Background
Prostate cancer (PCa) is the most frequently diagnosed cancer and second leading cause of cancer death in men in the United States [
1]. PCa is a chronic type of tumor that requires a long time for small lesions to become clinically manifested compared to some other cancers [
2]. Inflammation has been thought to be one of the key pathogenic factors for PCa and there is an association between chronic inflammation and increased prevalence of PCa [
3‐
6]. Furthermore, tumor associated macrophages (TAM) form a major component of the inflammatory infiltrates in both primary and secondary tumors [
7] and can release growth factors, cytokines and chemokines to regulate tumor growth and invasion [
8]. However, the detailed mechanisms how the interactions among stromal cells, TAM, and PCa cells could influence the growth and metastasis of PCa remain to be further elucidated.
An earlier study suggested that cancer associated fibroblasts (CAF) may play important roles to influence PCa progression and invasion [
9]. In the prostate tumor microenvironment (TME), PCa epithelial cells can produce some growth factors, such as TGF-β, PDGF and FGF, to influence/activate peripheral stromal cells that result in transformation of normal fibroblasts into CAF. Subsequently, CAF can then increase in population through transforming from normal fibroblasts [
10], differentiation from bone marrow-derived mesenchymal stem cells [
11] or by epithelial to mesenchymal transition (EMT). The important functions of CAF include the regulation of deposition of extracellular matrix (ECM), epithelial differentiation, tumor inflammation, and wound healing [
12]. Ezer et al. demonstrated that CAF could mediate inflammation and angiogenesis by recruiting macrophages to stimulate angiogenesis, which may then promote tumor growth [
13].
The existence of aromatase (to convert testosterone to estrogen) [
14] and the finding of an increase in estrogen-to-androgen ratio in aging men [
15] indicated that estrogens, in addition to androgens, could play important roles in PCa initiation and progression. Animal studies also demonstrated that 100 % of rats being treated with 17β-estradiol (E
2) plus testosterone for around 44 weeks had prostatic adenocarcinomas [
16].
Estrogen action is mainly mediated through its specific nuclear receptors that regulate transcription of target genes
via binding to the estrogen response element (ERE) or non-ERE mediated transactivation, as well as non-genomic regulations [
17]. There are two major types of estrogen receptors (ERs), ER alpha (ERα) and ER beta (ERβ) [
18,
19]. The two ER subtypes are structurally similar, consisting of the six common domains (A–F), but encoded by separate genes (
ESR1 and ESR2). Immunostaining indicated that ERα positive [ERα(+)] staining was present in normal prostate stromal cells nuclei [
20]. The function of stromal ERα, however, remains largely unknown.
It has been well demonstrated that cancer related inflammation promotes cancer cells proliferation, migration and invasion through several pathways, including signal transduction activation, cytokines secretion and immune cells infiltration [
21]. The TAM, M2 type, are the major players that link tumor related inflammation and tumor progression [
22]. A variety of chemokines, like CCL2 and CCL5, have been detected in neoplastic tissues and associated with tumor associated immune cells formation and recruitment [
23].
Using the in vitro co-culture system and in vivo mouse models, we studied CAF ERα roles in PCa invasion and found CAF ERα could inhibit PCa metastasis
via suppression of macrophage infiltration and M2 type macrophages formation. This CAF.ERα(+) → macrophages → PCa invasion pathway involves the modulation of CAF CCL5 and macrophages IL6 gene expressions. This finding supports the clinical observation that PCa patients with stromal ERα have better PSA free survival rates [
24].
Discussion
In the TME, chronic inflammation has been proven to promote cancer progression [
34]. Tumor cells can secrete chemokines, cytokines and prostaglandins for inflammatory cells recruitment in order to sustain the inflammatory response. Nelson et al. [
35] indicated that inflammation plays an important role in the development and progression of PCa. The chronic inflammation mainly occurred in the area directly adjacent to PCa lesions and induced inflammatory cell infiltration/accumulation. After immune cells accumulated at the sites, the tumor consequently increased prostate epithelial cells proliferation by inflammatory oxidants secretion [
36]. Furthermore, cancer related inflammation may affect tumor cell migration, invasion, angiogenesis, etc. Not only epithelial cells, but also CAF can produce inflammatory factors and affect immune cells recruitment. Among several chemokines, CCL1, −2, −4, −5, −7, −8, −12, −13, and IL6 might influence the interaction of inflammation with cancer malignancy, and CCL2, −3, −5, −7, CXCL12, −14, and IL6 were found to be able to affect the macrophage infiltration [
37]. In our study, we found that expression of ERα in CAF can reduce the number of infiltrated macrophages recruited by CAF and PCa cells and subsequently suppress cancer invasion.
