Targeting CDC25C, PLK1 and CHEK1 to overcome Docetaxel resistance induced by loss of LZTS1 in prostate cancer

Abstract

Docetaxel is used as a standard treatment in patients with metastatic castration-resistant prostate cancer. However, a large subset of patients develops resistance. Understanding resistance mechanisms, which are largely unknown, will allow identification of predictive biomarkers and therapeutic targets. We established resistant IGR-CaP1 prostate cancer cell lines for different doses of Docetaxel. We investigated gene expression profiles by microarray analyses in these cell lines and generated a signature of 99 highly differentially expressed genes potentially implicated in chemoresistance. We focused on the role of the cell cycle regulator LZTS1, which was under-expressed in the Docetaxel-resistant cell lines, its inhibition resulting from the promoter methylation. Knockdown of LZTS1 in parental cells with siRNA showed that LZTS1 plays a role in the acquisition of the resistant phenotype. Furthermore, we observed that targeting CDC25C, a partner of LZTS1, with the NSC663284 inhibitor specifically killed the Docetaxel-resistant cells. To further investigate the role of CDC25C, we used inhibitors of the mitotic kinases that regulate CDC25C. Inhibition of CHEK1 and PLK1 induced growth arrest and cell death in the resistant cells. Our findings identify an important role of LZTS1 through its regulation of CDC25C in Docetaxel resistance in prostate cancer and suggest that CDC25C, or the mitotic kinases CHEK1 and PLK1, could be efficient therapeutic targets to overcome Docetaxel resistance.

Figure 1. Characterization of Docetaxel-resistant cell lines

A: Parental and resistant IGR-CaP1 cell lines were exposed to increasing concentrations of Docetaxel for 48h and cell survival was determined. Dose-response curves in IGR-CaP1-R5 (■) (IC₅₀=24nM), IGR-CaP1-R50 (▲) (IC₅₀=100nM) and IGR-CaP1-R100 cells (♦) (IC₅₀=148nM) compared to parental IGR-CaP1 cells (●) (IC₅₀=0.34nM). B: Representative cell cycle distributions of parental IGR-CaP1 and IGR-CaP1-R100 cells in the absence (untreated) or presence of 100nM of Docetaxel for 48h. X-axis: PI nucleic acid stain (DNA content); Y-axis: cell number per channel (counts). The percentage of cells in the different phase of the cycle is indicated. C: Immunofluorescence for γ-tubulin (green) showing the centrosomes. Nuclei were counterstained with Dapi (blue).

Figure 2. Inhibition of LZTS1 gene expression in Docetaxel-resistant IGR-CaP1-R cells

A: Real-time qRT-PCR showing a high decrease of LZTS1 gene expression in the resistant cell lines. B: Whole cell extracts of parental and drug-resistant cells were subjected to immunoblotting with antibodies specific for LZTS1 or β-actin (loading control). C: Immunoblot realized as in B, after a 48h- treatment with 100nM of Docetaxel (Dtx) or without treatment (NT). D: Representative scheme of the larger form of the LZTS1 gene (LZTS1-001, total length 58999 nucleotides, transcript ID: ENST00000381569). The two regions of interest are indicated. Region 1 encompasses the sequence encoding exon 1 (601-824), region 2 encompasses the sequence encoding the exon 2 (49284-49727). Comparison of the mean methylation identified at 20 CpGs in region 1 and at 32 CpGs in region 2 was shown in parental IGR-CaP1 cells (S) and in Docetaxel-resistant (R100) cells.

Figure 3. LZTS1 down-regulation enhances survival of IGR-CaP1 cells

A: Inhibition of LZTS1 expression in IGR-CaP1 cells after a 48h-transfection with siRNA targeting LZTS1 (siLZTS1). Cells transfected with either a non-targeted siRNA (siNT) or a siRNA targeting GAPDH (siGAPDH) were used as control. Western-blot analysis was performed using specific antibodies for LZTS1, GAPDH or β-actin. These data are representative of three separate experiments. B: Growth curves comparing LZTS1-depleted cells (siLZTS1) with control IGR-CaP1 cells (siNT). 12nM of Docetaxel was added 24h after siRNA-transfection (hatched lines). C: Inhibition of LZTS1 reduced the number of IGR-CaP1 cell clones. D: Representative cell cycle distributions of LZTS1-depleted IGR-CaP1 cells (siLZTS1, grey) or in controls (siNT, black), in absence or presence of 100nM of Docetaxel as in Fig. 1 E: Analysis of polynucleation in LZTS1-depleted IGR-CaP1 cells or in controls in absence or presence of 12nM Docetaxel. In 3B, 3C and 3E, the two-way anova statistical analysis showed a significant interaction between siRNA and Dtx effects (***:P-value <0.001).

