chemotherapy (Cx) limits therapeutic options for patients with metastatic hormone-sensitive (mHSPC) and metastatic castration-resistant (mCRPC) prostate cancer. In this context, up-regulation of the glucocorticoid receptor is identified as a potential bypass mechanism in mCRPC. A combination of docetaxel and mifepristone (Doc + RU-486), an inhibitor of the glucocorticoid receptor, re-sensitizes docetaxel-resistant cell models to Cx. This study was designed to elucidate the molecular mechanisms responsible for this phenomenon. RNA sequencing was performed in docetaxel-resistant prostate cancer cell models after Doc + RU-486 treatment with consecutive functional assays. Expression of selected proteins was verified in prostatic tissue from prostate cancer patients with progressive disease. Treatment with Doc + RU-486 significantly reduced cancer cell viability, and RNA sequencing revealed sterol regulatory element of binding transcription factor 1 (SREBF-1), a transcription factor of cholesterol and lipid biosynthesis, as a significantly down-regulated target. Functional assays confirmed that SREBF-1 down-regulation is partially responsible for this observation. In concordance, SREBF-1 knockdown and pharmacologic sterol regulatory element binding protein inhibition, together with other key enzymes in the cholesterol pathway, showed similar results. Furthermore, SREBF-1 expression is significantly elevated in advanced prostate cancer tissues, showing its potential involvement in tumor progression and emerging therapy resistance. Therefore, specific inhibition of cholesterol and lipid biosynthesis might also target Cx-resistant cancer cells and represents a potential additive future therapeutic option to improve mCRPC therapy.

Figure 1

A: Significantly reduced cell viability in the docetaxel (Doc)-resistant cell models PC3-DR, DU145-DR, and CWR22Rv1-DR after 3 days of treatment with RU-486 (10 μmol/L) in combination with Doc (12.5 nmol/L) compared with single treatment of Doc (12.5 nmol/L) and RU-486 (10 μmol/L). B: Flow cytometry analysis shows increased apoptosis after combination treatment in all three Doc-resistant cell lines. C: Microscopic images following the respective treatment after 3 days. Data are expressed as means ± SEM from at least three independent experiments. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001. Scale bars = 200 μm. Original magnification, ×4.

Figure 2

A: Measurement of cell viability after treatment with different concentrations of docetaxel (Doc) in all three cell lines. B: Western blot analysis of apoptotic and antiapoptotic cell cycle proteins in each cell line. C: Analysis of the apoptosis marker cPARP. All experiments have been performed at least three times. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Figure 3

A: Results from RNA sequencing analyses [z score of log2 (normalized_counts+1)] represented as heatmaps after treatment with dimethyl sulfoxide (DMSO), docetaxel (Doc; 12.5 nmol/L), RU-486 (10 μmol/L), and the combination of Doc (12.5 nmol/L) and RU-486 (10 μmol/L) in PC3-DR and DU145-DR of significantly differentially expressed genes between RU-486 + Doc versus DMSO [more than twofold change; false discovery rate (FDR) <0.1] (left). Log2-fold changes of top 30 up- and down-regulated genes are indicated as heatmap (right). Venn diagram (below) showing overlap of significantly up-regulated and down-regulated genes of RU-486 + Doc versus DMSO treatment between PC3-DR and DU145-DR. B: Gene set enrichment analyses show a significantly elevated down-regulation of cholesterol homeostasis signature [normalized enrichment score (NES); FDR]. C and D: SREBF-1 was among the most significantly down-regulated genes with a significantly decreased mRNA expression in both cell lines (C) and significantly reduced protein levels in Western blot analysis confirming the results from the RNA sequencing data (D). ∗P < 0.05; ∗∗∗P < 0.001.

Figure 4

A: Significantly increased expression of sterol regulatory element of binding transcription factor 1 (SREBF-1) in 40 macro-dissected primary patients with prostate cancer (PCa) compared with tissue from 40 benign prostate samples as well as significantly increased expression of SREBF1 of 949 patients with PCa and 50 PCa metastasis (Mets) samples compared with 269 benign tissue samples analyzed in public transcriptome data set. B: Representative microscopy images of negative and high expression of SREBF-1 in the overall cell compartment, the cytoplasm, and the nucleus as well as the corresponding statistical analysis between benign tissue and PCa tissue. In addition, patients with PCa were dichotomized into metastatic hormone-sensitive prostate cancer (mHSPC) and metastatic castration-resistant prostate cancer (mCRPC) groups in which SREBF-1 was more intensely expressed in mHSPC than mCRPC. However, this was not statistically significant. C: SREBF-1 significantly correlates with androgen receptor (AR) activity within the publicly available TCGA-PRAD (547 samples) data set. In the SU2C-PRAD data sets, AR and glucocorticoid (GR) activities were significantly positively correlated with SREBF1. Primary PCa is defined as mostly untreated PCa, whereas the SU2C database consists of heavily pretreated castration-resistant (CRPC) and metastatic PCa (mCRPC). A: unpaired t-test, ∗P < 0.05, ∗∗∗P < 0.001, box whisker plot with 10 to 90 percentile. B: ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001. Scale bars = 100 μm. Original magnification: ×20 (main images); ×40 (insets) (B). BPH, benign prostatic hyperplasia; TCGA, The Cancer Genome Atlas.

Figure 5

Confirmation of functional inhibition of the target gene SREBF-1 with siRNA knockdown in PC3-DR and DU145-DR. A and B: Viability assay (A) and flow cytometry analysis (B) show increased apoptosis after siRNA knockdown of the target gene SREBF-1. C: Diagram of sterol regulatory element binding proteins (SREBPs) and their involvement in the cellular processes of cancer as well as point of action for fatostatin and simvastatin. Simavastatin and fatostatin and their primary target of inhibition are marked in red. D: Viability assays with pharmacologic inhibition of fatostatin and simvastatin show decreased viability with increasing concentrations of fatostatin and simvastatin. ∗P < 0.05; ∗∗∗P < 0.001. CoA, coenzyme-A; HMGCoA, 3-hydroxy-3-methylglutaryl-coenzyme-A; mTOR, mammalian target of rapamycin; SREBF-1, sterol regulatory element of binding transcription factor 1.