**1. Introduction**

Prostate cancer is among the most frequently diagnosed malignancies worldwide in men [1]. The five-year survival rate for localized prostate cancer is close to 100%, and the prognosis for localized prostate cancer is the best among all types of cancers; however, metastatic prostate cancer is associated with a very poor prognosis, with no curative treatments currently available [1,2]. Androgen/androgen receptor (AR) signaling is known to be a significant driver of prostate cancer progression, therefore androgen-deprivation therapy (ADT)—with or without anti-androgens—is often used as a standard form of care for patients with advanced and metastatic prostate cancer [3,4]. ADT has been demonstrated to improve not only serum prostate-specific antigen levels, but also patient survival, however prostate cancer generally progresses to castration-resistant prostate cancer (CRPC) following several years of ADT [5]. Several potential mechanisms underpinning CRPC progression that relate to AR function have been identified, including androgen hypersensitivity, AR mutation, ligand promiscuity, and AR variants. Nonetheless, no radical treatments exist at present and all AR-targeting agents for CRPC eventually fail to suppress cancer cell activity [6]. Recently, some studies have reported suppressive effects of androgen/AR signaling in prostate cancer cells, therefore suppression of AR function itself may cause CRPC [7,8]. Previously, we demonstrated that androgen/AR signaling

increases prostate cancer cell proliferation, while simultaneously inhibiting cancer cell migration, which is induced by the activation of several C-C motif ligand (CCL)-receptor (CCR) axes downstream or upstream of androgen/AR signaling [9–12]. This review focuses on such suppressive effects of androgen/AR signaling on prostate cancer cells through CCL-CCR axes.

### **2. The Role of CCL2 as a Downstream Mediator of Androgen/AR Signaling**

Therapeutic approaches that solely target androgen/AR signaling are insufficient to control prostate cancer cell activity [13–15]. Genetic ablation of AR in prostate epithelial cells promotes the development of invasive prostate cancer [7], suggesting that therapeutic suppression of androgen/AR function induces unwanted signals that may promote the progression of surviving prostate cancer cells to an advanced metastatic stage. When AR function of C4-2 (a human prostate cancer cell line) cells were silenced with AR-siRNA (siAR), using scramble RNA (scr) as a control, siAR cells were observed to possess an increased migratory capacity [8]. Cytokine array analysis of conditioned media from siAR and scr cells revealed increased CCL2 expression in siAR cells, supporting a potential role for prostate cancer cell-derived CCL2 in mediating local inflammatory responses during suppression of AR [8]. CCL2 is reported to play a potential role in stimulating capillary network formation of human microvascular endothelial cells in the microenvironment of prostate cancer [16]. C4-2 siAR cells were also observed to express increased levels of epithelial-mesenchymal transition (EMT) markers and pSTAT3 via the CCL2-CCR2 axis in an autocrine manner. In addition, C4-2 siAR cells were observed to possess significantly reduced levels of PIAS3 (the endogenous protein inhibitor of activated STAT3), which is controlled by androgen/AR signaling [17]. Notably, STAT3 activation was also observed to increase CCL2 expression levels in C4-2 siAR cells. These results sugges<sup>t</sup> that androgen/AR signaling in prostate cancer cells may inhibit CCL2 and pSTAT3 expression through upregulation of PIAS3 [8,9]. EMT is believed to be an essential cancer cell characteristic for invasion and metastasis to distant sites [18]; pSTAT3 activation has been reported to play an important role in EMT induction, as well as inflammation and cancer progression [19,20]. Furthermore, ADT is known to be linked to EMT induction [21]. In summary, prostatic epithelial AR silencing via siAR promotes STAT3 activation and EMT in prostate cancer cells via CCL2 induction, which may be associated with a secretory phenotype and pro-invasive characteristics of prostate cancer cells [8,9].