In the cancer initiation stage, epithelial cancer cells can activate and differentiate fibroblasts into myofibroblasts and the activated fibroblasts consequently promote tumor growth [
38,
39]. When tumors progress, the ratio of cancer cells to CAFs may vary depending on the stages of the disease. A previous study showed that epithelial and CAF cells were set at different ratios to study the interaction between fibroblasts and different breast cancer cells [
40]. In a prostate cancer study, Camps et al. co-injected PC-3 cells with CAFs (PCa:CAF = 10:1; 1 × 10
6:1 × 10
5) into mice and successfully promoted tumor growth [
41]. In another of our studies, we co-injected CWR22Rv-1 cells and CAFs (22Rv-1:CAFs = 9:1; 9 × 10
5:1 × 10
5) into each lobe of mouse anterior prostates and tested whether the ERα status in CAFs could promote or inhibit tumor invasion [
24]. When we changed the PCa:CAF ratio from 9:1 to 5:1, we could also see the similar effects (data not shown). The data presented in this study was collected from PCa:CAF at ratio 9:1. In addition to determining the CAF.ERα-regulated PCa invasion, in another of our projects studying CAF ERα role in PCa growth, we found the differential roles of CAF.ERα(+). CAF cells with higher ERα expression could promote the growth, but inhibit the invasion of PC3, LNCaP, C4-2 and CWR22Rv-1 cells. The in vivo model also demonstrated mice co-injected with CWR22Rv-1 and CAF.ERα(+) cells can develop bigger tumors yet lower metastasis rates as compared to mice co-injected with CWR22Rv-1 and CAF.ERα(−) cells (Da and Yeh et al., paper in preparation).
CAF have been demonstrated to play important roles in cancer progression through promoting tumor initiation, growth and invasion
via promotion of the extracellular matrix (ECM) remodeling and release of growth factors and cytokines. CAF are a source of ECM-degrading proteases such as the MMPs [
42], which might allow cancer cells to escape the primary tumor site. Our previous study also indicated CAF.ERα(+) suppressed PCa metastasis through decreased Thbs2 and MMP3 expression [
24]. Other studies demonstrated liver CAF could induce metastases through secreting inflammatory factors, like IL6 and MCP-1 [
43,
44]. In addition, CAF have the capability to recruit immune cells into the tumor region
via altering the expression of IL6, CCL2 [
45], or NF-kB signals [
13].
Our findings indicated CAF cells expressing ERα have a lower capability to recruit macrophages. Further mechanism dissection showed that both CCL5 and IL6 secretions are decreased in CAF.ERα(+), with CCL5 subsequently related to macrophage recruitment, but not IL6. We hypothesized that CCL5 may play a key role for recruiting the infiltrating macrophages to PCa cells. Robinson et al. also demonstrated that CCL5 plays an important role in attracting macrophage migration and may become a target for breast cancer therapy [
46]. In a breast cancer murine model, those murine cells treated with Met-CCL5 (receptor antagonist) had a decreased number of infiltrating macrophages associated with a significantly reduced tumor size. The development of “anti-macrophages” may become one option for cancer therapy in the future. M2 type macrophages, one type of inflammatory cells that are differentiated by IL-4 and IL-13 stimulations, are known as major mediators linking cancer and inflammation [
22,
47]. Recent data showed CAF, through stromal-derived growth factor-1 secretion, promote M2-type macrophages expression and PCa progression [
48]. We examined M2 macrophages related markers expression in macrophages after CAF CM treatment. Surprising, after co-culture with the CAF.ERα(+)CM, the macrophages expressed less M2 macrophage markers, including IL-10, Fuzz1 and Ym1, but not arginase-1 (Additional file
5: Figure S5A), suggesting CAF.ERα(+) may be able to suppress M2-type macrophages in the PCa TME. This conclusion is further supported by the finding of higher IL-4 and IL-13 expression in CAF.ERα(−) than in CAF.ERα(+) cells (Additional file
5: Figure S5B). This suggests CAF.ERα(+) cells can release less IL-4 and IL-13 and may induce less M2-type macrophages than CAF.ERα(−) cells.
