Cathepsin D stimulates the activities of secreted plasminogen activators in the breast cancer acidic environment

Two proteases cathepsin D (cath D) and urokinase plasminogen activator (uPA) are tissue markers associated with an increased risk of metastasis in breast cancer. We investigated whether cath D, the major aspartyl protease overexpressed by breast cancer cells can trigger a proteolytic cascade via activation of plasminogens at the extracellular pH measured in hypoxic tumors. The effects of the aspartyl protease inhibitor pepstatin on the plasminogen activator (PA) system were analysed by conditioning media of human MDA-MB231 breast cancer cells at pH 6.6 and pH 7.4. Zymography analysis of culture media showed that pepstatin inhibited the secreted activity of tissue-type plasminogen activator (tPA) but not that of uPA. tPA was identified on the basis of the molecular weight, the immunoreactivity with relevant antibodies and the resistance to amiloride, a specific uPA inhibitor. The secreted tPA activity measured by a chromogenic assay in the presence of amiloride was also inhibited by pepstatin at pH 6.6. Surprisingly, pepstatin did not affect secreted tPA protein concentration but markedly increased the amount of the secreted plasminogen activator inhibitor-1 (PAI-1). We conclude that cath D overexpressed by these cells, stimulates at pH 6.6, but not at neutral pH, the extracellular PA proteolytic activity indirectly via PAI-1 proteolysis. This suggests that cath D at acidic pH close to the hypoxic regions of solid tumors, contributes to trigger a proteolytic cascade facilitating cancer cell invasion and metastasis.