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Characterization of Expression of Matrix Metalloproteinases and Tissue Inhibitors of Metalloproteinases in Prostate Cancer Cell Lines

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Characterization of Expression of Matrix Metalloproteinases and Tissue Inhibitors of Metalloproteinases in Prostate Cancer Cell Lines Prostate Cancer and Prostatic Diseases (2003) 6, 15–26 ß 2003 Nature Publishing Group All rights reserved 1365–7852/03 $25.00 www.nature.com/pcan Characterization of expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases in prostate cancer cell lines MM Daja1, X Niu,1,2, Z Zhao1, JM Brown1 & PJ Russell1* 1Oncology Research Centre, Prince of Wales Hospital, Sydney, and Department of Medicine, University of New South Wales, NSW, Australia; and 2School of Biochemistry and Molecular Genetics, University of New South Wales, Sydney, NSW, Australia Stromal expression of some matrix metalloproteinases (MMPs) has been asso- ciated with increasing tumour burden in prostate cancer. We investigated the expression of mRNA (by RT-PCR) and protein (by zymography and western blotting) of MMPs and endogenous inhibitors (tissue inhibitors of metallopro- teinases, TIMPs) in two parent epithelial prostate cancer cell lines and sublines of increasing invasive/metastatic potential. Expression of membrane type MMPs, MT1-MMP and MT3-MMP mRNA was higher in PC3-derived than in LNCaP- derived lines, whereas MT2-MMP mRNA expression was higher in the LNCaPderived than in PC3-derived cell lines. Active MT1, MT2 and MT3-MMP protein levels were similar in all lines, but processed MT-MMPs, indicative of latent MMP activation, were increased in more aggressive sublines. Expression of MMP-1, MMP-13 and TIMP-1 was higher in the more aggressive sublines and may be implicated in invasive/metastatic ability. Regulation of MMP-1 and MMP-13 expression may offer important therapeutic options for treating patients with prostate cancer. Prostate Cancer and Prostatic Diseases (2003) 6, 15–26. doi:10.1038/sj.pcan.4500609 Keywords: matrix metalloproteinasaes (MMPs); membrane type MMPs (MT- MMPs); human prostate cancer cell lines; LNCaP-derived cell lines; PC-3 derived cell lines; tissue inhibitors of metalloproteinases (TIMPs) Introduction 5 y survival).1,2 Despite extensive studies of prostate cancer progression, it is still not understood why some Prostate cancer is the most common male cancer and cancers remain localized to the prostate, whilst others are second highest cause of cancer mortality in men in highly invasive and metastatic. Such an understanding Western society. It is a heterogeneous disease, which would facilitate advances in diagnostic and therapeutic may be indolent and therefore not require intervention, outcomes. may remain localized to the prostate gland but be clini- A key step in the complex process of cancer progres- cally significant ( > 95% 5 y survival), or may be invasive sion and the establishment of bony metastases is the and metastatic, primarily to lymph nodes and bone (5% degradation of the extracellular matrices. This is achieved by a range of proteases including the matrix metallopro- teinases (MMPs). MMPs form an increasingly diverse family of zinc-dependent endopeptidases3 that can *Correspondence: Professor P Russell, Oncology Research Centre, degrade the basement membrane and extracellular Prince of Wales Hospital, Level 2, High Street, Randwick, Sydney, matrix (ECM) during physiological tissue remodelling, NSW 2031, Australia. E-mail: [email protected] as well as in pathological conditions such as tumour Received 28 February 2002; revised 16 May 2002; accepted invasion and metastasis. Most MMPs are secreted as 28 May 2002 inactive pro-enzymes; their proteolytic activity is regu- MMP and TIMP expression in prostate cancer MM Daja et al 16 lated by zymogen activation by extracellular proteases or of nude mice, and PC3-MM2 was grown from a bone other MMPs or by inhibition of specific inhibitors, tissue metastasis after injection of PC3-M cells.46,47 Using inhibitors of metalloproteinases (TIMPs) or other non- reverse-transcriptase polymerase chain reaction (RT- specific protease inhibitors.4 – 6 This implies that the bal- PCR) and western blotting, we detected differential ance between MMP and TIMP levels is a critical determi- expression of mRNA and protein for a range of MMPs nant of the net proteolytic activity at any given time. and TIMPs in these prostate cancer cells. Of particular MMPs are classified on the basis of their substrate speci- interest, we report for the first time the expression of ficity and include collagenases (e.g. MMP-1, -8 and -13), MMP-13 by prostate cancer epithelial cells and show that gelatinases (eg MMP-2, -9), stromelysins (eg MMP-3, -7, MMP-13, MMP-1 and TIMP-1 expression is relatively -10, -12), membrane-type MMPs (MT-MMPs, eg MT- increased in the PC3 sublines compared with the MMPs -1 to -5, also known as MMP-14, -15, -16, -17, LNCaP-derived lines. MMP-1 and MMP-13 are