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. 2016 May 28;5(2):13.
doi: 10.3390/microarrays5020013.

Small-Molecule Inhibition of Rho/MKL/SRF Transcription in Prostate Cancer Cells: Modulation of Cell Cycle, ER Stress, and Metastasis Gene Networks

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Free PMC article

Small-Molecule Inhibition of Rho/MKL/SRF Transcription in Prostate Cancer Cells: Modulation of Cell Cycle, ER Stress, and Metastasis Gene Networks

Chris R Evelyn et al. Microarrays (Basel). .
Free PMC article

Abstract

Metastasis is the major cause of cancer deaths and control of gene transcription has emerged as a critical contributing factor. RhoA- and RhoC-induced gene transcription via the actin-regulated transcriptional co-activator megakaryocytic leukemia (MKL) and serum response factor (SRF) drive metastasis in breast cancer and melanoma. We recently identified a compound, CCG-1423, which blocks Rho/MKL/SRF-mediated transcription and inhibits PC-3 prostate cancer cell invasion. Here, we undertook a genome-wide expression study in PC-3 cells to explore the mechanism and function of this compound. There was significant overlap in the genes modulated by CCG-1423 and Latrunculin B (Lat B), which blocks the Rho/MKL/SRF pathway by preventing actin polymerization. In contrast, the general transcription inhibitor 5,6-dichloro-1-β-d-ribofuranosyl-1H-benzimidazole (DRB) showed a markedly different pattern. Effects of CCG-1423 and Lat B on gene expression correlated with literature studies of MKL knock-down. Gene sets involved in DNA synthesis and repair, G1/S transition, and apoptosis were modulated by CCG-1423. It also upregulated genes involved in endoplasmic reticulum stress. Targets of the known Rho target transcription factor family E2F and genes related to melanoma progression and metastasis were strongly suppressed by CCG-1423. These results confirm the ability of our compound to inhibit expression of numerous Rho/MKL-dependent genes and show effects on stress pathways as well. This suggests a novel approach to targeting aggressive cancers and metastasis.

Keywords: Rho; cell cycle; metastasis; transcription.

Figures

Figure 1
Figure 1
Microarray analysis of regulated genes at 24 h. PC-3 cells were treated with 3 µM of CCG-1423, 0.5 µM of Lat B, and 50 µM of DRB under serum-starved conditions (0.5% FBS) for 24 h and gene expression assessed with an Affymetrix gene chip. (A) The Venn diagram shows the number of genes regulated (stimulated or inhibited) by CCG-1423, Lat B, and DRB with ≥2-fold change. The percentages of genes coordinately regulated by CCG-1423 and Lat B, or Lat B, and DRB are indicated; (B) A principal components analysis of the differentially regulated genes shows distinct patterns for Lat B and CCG-1423 vs. DRB.
Figure 2
Figure 2
Magnitude of expression changes observed with compounds compared to effects of MKL/MTRF suppression. Gene sets and the fold-change (log2) were compiled from our microarray data sets and from the data of Medjkane et al. [22]. For the overlapping genes, the magnitude of the change by CCG-1423 is plotted relative to the magnitude of the change induced by combined shRNA suppression of MRTF-A and MRTF-B (also referred to as MKL-1 and MKL-2). Linear regression analysis was performed to provide an indication of the extent to which the trends were similar for the two different interventions. Numbers in lower left quadrant are mean ± SD of slope and that in the lower right quadrant is the p value when significant. (AC) Comparison of compound addition on gene expression to the fold change observed in MDA-MB-231 breast carcinoma cells with shRNA against MRTF-A and MRTF-B; (DF) Same analysis but compared to B16F2 melanoma cells with shRNA against MRTF-A and MRTF-B.
Figure 3
Figure 3
Time-course of CCG-1423 effect on PC-3 cell gene expression. PC-3 cells were treated with 3 µM of CCG-1423 under serum-starved (0.5% FBS) conditions for 1, 2, 6, 12, and 24 h. Four genes: RGS4 (A); RGS7 (B); SOX9 (C); and CTGF (D), identified as metastasis candidates, were tested using qRT-PCR as described in the Materials and Methods. (AD) Data represent mean ± SEM fold-change values (over DMSO Control) for three separate experiments.
Figure 4
Figure 4
Concept map of gene expression changes in PC-3 cells induced by three treatments. KEGG pathways, defined by MSigDB (Broad Institute version 3.0), were examined using Gene set enrichment analysis (GSEA) and the entire microarray data set. The top 25 KEGG pathways significantly associated with changes induced by CCG-1423 (3 µM) are shown along with the related changes induced by Lat B (0.5 µM) and DRB (30 µM). All three compounds produced significant effects on genes involved in purine and pyrimidine metabolism while CCG-1423 and Lat B had more selective effects on cell cycle and DNA replication. Colors represent Z-scores with blue indicating that genes of the set were downregulated and red indicating that genes of the set were up-regulated. The darkness represents the level of significance. White means that the adjusted p value is >0.05. One of the 24-h Lat B samples was lost during processing for the microarray so there are only two replicates.
Figure 5
Figure 5
qRT-PCR confirmation of ER Stress-related gene expression. PC-3 cells were treated with 3 µM CCG-1423 for the indicated times. RNA was isolated and qRT-PCR performed as described. Expression of (A) ATF3, (B) ATF4, and (C) CHOP (also referred to as DDIT3) is expressed as the fold change compared to the DMSO control samples (24-h with DMSO). All expression levels were normalized relative to the expression of GAPDH.

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