### **3. The Role of CCL22 as a Further Downstream Mediator of CCL2**

CCL2 is a powerful chemotactic protein for macrophages and tumor-associated macrophages (TAMs), which infiltrate into tumors and contribute to cancer progression via immune suppression [22,23]. CCL17 and CCL22, which are high-affinity ligands for CCR4, have both been reported to be secreted by TAMs, with immunosuppressive functions [24]. Correlations have previously been reported between CCR4 expression levels and metastasis in cancer cells [25,26]. Therefore, we aimed to elucidate the relationship between the CCL2-CCR2 axis and CCL17/22-CCR4 axis in prostate cancer progression. Both CCR2 and CCR4 were observed to be expressed in human prostate cancer cell lines and prostate cancer tissues; furthermore, in vitro co-culture of prostate cancer cells and macrophages resulted in increased CCL2 and CCR2 levels in prostate cancer cells [11]. Notably, addition of CCL2 induced both CCL22 and CCR4 expression in prostate cancer cells; CCL22 subsequently promoted the migration and invasion of prostate cancer cells in an autocrine manner, via enhanced phosphorylation of Akt [11]. The CCL22-CCR4 axis is known to chemo-attract regulatory T cells (Tregs) into tumor tissues; Tregs recognize self-antigens, including tumor antigens present in tumor tissues, and efficiently suppress the activation of tumor antigen-specific effector T cells [27]. In summary, CCL2 and CCL22 secretion in the prostate cancer tumor microenvironment may induce not only direct metastasis of prostate cancer cells, but also promote the activation of TAMs and Tregs, which facilitate a suitable environment for cancer progression.

### **4. The Role of CCL5 as an Upstream Mediator of Androgen/AR Signaling**

Skeletal metastases occur in approximately 80% of patients with advanced prostate cancer, for which no curative treatment is available [28]. We previously reported that bone stromal cells and SaOS-2 osteoblast-like cells promote prostate cancer metastasis via activation of transforming growth factor-β1 (TGF β1) [29], which in turn induces the development of an immune suppressive microenvironment [30]. CCL2 is reported to increase bone metastasis through recruitment of TAMs and osteoclasts to the tumor site and blood vessel formation through vascular endothelial growth factor-A [31,32]. Therefore, we investigated whether further chemokines could be involved in the activation of prostate cancer cells within prostate cancer bone metastases. Migration of LNCaP cells (an AR-positive prostate cancer cell line) increased significantly when co-cultured with bone stromal cells isolated from prostate cancer bone metastases. Cytokine array analysis of conditioned media from bone stromal cell cultures subsequently identified CCL5, a high-affinity ligand of CCR5, as a concentration-dependent promoter of LNCaP cell migration [12]. LNCaP cell migration was observed to be suppressed by the addition of a CCL5-neutralizing antibody to cocultures with bone stromal cells, while AR knockdown using siRNA was observed to increase LNCaP cell migration compared with control cells [12]. As CCL5 was unable to promote migration of LNCaP siAR cells, it was concluded that elevated CCL5 secretion by bone stromal cells from metastatic lesions induced prostate cancer cell migration in a CCL5-dependent manner, upstream of AR signaling [12]. Upregulation of CCL5 has previously been reported to increase the aggressive potential of breast cancer cells and the invasiveness of prostate cancer cells [33–35]. In addition, Luo et al. found that CCL5 upregulation in bone marrow mesenchymal stem cells increased the metastatic potential of prostate cancer cells, and subsequently downregulated AR signaling, due to inhibition of AR nuclear translocation [36]. Furthermore, CCL5 has been found to suppress prolyl hydroxylase expression, leading to suppression of VHL-mediated HIF2 α ubiquitination and suppression of AR signaling [37]. Results obtained using LNCaP siAR cells indicate that CCL5 activity is located upstream of AR signaling. Moreover, SaOS-2 did not promote the migration of PC-3 AR-negative prostate cancer cells [12]. These results sugges<sup>t</sup> that the migratory potential of AR-positive prostate cancer cells in bone metastases is increased by CCL5, secreted by bone stromal cells via the suppression of androgen/AR signaling. CCL5 is also secreted by prostate cancer-associated fibroblasts and recruited macrophages into the prostate cancer microenvironment [38]. Estrogen receptor α could reduce prostate cancer cell invasion through reduction of CCL5 secretion from fibroblasts and macrophage infiltration prostate cancer [38].

### **5. Treatment Strategies Targeting CCL-CCR Axes and Androgen/AR Signaling**