Figure 4. Implication of CDC25C in the Docetaxel resistance mechanism

A: CDC25C expression is inhibited when LZTS1 is depleted. IGR-CaP1 cells were transfected with siRNA targeting LZTS1 or GAPDH or non-specific target. Whole cell extracts were subjected to immunoblotting using antibody specific for CDC25C. B: Expression of the three CDC25 phosphatases in parental and resistant cells. Whole cell extracts from IGR-CaP1 and IGR-CaP1-R100 were subjected to immunoblotting using antibodies for CDC25A, CDC25B, CDC25C. C: LZTS1 and CDC25C interaction. Cell extracts from IGR-CaP1 transfected with Myc-CDC25C were immunoprecipitated with anti-CDC25C and were subjected to immunoblotting using LZTS1 antibody. Actin was used as a loading control. D: Representative cell cycle distributions of IGR-CaP1 and IGR-CaP1-R100 cells in absence (Ctrl) or presence of 10µM NSC663284 inhibitor E: Dose-response curves showed a very significant difference in relative resistance to NSC663284 inhibitor in IGR-CaP1-R100 cells (black line) (IC₅₀=0.2µM, range [0.15-0.28]) compared to IGR-CaP1 cells (grey line) (IC₅₀=6.59µM, range [4.2-11]).

Figure 5. PLK1 inhibition induces cell death in Docetaxel-resistant cells

A: Cell proliferation assay. IGR-CaP1-R100 cells were treated for 4 days with the PLK1 inhibitor BI2536 at 200nM in the absence or presence of Docetaxel and with Docetaxel alone (100nM). Cell growth was assessed every day using WST1. Data are represented as mean ± SEM. B: Colony formation assay. IGR-CaP1-R100 cells were seeded in 6-well plates in triplicate and treated with 200nM BI2536 in the absence or presence of Docetaxel and with Docetaxel alone (100nM). Cells were stained with crystal Violet 3 weeks later. Data are represented as mean ± SD. P value was derived from the two-tailed Student's t test, significantly different (*P<0.05,). C: Apoptosis. Cells were treated for 48h with 200nM BI2536 in the absence or presence of Docetaxel and with Docetaxel alone (100nM). Apoptosis was assessed using annexinV and propidium iodide staining. P value was derived from the two-tailed Student's t test, significantly different (*P<0.05,). D: PARP and Caspase-3 cleavage. Cells were treated as in (C). Western-blot analysis was performed using specific antibodies for PARP1, Caspase-3 or β-actin. Ratio of cleaved Caspase-3 vs full-length is indicated.

Figure 6. CHEK1 inhibition induces cell death in Docetaxel-resistant cells

A: Dose-dependent growth of IGR-CaP1 and IGR-CaP1-R100 cells. Cells were treated with various concentrations of CHIR-124 for 48h. Cell proliferation was assessed with WST1. B: Cell proliferation of IGR-CaP1-R100 cells. Cells were treated with 100nM CHIR-124 in the presence or absence of 100nM Docetaxel or with Docetaxel alone during 4 days. Proliferation was assessed using WST1. C: Colony formation assay. IGR-CaP1-R100 cells were seeded in 6-well plates in triplicate and treated with CHIR-124 in the absence or presence of Docetaxel and with Docetaxel alone (100nM). Cells were stained with crystal Violet 3 weeks later. Data are represented as mean ± SD for >3 independent experiments. P value was derived from the two-tailed Student's t test, significantly different (*P<0.05, **P<0.01, ***P<0.001). D: Apoptosis. Cells were treated for 48h with 100nM CHIR-124 in the absence or presence of Docetaxel and with Docetaxel alone (100nM). Apoptosis was assessed using annexinV and propidium iodide staining. Data are represented as mean ± SEM. P value was derived from the two-tailed Student's t test, significantly different (*P<0.05). E: PARP and Caspase-3 cleavage. Cells were treated as in (C). Western-blot analysis was performed using specific antibodies for PARP1, Caspase-3 or β-actin. Ratio of cleaved Caspase-3 vs full-lenght is indicated.