In prostate development, using Cre-loxP gene knockout strategy, reports have shown that ERα plays different roles in prostate epithelial as well as different types of prostate stromal cells [
26,
49]. In the PCa mouse models, both ERα knockout [
50] and ERα agonist treatment [
51] showed mice with activated ERα can develop high-grade PIN, suggesting ERα might play important roles in PCa progression. Early studies indicated the expression of epithelial ERα, but not stromal ERα, was increased in PCa [
52]. Celhay et al. demonstrated stromal ERα may also play an important role in recurrence of hormone refractory PCa. They compared ERα expression by IHC in 55 paired patient PCa samples collected before androgen deprivation therapy and after hormonal relapse. They found a shorter time to hormonal relapse was associated with low staining for ERα in stromal cells and correlated to shorter patient survival rate [
53]. Daniels et al. [
28] reported that ERα positive rates reduced in the cancer associated stromal cells compared to the adjacent benign prostate tissue. Although the expression level of ERα in cancer associated stromal cells is relatively weak, the intensity of ERα expression in tumor-associated stroma shows a positive correlation with cancer progression. The reduced CAF ERα IHC staining by Daniels et al. [
28] supports our finding that CAF ERα plays a protective role in cancer invasion. Furthermore, PCa patients with CAF.ERα(+) expression have a better PSA free recurrence survival rate [
24]. Our data demonstrated stromal ERα can inhibit PCa invasion through suppressing macrophage infiltration into tumor sites and directly decrease cytokine secretion in PCa cells.
Platz et al. indicated chronic inflammation could be an epidemiologic factor for PCa [
54], and De Marzo et al. also linked the PCa progression to inflammation related dietary factors [
4]. Prins et al. [
55] demonstrated that estrogen induced inflammation is specifically mediated by epithelial ERα. The epithelial inflammatory cell infiltrates were observed with aging in wild type and ERβ knock out (ERβKO), but not in ERαKO, mice after DES (Diethylstilbestrol) treatment. Van Laere et al. demonstrated that activation of NF-kB in inflammatory breast cancer was associated with loss of ERα expression, suggesting ERα might play a positive role in anti-inflammation [
56]. In autoimmune encephalomyelitis, ERα-ligands mediated anti-inflammation is important in neuroprotection for reducing the levels of central nervous system inflammation [
57]. ERα has been proven to have an anti-inflammatory function in macrophages. However, the ERα roles in inflammation-mediated PCa progression may depend on the ERα location. Our data showed stromal ERα can decrease macrophage infiltration, but may also suppress CAF-mediated inflammation response.
Our results showed ERα in CAF not only decreases IL6 expression in CAF cells, but also regulates macrophages activity to decrease IL6 secretion, although the mechanisms by which CAF.ERα(+) cells affect macrophage activity are still unclear. Previous studies indicated IL6 and leukemia inhibitory factor (LIF) secretion increases in tumor tissues can promote TAM generation. Deprivation of IL6 and LIF can suppress TAM induction. Early studies indicated that inflammatory cytokines, such as IL6, might play major roles in the metastasis of breast and neck cancers [
58,
59]. Michalaki et al. [
60] measured serum IL6 concentration from patients and found it is higher in patients with metastatic disease than localized disease. Lou et al. [
61] also determined IL6 plays an important role in the PCa metastatic Stat3 signaling transduction pathway. But, after CAF CM treatment, we found IL6 expression in PCa cells shows no significant difference between CAF cells with/without ERα. Hsu et al. also found anti-IL6 might suppress the MMP2 and MMP9 expressions in a colon cancer model [
62]. Importantly, Karin et al. demonstrated estrogen and propyl pyrazole triol (PPT, ERα specific agonist) could suppress metastasis of hepatocellular carcinoma
via inhibition of IL6 expression [
63]. They also indicated that the gender difference in tumor susceptibility resulted from a downregulation of IL6 production by macrophages in response to estrogens.
Competing interests
The authors confirm that there are no conflicts of interest.
Authors’ contributions
CR Yeh and SY Yeh developed the original hypothesis, experimental design and draft of manuscript. CR Yeh carried out in vitro studies, especially macrophage recruitment and PCa invasion. CR Yeh, S Slavin and J Da carried out animal studies. I Hsu prepared plasmid constructions. FJ Chou and J Ding worked on data collecting and paper revision. GQ Xiao preformed immunochemical staining. J Luo carried out in vitro studies especially the IL6 and CCL5 neutralizing antibody study. All authors read and approved the final manuscript.