Reactive stroma and tumor evolution
Authors: David R. Rowley, Feng Yang, Jennifer A. Tuxhorn, Steven J. Ressler, Stephanie Jones McAlhany, Doug Strand, Isaiah Schauer, Dang D. Dang
Journal: Cancer Research (2005)

SY12-2  Our previous studies have shown that reactive stroma in prostate cancer is characterized by progressive changes in stromal cell phenotype from differentiated smooth muscle to an activated myofibroblast / fibroblast population. Reactive stromal cells also exhibit elevated synthesis of pro-collagen I, tenascin, and fibroblast activation protein (FAP). In addition, we have shown that reactive stroma originates immediately adjacent to premalignant prostatic intraepithelial neoplasia (PIN) foci in human prostate cancer. Many PIN foci also overexpress TGF-β1 suggesting this factor is a mediator of reactive stroma. In addition to stimulating stromal cell wound repair responses, TGF-β1 functions to stimulate angiogenesis by regulating endothelial-pericyte interactions, differentiation of pericytes, and vessel stability. Moreover, TGF-β1 regulates expression of stromal growth factors important to wound repair stroma and angiogenesis, including connective tissue growth factor (CTGF) and fibroblast growth factor-2 (FGF-2). Reactive stroma in human PIN and cancer is associated with both elevated microvessel density and FGF-2 expression. Together, these data suggest that TGF-β1 and downstream regulators are critical for reactive stroma biology and angiogenesis in the premalignant PIN cancer microenvironment. A central hypothesis is that epithelial overexpression of TGF-β1 in PIN induces a typical stromal wound repair response via both direct and indirect actions mediated through downstream factors such as FGF-2. This stromal response continues to co-evolve with the foci of epithelial carcinoma cells during cancer progression. It is likely that the co-evolution of a proper reactive stroma is required for organ-confined progression of the carcinoma. Accordingly, it is becoming clearer that the reactive stroma microenvironment is an important component of overall tumorigenesis and is likely to involve a complex interplay and feedback loops between key stromal growth factors. We have developed the differential reactive stroma (DRS) xenograft model to address the role of reactive stroma, TGF-β1, CTGF and FGF-2 in prostate cancer. Differential tumorigenesis and angiogenesis was observed using different human prostate stromal cell lines co-inoculated with LNCaP human prostate cancer cells into nude mice. The inclusion of either human or mouse prostate stromal cells with LNCaP cancer cells resulted in a rapid and consistent angiogenesis and tumorigenesis as compared to LNCaP xenografts without the inclusion of stromal cells. Inoculated stromal cells were localized in reactive stroma of resulting tumor and were also observed in the vessel wall in a perictye position, suggesting a direct role in angiogenesis. Gene expression analysis showed several TGF-β1 regulated genes (including CTGF) were differentially expressed in human prostate stromal cells capable of supporting and promoting LNCaP xenograft tumors. Of interest, TGF-β1 was also shown to stimulate release of FGF-2 from prostate stromal cells in vitro in a Smad3 dependent manner. Neutralization of TGF-β1 activity in xenograft LNCaP / stromal cell tumors resulted in inhibited tumorigenesis and a significant (3.5 fold) reduction in microvessel density. Similarly, xenograft LNCaP tumors co-inoculated with TGF-β receptor II null or Smad3 dominant negative prostate stromal cell lines resulted in significantly decreased tumorigenesis and angiogenesis. These data suggest that TGF-β1 is a key mediator of the immediate reactive stroma microenvironment and functions directly and indirectly to stimulate epithelial tumorigenesis via actions in the stromal compartment including the regulation of angiogenesis. To address the role of CTGF, FGF-2 and other TGF-β1 regulated genes, DRS tumors were constructed with stromal cells engineered to overexpress genes of choice, either in a regulated manner (GeneSwitch) with Miphepristone (RU 486) or by transducing cells via retroviral infection. Prostate stromal cells engineered to over express CTGF resulted in elevated tumorigenesis and a stimulation of angiogenesis during early LNCaP / stromal cell xenograft tumor formation. Overexpression of FGF-2 in prostate stromal cells resulted in an elevated stromalization leading to fibrosis and differential tumor growth. Of interest, overexpression of FGF-2 in stromal cells null for TGF-β receptor type II or positive for dominant negative Smad3, resulted in an overt stromalization fibrosis that appeared to inhibit carcinoma cell survival in LNCaP / stromal cell xenograft tumors. In addition, the overexpression or inclusion of recombinant ps20 (an additional TGF-β1 regulated gene) in stromal cells resulted in increased tumorigenesis and angiogenesis. The ps20 protein also functioned to induce vascular cell adhesion molecule-1 (VCAM1) and induce the migration of human umbilical vein endothelial cells in vitro. These studies suggest that a principal function of carcinoma-expressed TGF-β1 in early prostate cancer is to induce a cascade of downstream regulatory factors in reactive stroma, including CTGF, FGF-2, and ps20 which leads to a wound repair type of reactive stroma response and induction of angiogenesis in the immediate tumor microenvironment. Our data suggests that the balanced actions of TGF-β1 and FGF-2 in the stromal compartment may be what regulates the ratio of fibroblasts to myofibroblast cell types in carcinoma associated reactive stroma. Moreover, it is appears that unbalanced action of FGF-2 in a stromal environment that is restricted from TGF-β1 signaling, results in a non wound repair fibrosis type of stromal response that might actually be tumor inhibitory. Together, these data suggest a complex feedback loop between TGF-β1 and FGF-2 signaling in tumor reactive stroma and point to how significant the balanced stromal response is to tumor progression. The reactive stroma microenvironment might play a role in metastatic progression as well. A hallmark of more advanced prostate cancer progression is epithelial to mesenchymal transition (EMT) as evidenced by expression of mesenchymal markers and acquisition of a more migratory and invasive phenotype. These changes include a splice switch of the FGF receptor 2 IIIb to the IIIc isoform (FGFR2IIIc) and expression of FGF receptor 1 (FGFR1) in carcinoma cells. Both FGFR2IIIc and FGFR1 are cognate receptors for FGF-2. Carcinoma cells expressing these receptors would be expected to have a growth advantage in this reactive stroma microenvironment, which is elevated in FGF-2 expression and possibly the release of FGF-2 protein. Accordingly, elevated FGF-2 in the adjacent reactive stroma microenvironment might provide a selection advantage for carcinoma epithelial cells undergoing EMT and differential expression of these FGF receptors. This would be expected to lead to tumors that are composed of carcinoma cells expressing mesenchymal proteins with a more aggressive and invasive behavior. Together, these data suggest that reactive stroma is important both in early progression of prostate cancer and during EMT changes, which ultimately lead to a stromal-independent progression and metastasis. Our future studies will continue to be focused on dissecting key downstream signaling mechanisms and co-regulatory loops of TGF-β1, CTGF and FGF-2 action in reactive stroma and carcinoma cell biology during prostate cancer progression.