collage- -24, respectively) and other MMPs (eg MMP-19, -20, -23, nases that preferentially degrade type I and type II -26 and -28).7 – 9 The MT-MMPs contain a transmembrane collagen, respectively. We propose that the overexpression domain anchoring the enzyme to the cell surface.10 Of five of MMP-1 and MMP-13 in invasive prostate cancer cells MT-MMPs (MT1 – MT5-MMP) identified so far,11 – 15 MT1- may contribute to their ability to metastasize, and could MMP is the most characterized and, in association with therefore potentially be used as markers of aggressive TIMP-2, is involved in the activation of pro-MMP-216,17 prostate cancers. and pro-MMP-13.18 MT2- and MT3-MMP can also acti- vate pro-MMP-2 but with lower efficiency. MT-MMPs are synthesised in a latent pro-form (MT1-MMP 63 kDa, MT2- MMP 68 kDa, MT3-MMP 64 kDa) that contains a putative Materials and methods furin or proprotein convertase motif (RXKR). Cleavage at this site produces an active MT-MMP (MT1-MMP 60 kDa, Cell culture MT2-MMP 62 kDa, MT3-MMP 60 kDa), which is further processed to a 45 – 50 kDa form once activation of pro- The human prostate cancer cell line, LNCaP, was from Dr MMP2 has been achieved.19,20 As well as being respon- L Chung (University of Virginia, VA, USA) and LNCaP sible for activating latent MMPs, MT-MMPs also exhibit sublines, LNCaP-C4 and LNCaP-C4-2 cells, from UroCor, proteolytic activity against components of the ECM, USA. PC3 cells were from the American Type Culture potentially facilitating egress of cancer cells from a Collection (ATCC, Rockville, MD, USA) and PC3 sub- tumour deposit. MMP activities are regulated by specific lines, PC3-M and PC3-MM2, from Dr C Pettaway, MD inhibitors, TIMPs, four of which (TIMP-1 – 4) have been Anderson Cancer Centre (TX, USA). LNCaP, LNCaP-C4 identified thus far,22,23 and LNCaP-C4-2 cells were grown in complete T medium In prostate cancer patients, analysis of MMP mRNA (80% v/v Dulbecco’s Modified Eagle Media, 20% v/v F12 and protein levels in patient serum and tissue samples nutrient mixture (both from Gibco BRL Life Technologies, has shown that increased expression of MMP-1,24,25 Melbourne, VIC, Australia), supplemented with 5% fetal MMP-2,26 – 32 MMP-3,24,29 MMP-7 and MMP-927,34 is cor- calf serum (Trace Biosciences, Sydney, NSW, Australia), m related with advanced or metastatic disease. More MT1- insulin (5 g/ml; Novo Nordisk, Denmark), triiodothyr- m MMP is expressed in high-grade prostatic intraepithelial onine (13.65 pg/ml), apo-transferrin (5 g/ml), biotin m m neoplasia (HGPIN) and prostate cancer compared with (0.244 g/ml), adenine (25 g/ml; all from Sigma Chemi- benign prostate tissue.35 However, MMP expression cal Co., St Louis, MO, USA), penicillin (50 U/ml) and during cancer progression is often the result of the streptomycin (0.05 mg/ml; Gibco BRL Life Technologies). stromal microenvironment stimulating MMP production PC3, PC3-M and PC3-MM2 cell lines were maintained in in tumour cells, or stromal cells themselves producing RPMI 1640 media (Gibco BRL Life Technologies), supple- MMPs as they surround an invasive tumour front.36 – 38 It mented with 10% fetal bovine serum (Trace Biosciences), is therefore difficult to ascertain from the literature penicillin (50 U/ml), streptomycin (0.05 mg/ml) and 0.2 mM L-Glutamine (Gibco BRL Life Technologies). All whether increased MMP production is a function of invasive prostate cancer cells per se and whether this cells were maintained at 37 Cin5%CO2 and passaged could potentially be used as a marker to differentiate with trypsin/EDTA (Gibco BRL Life Technologies) when between non-invasive and invasive prostate cancer. confluent. This study aimed to investigate the production of MMPs and TIMPs, in the absence of stroma or ECM, by In vitro invasion assay prostate cancer epithelial cells and their sublines that have been developed for their ability to metastasize in nude The ability of the cell lines to invade a Matrigel2 (Inte- mice. Two parent lines were used. LNCaP39 is androgen- grated Sciences P/L, Willoughby, NSW, Australia) coat- sensitive and non-invasive when implanted subcuta- ing in a transwell chamber assay was assessed using neously (s.c.) in nude mice; an androgen-independent serum-free, antibiotic-free medium in triplicate by the (AI) cell line, LNCaP-C4, obtained from a tumour-bearing method of Repesh.48 Cells were applied at 5000 cells/ mouse 4 weeks after castration, produces a soluble, non- 200 ml (700 ml was added to the outer well to prevent androgenic factor that stimulates PSA production in an dehydration), incubated at 37 C, 5% CO2 for three dou- autocrine fashion. Further characterization of LNCaP-C4 bling times before removing medium from the transwell produced a subline, LNCaP-C4-2, that metastasizes to and outer well, rinsed with PBS and stained with 0.5% lymph node and bone after s.c. or orthotopic implanta- crystal violet in 50% methanol/water for 10 min at room tion.40 – 44 PC345 is AI; PC3-M was derived from a liver temperature.
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