The expression and regulation of membranetype matrix metalloproteinases (MT-MMPS) in prostate cancer

Prostate cancer (PCa) represents the most frequently diagnosed cancer and the second leading cause of cancer death in males. Initial development and progression of the disease is mainly regulated by androgens. However, the pathology of the disease may progress to a loss of hormone dependence, resulting in rapid growth and a metastatic phenotype. Invasion and metastasis of tumour cells results from the degradation of the basement membrane (BM) and extracellular matrix (ECM). The degradation of the BM and ECM is in part mediated by a family of proteinases called the matrix metalloproteinases (MMPs). Currently more than 20 members of the MMP family have been identified and they are further divided in to sub-classes according to their protein structure. Collectively, MMPs are capable of degrading essentially all ECM components. High expression of some MMPs correlates with a malignant phenotype of various tumours. This study focused on the expression and regulation of a sub-class of MMPs called the membrane-type MMPs (MT-MMPs) in PCa. To date 6 MT-MMPs have been identified and they are characterized by a transmembrane domain, followed by a short cytoplasmic tail (MT1-, MT2-, MT3- and MT5-MMPs) or a glycosylphosphatidylinositol (GPI) moiety (MT4- and MT6-MMPs). MT-MMPs are thought to play a key role in tumour cell invasion by virtue of their ability to activate MMP-2 (a secreted MMP, which is implicated in many metastatic tumours) and their direct degradation activity on ECM components. Elevated MT-MMP expression has been shown in breast, colon, skin, stomach, lung, pancreas and brain cancers. Until very recently there had been no studies conducted on MT-MMPs in PCa. The few studies preceding or occurring in parallel with this one, have mainly reported the mRNA expression of these enzymes in PCa. Most studies have focused on MT1-MMP. Thus, at the commencement of this project there were many unexplored aspects of the expression and regulation of the broader MT-MMP family in PCa. The aims of this study were to examine: 1 a) The expression of MT-MMPs in prostate cancer cell lines using RT-PCR and western blot analysis and b) expression of MT1-MMP and MT5-MMP in BPH (benign prostatic hyperplasia) and PCa clinical tissue sections by immunohistochemistry. 2) The regulation of MT1-MMP, MT3-MMP and MT5-MMP in PCa cell lines by Concanavalin A (Con A), phorbol-12-myristate 13-acetate (PMA), dihydrotestosterone (DHT) and insulin-like growth factors I and II (IGF I and IGF II) using western blot analysis. In this study RWPE1, a transformed but non-tumorigenic prostate cell line was used as a "normal" prostate cell model, ALVA-41 and LNCaP as androgen-dependent PCa cell models and DU-145 and PC-3 as androgen-independent PCa cell models. The mRNA expression for the 6 MT-MMPs was determined by RT-PCR. The results indicate that MT1- and MT3-MMP were detected in all cell lines. This is the first study to report MT1-MMP mRNA expression in LNCaP cells and MT3-MMP mRNA in DU-145 cells. MT2-MMP mRNA was detected in only LNCaP and DU-145 cells, whilst MT5-MMP was detected in PC-3, DU-145 and LNCaP cells. nterestingly, MT2-, MT4-, MT5- or MT6-MMP mRNA expression was not detected in the "normal" cell line RWPE1, perhaps indicating an induction in gene transcription in tumour cells. MT4-MMP mRNA was only detected in the androgen-independent cell lines, indicating a potential role in the invasion and metastasis processes of the aggressive androgen-independent PCa. In this study, very low expression of MT6-MMP was detected only in LNCaP and DU-145 cells. Previously there had been no reports on the expression of MT6-MMP in the normal or cancerous prostate. Due to the mRNA of MT1-, MT3- and MT5-MMPs being the predominant MT-MMPs expressed in the current study, and the availability of suitable antibodies against them, the protein expression of these three MT-MMPs was studied by western blot analysis. MT1-, MT3- and MT5-MMP protein expression was detected in the cell lysates and conditioned medium (CM) of RWPE1, LNCaP and PC-3 cells. For each MT-MMP, various protein species were detected including putative proforms, mature (active) forms, processed or fragmented forms as well as soluble or shed forms. The presence of soluble or shed forms of MT-MMPs in the CM of cultures of "normal" and PCa cells could imply one of the following mechanisms: ectodomain shedding by either extracellular sheddases, the secretion of intracellular processed proteins without the transmembrane domain, the release of membrane vesicles containing membrane-bound enzymes, or the presence of alternatively spliced mRNA, which gives rise to MT-MMPs without a transmembrane domain. Further characterization of these various forms, including their amino acid sequence, is required to fully elucidate their structural composition. Despite the detection of the mRNA, we did not detect the cell-associated proteins of MT1-MMP and MT5-MMP and only very low expression of MT3-MMP in DU-145 cells (CM of DU-145 cells were not screened for soluble forms of the enzymes). This is the first study to report MT5-MMP expression at the protein level in prostate derived cell lines. Immunohistochemistry was carried out on benign prostatic hyperplasia (BPH) and PCa clinical tissues using MT1- and MT5-MMP antibodies to determine their cellular localisation in benign and cancer glands. MT1- and MT5-MMPs were expressed in BPH and moderate and high grade PCa. MT1-MMP expression was highest in moderate grade cancer compared to BPH and high grade cancer. MT1-MMP expression was predominantly observed in the cytoplasm of secretory epithelial cells of both benign and cancer glands, although in cancer glands, some nuclear staining was also observed. Stromal expression of MT1-MMP was only observed in high grade cancer. This study is the first to report the immunolocalization of MT5-MMP outside the brain and in kidneys of diabetic patients. MT5-MMP was predominantly expressed in the cytoplasm of the secretory cells in benign glands. In the cancer glands, staining was heterogeneous with low to intense staining, mainly in the nuclei, plasma membrane and cytoplasm of secretory epithelial cells. Stromal expression of MT5-MMP was only observed in cancer tissues, particularly in high grade cancer. To study the regulation of MT-MMPs in PCa, we treated LNCaP and PC-3 cells, with either Con A, PMA, DHT or IGF-I and -II and studied the protein expression of MT1-, MT3- and MT5-MMPs by western blot analysis. Con A and PMA have been shown to stimulate MMP expression in other cell systems. Con A treatment showed a general increase in the protein expression of MT1-, MT3- and MT5-MMPs. By far the greatest induction by Con A observed was the nearly 4 fold increase in MT5-MMP expression caused by 40μg/mL Con A treatment of PC-3 cells. PMA treatment of LNCaP and PC-3 cells appeared to increase shedding or secretion of all three MT-MMPs in to the CM. This increase in the soluble forms corresponded to a decrease in cell-associated forms in LNCaP cells. Treatment of LNCaP with DHT alone and treatment of LNCaP and PC-3 cells with IGF-I and -II alone failed to detect any change in expression of MT1-MMP. The information gathered in this study on MT-MMPs with respect to cellular localization, expression levels and regulation by growth factors or chemicals that mimic their actions, will aid in our understanding of the role of MT-MMPs in PCa. This study provides strong preliminary data for further research, particularly with respect to functional studies of MT-MMPs in PCa. Understanding the processes which govern the actions of such proteins as these will provide potential insights into development of new management and therapeutic regimens to prevent cancer progression.