Comprehensive quantitative profiling of the matrix metalloproteinases in bladder cancer

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This work represents the first comprehensive, quantitative profile of the expression of the Matrix Metalloproteinases (MMPs) and their endogenous tissue inhibitors (TIMPs) in a large group of human bladder tumor samples using real-time Polymerase Chain Reaction (PCR). The MMPs are a family of extracellular, zinc dependent endopeptidases with a broad spectrum of enzymatic activity against most components of the extracellular matrix. They are involved in organogenesis and remodeling in normal tissue, and are over expressed in a number of malignant processes. There are 24 known members of the human MMP family, and 4 primary TIMPs. From a tumor bank consisting of 167 human Transitional Cell Carcinoma (TCC) samples and 20 samples of normal bladder tissue, total RNA was extracted and 1μg of complimentary DNA was reverse transcribed. In addition to spectrophotometry and gel electrophoresis, the 18s rRNA gene was used as an endogenous control to assess RNA quality and normalize for slight variations in the amount of total RNA in each sample. Real-time PCR was performed for each of the MMP and TIMP genes; the cycle threshold (C_T), at which amplification became exponential was used as a marker of the quantity of initial target RNA. Spearman’s Rank Correlation was performed to define the relationship between tumor grade and MMP/TIMP RNA expression (P values <0.001 considered significant). MMPs 1, 2, 7, 11, and 28, and the membrane bound MMPs 14 and 15 were all highly expressed in TCC samples. MMPs 3, 9, 10, 12, 13, 16, 17, 19, 23, 24, and 25 were moderately expressed, whilst there was little or no expression of MMPs 8, 20, 21, 25, 26 and 27. TIMPs 1 and 3 were very highly expressed, and TIMPs 2 and 4 were highly expressed. The following MMPs demonstrated a highly significant positive correlation between tumor grade and expression: MMPs 1, 3, 8, 9, 10, 11, 13, 14, 15, 25 and 27. MMP19 showed a significant negative correlation, as did TIMP 4. The other TIMPs, whilst highly expressed, did not correlate with tumor grade. Whilst collation of complete clinical information is currently ongoing, initial analysis of MMP expression and tumor grade demonstrate highly significant correlation for many MMPs including 3, 10, 15, 25 and 27, which have not previously been shown to correlate with an aggressive bladder tumor phenotype. It is the localization and function assessment of these key, up regulated MMPs and TIMPs which will form the basis of further investigation.