From DEPARTMENT OF ONCOLOGY-PATHOLOGY CANCER CENTRUM KAROLINSKA Karolinska Institutet, Stockholm, Sweden ANALYSIS OF STROMA-DERIVED FACTORS WITH POTENTIAL PROGNOSTIC AND THERAPEUTIC SIGNIFICANCE IN PROSTATE CANCER

Prostate cancer is the most common malignancy in Sweden with about 9000 new cases diagnosed every year. New markers are needed to improve diagnostic accuracy. The most commonly used tissue-biomarkers are basal cell markers and AMACR, often used in combination. We identified three potential tissue biomarkers, CYCS, ICK and IKBKB, by using the Human Protein Atlas database and investigated their diagnostic accuracy. The potential of these biomarkers was also compared with AMACR. Immunohistochemical analysis of the markers was performed on a tissue microarray (TMA) consisting of tissue from 40 prostate specimens, including benign prostatic tissue, atrophy, high-grade prostatic intraepithelial neoplasia (HGPIN) and prostate cancer. In addition, qRT-PCR analysis of malignant and benign frozen tissue samples from 32 radical prostatectomy specimens was performed. All four biomarkers showed a higher protein expression in prostate cancer and HGPIN than in benign tissue. The prognostic accuracy was highest for AMACR, but the results indicate that in some cases CYCS, ICK and IKBKB may serve as additional diagnostic markers. It is known that prognostic information can also be derived from tumor stroma. The prognostic value of stromal expression of PDGFRβ was therefore evaluated by immunohistochemical analysis of PDGFRβ on a TMA containing cancer and nonmalignant tissue from more than 300 prostate cancer patients. The association between stromal staining intensity and a number of clinical characteristics were then analyzed. Expression of PDGFRβ in non-malignant and tumor stroma was associated with high Gleason grade and reduced cancer specific survival. Cancer associated fibroblasts (CAFs) are found in many solid tumors and promote tumor growth and progression. Identification and inhibition of molecules mediating these interactions constitute an attractive strategy for development of new cancer therapies. By comparative analyses of CAFs and normal fibroblasts from prostate tissue we have identified a number of genes upregulated in prostate CAFs. CXCL14, an orphan chemokine, was the most upregulated transcript. Overexpression of CXCL14 in fibroblasts increased their proliferation and migratory capacity. Also over-expression in fibroblasts of CAF led to increased ability of these cells to stimulate proliferation and migration of prostate cancer cells. Furthermore, fibroblasts overexpressing CXCL14 enhanced tumor growth, vascularisation and macrophage infiltration in a stromadependent prostate cancer model. Another transcript identified to be upregulated in prostate CAFs was GDF15, a member of the TGFβ superfamily. GDF15 was shown to stimulate fibroblast proliferation and enhanced growth, migration and invasion of prostate cancer cells. Fibroblasts overexpresssing GDF15 was also able to stimulate prostate xenograft growth when coinjected with prostate cancer cells. Interestingly, these fibroblasts also increased the ability of tumor xenograft to promote growth at a distant site suggesting direct or indirect systemic pro-tumoral effects of fibroblast-derived GDF15. These studies thus identify a set of new diagnostic and prognostic markers for prostate cancer and stroma-derived potential therapeutic targets. LIST OF PUBLICATIONS I. Lars Häggarth, Christina Hägglöf, Sara Jonmarker Jaraj, Kenneth Wester, Fredrik Pontén, Arne Östman and Lars Egevad Diagnostic biomarkers of prostate cancer Manuscript II. Christina Hägglöf, Peter Hammarsten, Andreas Josefsson, Pär Stattin, Janna Paulsson, Anders Bergh and Arne Östman Stromal PDGFRβ expression in prostate tumors and non-malignant prostate tissue predicts prostate cancer survival PLoS One 2010 May 20;5(5):e10747 III. Martin Augsten*, Christina Hägglöf *, Eleonor Olsson, Claudia Stolz, Panagiotis Tsagozis, Tetyana Levchenko, Mitchell J. Frederick, Åke Borg, Patrick Micke, Lars Egevad and Arne Östman CXCL14 is an autocrine growth factor for fibroblasts and acts as a multi-modal stimulator of prostate tumor growth Proc Natl Acad Sci. 2009 Mar 3;106(9):3414-9 IV. Francesca Bruzzese, Christina Hägglöf, Alessandra Leone, Arne Östman, Alfredo Budillon and Martin Augsten Local and systemic pro-tumorigenic effects of fibroblast-derived GDF15 Manuscript * Authors contributed equally