CCL2 is induced by chemotherapy and protects prostate cancer cells from docetaxel-induced cytotoxicity

Abstract

Background: Metastatic prostate cancer is either inherently resistant to chemotherapy or rapidly acquires this phenotype after chemotherapy exposure. In this study, we identified a docetaxel-induced resistance mechanism centered on CCL2.

Methods: We compared the gene expression profiles in individual human prostate cancer specimens before and after exposure to chemotherapy collected from previously untreated patients who participated in a clinical trial of preoperative chemotherapy. Subsequently, we used the gain- and loss-of-function approach in vitro to identify a potential mechanism underlying chemotherapy resistance.

Results: Among the molecular signatures associated with treatment, several genes that regulate the inflammatory response and chemokine activity were upregulated including a significant increase in transcripts encoding the CC chemokine CCL2. Docetaxel increased CCL2 expression in prostate cancer cell lines in vitro. CCL2-specific siRNA inhibited LNCaP and LAPC4 cell proliferation and enhanced the growth inhibitory effect of low-dose docetaxel. In contrast, overexpression of CCL2 or recombinant CCL2 protein stimulated prostate cancer cell proliferation and rescued cells from docetaxel-induced cytotoxicity. This protective effect of CCL2 was associated with activation of the ERK/MAP kinase and PI3K/AKT, inhibition of docetaxel-induced Bcl2 phosphorylation at serine 70, phosphorylation of Bad, and activation of caspase-3. The addition of a PI3K/AKT inhibitor Ly294002 reversed the CCL2 protection and was additive to docetaxel-induced toxicity.

Conclusion: These results support a mechanism of chemotherapy resistance mediated by cellular stress responses involving the induction of CCL2 expression and suggest that inhibiting CCL2 activity could enhance therapeutic responses to taxane-based therapy.

Figure 1. The modulations of inflammatory genes in vivo and in vitro

A. The heatmap of 5 most significantly upregulated inflammatory cytokine gene expression in prostate cancer epithelium from 31 patients underwent chemotherapy. Tumor epithelium was laser-capture microdissected from post-treatment radical prostatectomy samples and pre-treatment needle biopsy samples. cDNA were synthesized as previously described. The expression level was expressed as post-treatment vs. pre-treatment. Rows represent genes and columns represent individual patients. Red: upregulation, Green: down-regulation, Black: no change, White: no data. B. Docetaxel induced the expression of inflammatory genes. LNCaP cells were treated with 0, 1, 5, and 25 nM of docetaxel for 48 hours. RNA was extracted, and gene expressions were measured by qRT-PCR, analyzed using the delta-delta Ct method and normalized to samples treated with 0 nM docetaxel.

Figure 2. JNK and NF-kappaB pathways mediated the docetaxel-induced CCL2 gene expression

A. The CCL2 upregulation in post-treatment cancer epithelium was confirmed by qRT-PCR. The cDNA of 31 patients from post-treatment radical prostatectomy samples and pre-treatment needle biopsy samples were used. P < 0.002, paired-t-test. B. Docetaxel induced CCL2 protein expressions in LNCaP cells. Cells were treated with 1 and 3 nM of docetaxel (DTX) for 72 hours. Whole cell lysates and proteins in LNCaP extracellular media were extracted and probed with an anti-CCL2 antibody by western blot. The intracellular and extracellular CCL2 loadings were controlled by tubulin and coomassie stain, respectively. C. LNCaP cells were treated with solvent, 5 nM of docetaxel, 1 μM of JNK inhibitor (SP600125), or combination of both for 48 hours. Gene expression was measured by qRT-PCR, normalized to solvent control, and expressed as relative mRNA. D. LNCaP cells were treated with solvent, 5 nM of docetaxel, 1 uM or 10 uM of NF-kB inhibitor (parthenolide), or combinations for 48 hours. Gene expressions of CCL2 were measured, normalized to solvent control, and expressed as relative mRNA. * P < 0.05, ANOVA.

Figure 3. CCL2 loss-of-function by siRNA enhanced prostate cancer cells sensitivity toward low-dose of docetaxel

A. LNCaP cells were transfected with scramble control (siControl) or siRNA oligo targeting CCL2 (siCCL2) for 48 hours. Then cells were treated with 3 nM docetaxel. The expressions of CCL2 were measured by qRT-PCR in cells and western blot in cell culture media. B. & C. Prostate cancer cells LNCaP (B) and LAPC-4 (C) were transfected with a CCL2 specific siRNA oligonucleotide. 24 hours later viable cells were counted, cultured in serum free media and treated with low-doses of 1 and 3 nM of docetaxel for 5 days. The viable cells of siRNA control and siCCL2 were counted and normalized to that of respective day 0 control. *, #, **, P < 0.05, t-test.

Figure 4. CCL2 gain-of-function decreased prostate cancer cell sensitivity to low-dose of docetaxel

A. LNCaP cells were transfected with either a CCL2 over-expression vector or an empty vector control. Then cells were treated with 3 nM of docetaxel for 5 days. The viable cell numbers of empty vector cells or CCL2 cells were counted at day 0, 2 5 of docetaxel treatment, and normalized to that of respective day 0 controls. * vs. Ev+DTX, P < 0.05, t-test. B. LAPC4 cells were cultured at serum-free condition, and viable cell numbers were counted as day 0 controls. Then cells were treated with 3 or 10 nM of docetaxel, in combinations with 0, 10 or 100 ng/ml of recombinant human CCL2. After 5 days of treatment, viable cells in each CCL2 group were counted and normalized to that of respective day 0 control. *, #, P<0.05, t-test. C. LAPC4 cells treated with control, 3nM docetaxel (DTX), 10 ng/ml CCL2 or combination in (B) were stained with PI and subjected to flow cytometry. The % of dead cells in sub-G1 phase was plotted. Mean and standard deviation. *, P < 0.05, t-test.

Figure 5. CCL2 modified signal transduction

A. CCL2 increased Erk and AKT signaling, and decreased docetaxel induced JNK signaling. LNCaP cells were treated with docetaxel, CCL2 or combination for 48 hours. Whole cell lysates were prepared, resolved on 12% SDS-PAGE gel, and probed with antibodies specific for JNK1, c-jun, Erk and AKT phosphorylation. Total Erk and AKT were used as loading controls. B. CCL2 activated Bcl2 and inactivated Bad. Cells and proteins were prepared as in A, and probed with antibodies specific for Bcl2 phosphorylation at serine 70, Bad phosphorylation at serine 112. The total Bcl2 and Bad were used as loading controls.

Figure 6. PI3K/AKT is utilized by CCL2 to confer survival advantage during chemotherapy

A. Inhibition of PI3K/AKT restored the docetaxel-induced Bcl2 phosphorylation and caspase 3 activation. Cells were treated with docetaxel or docetaxel with CCL2 as in figure 5A, plus PI3K/AKT inhibitor Ly294002 (5 μM) was added into the media of docetaxel and CCL2 co-treated cells. B. PI3K/AKT inhibitor enhanced the growth inhibitory effect of low-dose docetaxel. LNCaP cells were treated with solvent control, low-dose of docetaxel (1 nM), Ly294002 (5 μM), or combination for 72 hours. Viable cells were counted and expressed as % of solvent control. * P < 0.05 vs. docetaxel, Ly294002, t-test.