T58: An important role of proteoglycans in the interactions of normal and cancer prostate cells with fibroblasts

Abstract

Over time, study of malignant tumours was concentrated on cancer cells only. Now, however, an important role of cancer cell microenvironment and interactions cell with extracellular matrix (ECM) is becoming evident. Tumour cells modify normal ECM to cancer-associated matrix (CAM) thereby creating favorable conditions for the growth and development of tumors. One of the main cellular components of ECM are fibroblasts that synthesise extracellular matrix components including proteoglycans (PG) – complex glycosylated molecules consisting of core protein and one or more carbohydrate chains of glycosaminoglycans. PGs are expressed on the cell surface and in extracellular matrix for all mammalian cells and tissues, playing an important role in cell–cell and cell–matrix interactions and signaling. We have hypothesised that proteoglycans are related to transformation of normal ECM to cancer-associated matrix under the influence of prostate cancer cells, and understanding of the mechanism this transformation is very important issue. Thus, the aim of our work was to investigate a possible involvement of proteoglycans in the transformation normal fibroblasts to cancer-associated fibroblasts (CAFs). To study this problem, we co-cultivated fibroblasts with normal prostate cells (PNT2) and prostate cancer cells with different physiological characteristics (LNCaP, PC3, DU145) with subsequent separation of the cells and proteoglycans expression analysis. Firstly, we co-cultivated human fibroblasts with normal prostate epithelial cell PNT2. Expression patterns of main proteoglycans (syndecan-1, glypican-1, perlecan, aggrecan, versican, decorin, lumican, CSPG4/NG2, brevican) were determined in fibroblasts before and after co-culture with PNT2 cells using RT-PCR analysis. It was shown that after co-culture the total level of proteoglycans expression in fibroblasts was reduced by 2-fold, although the fibroblasts expressed a similar set of proteoglycans with the exception of CSPG4/NG2 (which was completely disappeared). The results suggest that normal prostate cells PNT2 affect composition and transcriptional activity of proteoglycans in PNT2-exposed fibroblasts compared with fibroblast monoculture, and this is a normal process of mutual adaptation of these cells to each other. At the next step, fibroblasts were co- cultivated with prostate cancer cells of various rate of aggressiveness (hormone-dependent non-metastatic LNCaP cells, hormone-independent metastatic PC3 and DU145 cells). Co-culture fibroblasts with LNCaP cells resulted in significant down-regulation of expression of glypican-1 and up-regulation of CSPG4/NG2 expression in the fibroblasts, changing overall proteoglycans expression pattern in the cells. Interestingly, CSPG4/NG2 expression is more characteristic for nerve cells and cancer stem cells, and up-regulation of the gene in the cancer cell-exposed fibroblasts may argue for their dedifferentiation. Co-culture fibroblasts with more aggressive DU145 and PC3 resulted in similar results with an additional down-regulation of versican expression. This data show, that prostate cancer cells affect normal fibroblasts by the other way than normal prostate cells and stimulate them to express another set of proteoglycans, which usually are expressed in poor-differentiated or morphologically different cells. Data obtained by immunocytochemical analysis confirmed RT-PCR data and showed specific activation of some proteoglycans at the region of contact between fibroblasts and normal but not cancer prostate cells in co-culture. Taken together, obtained results show that normal prostate epithelial cells affect surrounding fibroblasts and modify transcriptional activities of proteoglycan-coding genes in the cells, while cancer cells change the proteoglycans expression in cancer cells-exposed fibroblasts seems contributing to reprogramming of normal fibroblasts to CAFs. Understanding the mechanisms of this reprogramming may help to develop a new approach for cancer therapy based on the return reprogramming of CAFs to normal fibroblasts and correction of tumour microenvironment. The work was supported by the research Grant from Ministry of Education and Science of the Russian Federation, Scholarship of Russian Federation President for young scientists (Suhovskih A.V.), FEBS Short Term Fellowships (AVS) and UICC International Cancer Technology Transfer Fellowships (EVG, ICR/2015/353372).