MMPs Identified in Urine of Patients with Cancer

Gelatin zymography has been used to demonstrate the presence of several forms of MMPs in the urine of patients with cancer. The potential for using this test for early diagnosis or staging of cancer has been proposed.

Based on the molecular weight of MMP-2 and MMP-9 (72 and 92 kDa, respectively) and the glomerular filtration limit of ~45 kDa, it was not anticipated that MMPs would be detected in urine. Nonetheless, employing gelatin substrate zymography and immunoassays, Marguilies et al. (M6) demonstrated increased levels of type IV collagenase (MMP-2) in the urine of 70% of patients with transitional cell carcinoma of the bladder. The major form of the enzyme in urine was the amino terminal fragment of MMP-2 (45 kDa). A monoclonal antibody (CA-4001) recognized this major fragment of MMP-2 in all patients with transitional cell carcinoma of the bladder, but in none of the control cases. Tissue sections of normal urinary bladder epithelium were negative for immunoreactivity. In contrast, tissue sections of papillary transitional cell carcinoma of the bladder were positive. This result supported the assumption that the source of urinary MMP-2 as well as enzyme inhibitor complexes and enzyme fragments were at least partially derived from the carcinoma itself.

In a subsequent study, Moses et al. (M16) reported that MMP-2 and MMP-9 in urine correlated with the presence of malignant disease, not just limited to the genitourinary tract. The presence of biologically active MMP-2 or MMP-9 alone in the urine was an independent predictor of organ-confined cancer; the higher molecular weight MMP species (>150 kDa) in the urine served as independent predictors of metastatic cancer (M16). The frequency of detection of the three MMP species in the urine was as follows: normal or no evidence of disease (11-20%), cancer (71%), metastatic cancer (90%). The frequency of urinary MMPs was significantly higher in patients with prostate and breast cancer, 75 and 100%, respectively. Odds of metastatic cancer were ~30 times greater when the higher molecular weight MMP species were present in urine than when this marker was absent. A 125 kDa MMP was detected only in the urine of patients with breast cancer (M16). In-depth studies by Li et al. (Y2) demonstrated that the 125 and 115 kDa gelatinolytic bands represent complexes of MMP-9 and neutrophil gelati-nase-associated lipocalin (NGAL). They further demonstrated that lipocal-cin serves to protect MMP-9 from degradation, thereby preserving MMP-9 enzymatic activity.

Studies by other investigators have confirmed the detection of MMPs in the urine of patients with cancer. In normal subjects, Monier et al. (M13) reported that the level of TIMPs in urine samples was higher than active MMPs (as measured by ELISA). In cancer patients, increased urinary levels of proMMP-9 and active MMP-2 (measured by substrate zymography) with reduced TIMP-2 levels correlated with higher stage and histological grade of urothelial cancers. Contrary to expectation, reduced MMP-9 and NGAL (lipocalin complexes with MMP-9) levels in urine were initial hallmarks of clinical relapse. The imbalance between increased MMP-2 and MMP-9 and decreased TIMP-2 levels appears to be linked to tumor stage and grade and, more importantly, to clinical events. Changes in the MMP-9 activation state and a lack of neutrophil-associated lipocalin (NGAL) were proposed as novel markers of tumor progression (M13). Monier et al. (M14) also employed continuous elution electrophoresis for isolating urinary MMPs. Significantly increased levels of MMP-2 and MMP-9 and a high molecular weight MMP-9 band (115 kDa) were detected by zymography in the urine of patients with epithelial cancers including breast, colon, and prostate; even higher enzyme levels were detected in the urine of patients with bladder cancer. Bladder washings revealed increased levels of proMMP-9 but not MMP-2 in patients with evolving bladder cancer as compared to control subjects (M14).

Durkan et al. (D5) reported that urinary MMP-1 was detected in a higher percentage of patients with advanced stage (T2-T4) and grade (3) bladder cancers than in patients with early stage (cis/Ta/T1) or grade (1-2) bladder cancer. Patients with increased levels of urinary MMP-1 had higher rates of disease progression and death from bladder cancer. All patient groups also had higher levels of urinary TIMP-1 than the control group. Urine TIMP-1 levels strongly correlated with tumor size. Progression-free survival rates were lower for patients with high urine TIMP-1 concentrations.

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