Reciprocal signalling between tumour cells and adjacent stromal cells plays pivotal roles in orchestrating the oncogenic epithelial cellular and molecular mechanisms. The cross-talk between tumours and various stromal cells in the PCa are still not fully understood. We developed the stromal fibromuscular ARKO mice to examine PCa initiation in Pten+/− mice. Our experimental results with these compound mutant mice highlight the crucial roles of stromal fibromuscular AR on the regulation of adjacent epithelial lesion development through a paracrine manner and re-organization of a favorable microenvironment. In this study, we also observed a reduction in collagen deposition, neovasculature formation and immune cells infiltration in this dARKO/Pten+/− mouse as the features of tumour-promoting microenvironments (). Although we still did not have the direct evidence to support that those microenvironmental changes are exclusively due to the stromal AR deletion, it is likely that these changes are attributed to the intermixing effects of loss of stromal AR and reduction of PIN lesion in dARKO/Pten+/− mice. The oncogenic epithelium within the PIN lesion was also able to secrete growth factors and cytokines/chemokines to create a more favorable microenvironment including the ECM remodelling, angiogenesis and immune cell infiltration.
Stromal fibromuscular AR regulates PIN lesion through secretion of paracrine factors and alteration of surrounding microenvironments
Emerging evidence of risk factors such as infectious agents and environmental factors have been proposed to precede the development of PIN lesions (De Marzo et al, 1999
; De Marzo et al, 2007
). Epidemiological studies also suggested that chronic inflammation and/or recurrent infection might contribute to prostate carcinogenesis (Grivennikov et al, 2010
; Karin & Greten, 2005
; Karin et al, 2006
). Proliferative inflammatory atrophy (PIA) lesions of the glandular epithelial foci could be a connection between prostatitis and PIN or PCa (De Marzo et al, 2007
; Palapattu et al, 2005
). The inflammatory lesions in the aging prostate are frequently associated with atrophic epithelium and some fractions of epithelium undergo active proliferation, which may develop into PIN or PCa. In the molecular aspects, several genetic alteration such as glutathione S-transferase pi 1 (GSTP1), NKX3.1, p27 and PTEN have been regarded as the markers of PIN and PCa (Wagenlehner et al, 2007
). Collectively, this epidemiological and genetic evidence might suggest a causative role of inflammation in the complicated process of prostate carcinogenesis, implicating the important contribution of immune cells in the prostate pathogenesis.
Regarding the functions of tumour associated stromal cells, tumour associated fibroblasts (TAFs) are distinguishable from normal fibroblasts and a subset of TAFs has been characterized as myofibroblasts based on the expression of smooth muscle α-actin (SMAα; Santos et al, 2009
). TAFs in skin were demonstrated to be able to produce pro-inflammatory cytokines to foster epithelium growth, but the functions of TAFs in PCa are unclear (Erez et al, 2010
). In addition to TAFs, B-cells residing in the skin were able to ‘educate’ normal fibroblasts to evolve into TAFs, which can produce various pro-inflammatory cytokines in order to evoke tumour initiation (Erez et al, 2010
). Furthermore, the sensitivity to castration in PCa can be modulated through infiltrated B-cells derived lymphotoxin-epithelium engagement in the transgenic adenocarcinoma of the mouse prostate (TRAMP) mice (Ammirante et al, 2010
). Overall, inflammatory responses from either infiltrated immune cells or TAFs may contribute, in part, to the PCa development. In this report, we demonstrated that AR in CAFs could modulate cytokines/chemokines production to affect the immune cells recruitment. Although we have not identified whether T-cells or B-cells are more important in mediating this tumour-promoting inflammatory response in Pten+/−
mice, further studies by crossing T-cell or B-cell deficient mice with Pten+/−
mice may provide direct answers to address this question.
As noted, tissue recombination experiments conducted in immunodeficient mice, which lack functional T- and B-cell mediated immune responses. The elicited T-/B-cells inflammation has been associated with the tumour progression through pro-inflammatory cytokine production and recruitment of macrophages (Balkwill et al, 2005
; Coussens & Werb, 2002
; Karin et al, 2006
). Furthermore, subrenal capsule implementation of tissue recombinants provide enriched nutrition and ample oxygen supply for recombinants growth, however, this does not mimic the real in vivo
prostate microenvironment. The ECM composition, basement membrane components and matrix/metalloproteinase factors are distinct between renal capsules and prostate. Therefore, by generating conditional stromal fibromuscular ARKO mice with the cre-loxP system, we can breed with other transgenic/KO mice such as TRAMP, Nkx3.1 KO, p53 KO and c-Myc transgenic mice to further understand stromal AR roles in vivo
with intact immune systems.
Interestingly, a previous report has suggested that human prostate tumour samples with higher mesenchymal stem cells (MSCs) marker CD90 expression might contain the tumour-promoting potentials of CAFs (Zhao & Peehl, 2009
). To characterize the MSCs population in the Pten+/−
PrSCs and explore the potential AR regulation, we performed QPCR analysis to examine MSC markers (CD29, CD44 and CD90). The results showed that PrSCs isolated from Pten+/−
mice expressed these three MSC markers, with higher expression in CD90 (Supporting Information Fig S5D
). By using genetic knockout (WT vs. ARKO bone marrow-MSCs, BM-MSCs) or AR siRNA knockdown approach in Pten+/−
PrSCs to disrupt AR expression (Supporting Information Fig S5A
), we did not see ablation of AR could reverse these MSC marker expressions (Supporting Information Fig S5B–D
). However, we could observe that loss of AR in BM-MSCs could enhance self-renewal by CFU-F assay (Supporting Information Fig S5E
). Therefore, we reasoned that knockdown of stromal AR might increase MSCs population, which has the potential to promote PCa development, however, this may not happen in the dARKO-Pten+/−
mice because dARKO-Pten+/−
mice showed reduced PIN lesion.
Finally, the AR degradation enhancer, ASC-J9®, has been extensively examined in several types of cells such as motor neuronal cells, bladder cancer cells, hepatocarcinoma cells and macrophages in wound tissues (Lai et al, 2009
; Ma et al, 2008
; Miyamoto et al, 2007
; Yang et al, 2007
). The results demonstrated that ASC-J9® was able to degrade AR in those various cells to achieve therapeutic effects. Here, we evaluated the effects of ASC-J9® in Pten+/−
mice and demonstrated ASC-J9® can reduce PIN development partially through stromal AR degradation. Taken together, our study not only elucidates the roles of stromal fibromuscular AR in prostate tumourigenesis via characterization of dARKO-Pten+/−
mice, but also provides an alternative therapeutic candidate using implementation of ASC-J9® for early PCa intervention.