Infiltrating bone marrow mesenchymal stem cells (BM-MSCs) increase prostate cancer cell invasion via altering the CCL5/HIF2α/androgen receptor signals

Abstract

Several infiltrating cells in the tumor microenvironment could influence the cancer progression via secreting various cytokines. Here, we found the CCL5 secreted from BM-MSCs suppressed androgen receptor (AR) signals via enhancing the expression of hypoxia inducible factor 2α (HIF2α) in prostate cancer (PCa) cells. Mechanism dissection revealed that the increased HIF2α might alter the AR-HSP90 interaction to suppress the AR transactivation, and inhibition of HIF2α reversed the BM-MSCs-increased PCa stem cell population and PCa cells invasion. Importantly, CCL5 could suppress the prolyl hydroxylases (PHDs) expression, which might then lead to suppress VHL-mediated HIF2α ubiquitination. Together, these results demonstrated that the CCL5 signals from infiltrating BM-MSC cells to HIF2α signals within PCa cells might play a key role to increase PCa stem cell population and PCa metastasis via altering the AR signals. Targeting this newly identified CCL5/HIF2α/AR axis signal axis may allow us to develop a novel way to suppress PCa metastasis.

Keywords: CCL5; HIF2α; androgen receptor; bone marrow mesenchymal stem cells; prostate cancer.

Figure 1. BM-MSCs increase HIF2α expression in PCa cells

a. The cartoon demonstrating BM-MSCs and PCa cells co-culture 1 × 10⁵ BM-MSCs (with media as control) were placed in upper chambers of the transwell plates (0.4 μm membrane) while PCa cells (1 × 10⁶) were placed in lower chambers. b. Sphere formation assay. The PCa cells were co-cultured with the primary MBM-MSCs (media used as control) in 0.4 μM membrane transwell plates for 5 days. Cells were then mixed with Matrigel (1:1, v/v), plated in 24-well plates, and cultured for 10 days. Quantification was shown at right. c. Invasion assay result. The C4-2 cells (1 × 10⁵) were co-cultured with the mouse primary BM-MSCs for 3 days in transwell plates (0.4 μM membrane). The invaded cells were stained by toluidine blue, and the positively stained cells were counted from 5 random areas. Quantitation was shown at right. d. Western blot analysis of HIF2α expressions. The LNCaP and C4-2 cells were co-cultured with or without the primary mouse BM-MSCs and HIF2α expression was analyzed. e. qPCR analysis of HIF2α mRNA level in C4-2 cells with or without BM-MSCs co-culture. f. HIF2α IHC staining of the tumor tissues obtained from the CWR22RV1 (22RV1) xenografted mice, with or without co-implantation with the primary BM-MSCs.

Figure 2. HIF2α is essential for BM-MSCs increase of PCa invasion and stem cell population

a. qPCR analysis of expression of HIF2α in C4-2 and CWR22RV1 (22RV1) cells after infection by the scramble (Sc) and siHIF2α viruses. b. Sphere formation assay of the Sc or siHIF2α C4-2 cells co-cultured with BM-MSCs (media as control). c. Invasion assay of the Sc or siHIF2α C4-2 cells co-cultured with the BM-MSCs. d. qPCR analysis of expressions of the stem and metastasis marker genes. The Sc or siHIF2α C4-2 cells were co-cultured with or without the primary BM-MSCs. The total RNAs were extracted and the expressions of the CD133, ZEB-1, and CXCR4 genes were analyzed.

Figure 3. BM-MSCs secrete CCL5 to induce the HIF2α expression

a. Western blot analysis results of HIF2α expressions after CCL5 treatment. The PCa cells were treated with 10 ng/ML rCCL5 for 48 hrs and expressions of HIF2α were analyzed. b. C4-2 cells were co-cultured with BM-MSCs (MSCs) and then treated with either IgG or CCL5 neutralizing antibody. The HIF2α protein levels were then examined by Western blot analysis. c. Sphere formation assay of Sc or siHIF2α C4-2 and and CWR22RV1 (22RV1) cells treated by CCL5. The C4-2 and CWR22RV1 cells were infected by scramble or HIF2α siRNA, and then cells were treated with 10 ng/ml CCL5 for 10 days. d. Invasion assay of C4-2 and CWR22RV1 cells treated with CCL5. The cells were treated with 10 ng/ml CCL5 for 72 hrs and then the invasion assay was performed. e. The qPCR analysis of the mRNA level of CD133 and ZEB-1 in Sc/siHIF2α C4-2 cells treated with 10 ng/ml CCL5.

Figure 4. CCL5 suppresses the ubiquitination of HIF2α through VHL

a. qPCR analysis of mRNA level of HIF2α in PCa cells after treating with CCL5. b. The Western blot analysis of HIF2α in C4-2 cells treated with different doses of CCL5 for 48 hrs, and then the DMSO or 20 μM MG132 were added into the cells for 4 hrs. The expression of HIF2α was analyzed. c. The ubiquitinaion assay of HIF2α in 293T cells. The 293T cells were transfected with the GFP-Ub and HIF2α plasmids. The GFP-Ub proteins were immuno-precipitated by GFP antibody, the HIF2α proteins were analyzed by Western blot. d. The PHD1/PHD4 expression was regulated by BM-MSCs. The C2-4 cells were co-cultured with BM-MSCs and mRNA expression of PHDs was investigated by qPCR analysis. e. qPCR analysis of PHD1 and PHD4 after incubating C4-2 cells with 10 ng/ml rCCL5 for 48 hrs. f. The expression of HIF2α in Sc/siVHL C4-2 cells after adding 10 ng/ml CCL5. Left panel showed that the VHL was successfully knocked down in C4-2 cells. g. Co-IP experiment. 293 cells were transfected with HIF2α and VHL, immunoprecipitated with HA antibody, and the VHL protein binding was detected in Western blot analysis using GFP antibody.

Figure 5. HIF2α suppress AR transactivation activity

a. qPCR analysis of PSA, TMPRSS2 and HIF2α expression after treating C4-2 cells with CCL5. b. Sc or siHIF2α C4-2 cells were co-cultured with or without the BM-MSCs. The total RNAs were extracted and the expressions of the PSA genes were analyzed. c. HIF2α regulates AR signal. The luciferase assay was performed using MMTV-luc in 293 cells in the absence and presence of HIF2α. d. HIF2α regulates AR translocation. The 293 cells were transfected with the AR with or without HIF2α, the nuclear extraction were performed and detected by Western blot. Tubulin was used as the cytosol marker and the PARP was used as the nuclear marker. e. The qPCR analysis of PSA, TMPRSS2, CD133 and ZEB-1 expression in C4-2 and CWR22RV1 cells under normoxia and hypoxia (0.5% oxygen) condition. f. The immuno-fluorescent staining of AR under normoxia and hypoxia condition in C4-2 and CWR22RV1 cells. g. Hypoxia regulates the AR nuclear translocation. C4-2 cells were cultured under normoxia and hypoxia conditions, the nuclear extraction were performed and detected by Western blot. Tubulin was detected by Western blot as the cytosol marker; the cyclinD1 was detected as nuclear marker. h. Sc and siHIF2α 293T cells were cultured under normoxia or hypoxia condition. The AR localization was analyzed by western blot. i. The CoIP assay to detect the AR and HSP90 interaction was performed in 293 cells. Cells were transfected by HSP90, AR and HIF2α plasmids. The AR antibody was used for the IP of AR-HSP90 interaction complex and the pull-down protein complex was further detected by western blot.

Figure 6. The cartoon of the pathway