WNT5A induces castration-resistant prostate cancer via CCL2 and tumour-infiltrating macrophages

Abstract

Background: Although the standard treatment for the patients with recurrent and metastatic prostate cancer (CaP) is androgen deprivation therapy, castration-resistant prostate cancer (CRPC) eventually emerges. Our previous report indicated that bone morphogenetic protein 6 (BMP6) induced CRPC via tumour-infiltrating macrophages. In a separate line of study, we have observed that the WNT5A/BMP6 loop in CaP bone metastasis mediates resistance to androgen deprivation in tissue culture. Simultaneously, we have reported that BMP6 induced castration resistance in CaP cells via tumour-infiltrating macrophages. Therefore, our present study aims to investigate the mechanism of WNT5A and its interaction with macrophages on CRPC.

Methods: Doxycycline inducible WNT5A overexpression prostate cancer cell line was used for detailed mechanical study.

Results: WNT5A was associated with increased expression of chemokine ligand 2 (CCL2) in the human CaP cell line, LNCaP. Mechanistically, this induction of CCL2 by WNT5A is likely to be mediated via the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signalling pathway. Our in vivo experiments demonstrated that the overexpression of WNT5A in LNCaP cells promoted castration resistance. Conversely, this resistance was inhibited with the removal of macrophages via clodronate liposomes. When patient-derived CaP LuCaP xenografts were analysed, high levels of WNT5A were correlated with increased levels of CCL2 and BMP6. In addition, higher levels of CCL2 and BMP6 were more commonly observed in intra-femoral transplanted tumours as compared to subcutaneous-transplanted tumours in the patient-derived PCSD1 bone-niche model.

Conclusions: These findings collectively suggest that WNT5A may be a key gene that induces CRPC in the bone niche by recruiting and regulating macrophages through CCL2 and BMP6, respectively.

Figure 1

Role of WNT5A in castrated mice (N=5 mice per group). (A) LNCaP cells with inducible WNT5A were subcutaneously injected into nude mice. All animals were castrated and divided to two groups. When WNT5A was induced by administering doxycycline via drinking water, growth of tumour was increased significantly. Error bars represent average±SE. N=5 per group. (B) There is no significant difference between Doxy (−) and Doxy (+) tumours in haematoxylin staining. (C) WNT5A, CCL2, and BMP6 mRNA were analysed from tissues using RT-PCR. When WNT5A was overexpressed, expression of CCL2 and BMP6 were also increased. (D) A single-picture representative of IF for CCL2 and F4/80. (E) Quantification of IF demonstrated that CCL2 levels in WNT5A overexpressing tumours are increased and macrophages (F4/80) are more infiltrated. Mean fluorescence intensity (MFI) was measured by Image J. Values are means+SE. N=5 per group. *P < 0.05. BMP6=bone morphogenetic protein 6; CCL2=chemokine ligand 2; MFI=mean fluorescence intensity.

Figure 2

Induction of CCL2 expression in hormone sensitive CaP cells by WNT5A. (A) LNCaP and 22RV1 cells were treated with 0–100 ng/ml of WNT5A for 24 h. mRNA of CCL2 was increased. (B) The level of CCL2 protein was also increased by WNT5A. Upon treatment of WNT5A (100 ng/ml), CCL2 induction was increased at the (C) mRNA level and (D) protein level in a time-dependent manner. (E) A semi-quantitative RT-PCR revealed that cycloheximide (CHX) not actinomycin D (ActD) inhibited the expression of CCL2 by WNT5A in LNCaP cells. All study was repeated at least three times. *P < 0.05. CCL2=chemokine ligand 2.

Figure 3

Induction of CCL2 by WNT5A signalling. (A) LNCaP and 22RV1 cells expressed ROR2 mRNA (left) and protein (right). (B) When ROR2 was blocked with ROR2 shRNA, CCL2 expression was decreased. (C) When ROR2 was overexpressed with flag-ROR2 expression construct, CCL2 expression was significantly increased. (D) ERK inhibitor (U0126) significantly decreased CCL2 expression in LNCaP cells after treating WNT5A. (E) When WNT5A was treated on LNCaP cells, immunoblot of ERK demonstrated that ERK was phosphorylated. All study was repeated three times. *P < 0.05. CCL2=chemokine ligand 2; ERK=extracellular signal-regulated kinase.

Figure 4

WNT5A induced CRPC via macrophages. (A) Tumour growth rate is significantly decreased in clodronate liposomes injected group. After tumour injection, when the palpable tumours found, all mice got surgical castration. Then clodronate or control liposome was injected. (B) H&E staining showed that no significant difference between clodronate and PBS group. Error bars indicate average±SE. N=5 per group. (C)The expression of WNT5A and CCL2 in isolated tumours from both groups of mice were confirmed with semi-quantitative RT-PCR. (D) Representative pictures of IF for WNT5A, BMP6, CCL2 and F4/80. (E) Quantification of IF demonstrated that clodronate liposomes injected group showed decreased level of macrophage infiltration in tumour. MFI was measured by Image J. Values indicate means+SE. N=5 per group. *P < 0.05. CCL2=chemokine ligand 2; MFI=mean fluorescence intensity.

Figure 5

Analysis of patient-derived CaP xenograft samples. (A) The expression of BMP6, ROR2, and CCL2 was analysed in tumours from PCSD1 bone-niche model. PCR was repeated at least three times. (B) and a single-picture representative of IF demonstrated that high expression of CCL2 with high-Gleason tumour as compared with low-Gleason tumour. Macrophage infiltration also little bit higher in high-Gleason tumour than low-Gleason tumour. *P < 0.05. BMP6=bone morphogenetic protein 6; CCL2=chemokine ligand 2.

Figure 6

Proposed mechanism of WNT5A role in CRPC and bone metastasis. Bone stromal cells and CaP cells produced WNT5A which can induce BMP6 and CCL2 from CaP cells. Secreted CCL2 recruits macrophages into tumours and BMP6 induces CRPC in CaP cells. In turn, interaction between tumour cells and bone stromal cells would promote bone metastasis of CaP.