A great deal of evidence now supports bioactive sphingolipids as key players in multiple aspects of cancer biology. By its metabolism of ceramide, acid ceramidase has the potential to promote cancer by reducing the pro-apoptotic lipid ceramide and favoring generation of S1P, which has demonstrated roles in preventing apoptosis as well as promoting inflammation and cell invasion 15–18
. Our previous work supports an oncogenic role for AC in prostate cancer, including promoting prostate cancer cell invasion9, 19, 20
. In this study we investigated the mechanism of AC-induced cell invasion and discovered a prominent role for AC and S1P in matrix degradation by upregulation, pericellular localization, and secretion of cathepsin B.
Our initial investigation revealed that AC overexpression in both stable cell lines and in transiently transduced cells overexpressed cathepsin B at the protein level and had elevated cathepsin B mRNA by RT-PCR. Since we found that AC did not affect cathepsin B protein stability, we concluded that AC-induced cathepsin B upregulation was due either to increased transcription or post-transcriptional mRNA stability. It is reported in the literature that Ets1 is a key transcription factor for cathepsin B, a finding supported by the presence of three proposed Ets1 binding sites in the cathepsin B promoter region21, 22
. Our studies reveal that both AC and S1P promote both elevated nuclear Ets1 and increased Ets1 binding to the cathepsin B promoter by chromatin immunoprecipitaiton.
Cathepsin B has been implicated as a contributor to cell death including apoptosis and lethal autophagy23
. Thus, it was important for us to determine whether AC-induced cathepsin B was also a contributor to cell death. Supplementary Figure 8
demonstrates that while cathepsin B appears to be an important factor in ceramide-induced apoptosis, neither a reduction in cell viability nor an increase in apoptosis seems to be associated with overexpression of AC. These data are in keeping with our observations that AC promotes pericellular localization and membrane association of cathepsin B, which is associated with increased malignancy and invasion 24
Essential for new achievements in preventing morbidity and mortality associated with prostate cancer, and all solid cancers for that matter, will be a more comprehensive understanding of what promotes cancer invasion and metastasis, since exceptional cure rates are available to patients with localized disease whereas dismal prognoses accompany invasive and metastatic disease. In this study, we showed that cathepsin B is pericellularly redistributed when AC is overexpressed. Pericellular redistribution of cathepsin B is associated with cancer cell invasion, presumably by degradation of the extracellular matrix. Importantly, we demonstrate here that cathepsin B is actually localized to the outer leaflet of the cell membrane as flow cytometric analysis and confocal microscopy of non-permeabilized cells revealed antibody recognition of cathepsin B on the cell surface but not when first treated with trypsin. This finding is key as it demonstrates that cathepsin B is not only pericellular in distribution, but has direct access to the extracellular matrix where its role as a protease can contribute to tumor microenvironment remodeling and tumor cell invasion. AC overexpressing cells had distinct cathepsin B-rich invasive structures present with strong cathepsin B staining at the leading edge when plated on a synthetic matrix. These invasive structures were not present when cells were incubated with a specific cathepsin B inhibitor, strongly supporting a role for cathepsin B matrix degradation as a factor in AC-overexpression induced cell invasiveness.
These findings complement a growing body of literature asserting the role cathepsin B plays in extracellular matrix degradation and invasion of tumor cells. It is known that cancer cells express cathepsin B on the cell surface and secrete it into the extracellular space. Bellezza, et al, found increased cathepsin B and procathepsin B on the surface of secreted prostatomes from LNCaP and PC-3 compared to prostatomes from healthy ejaculate25
. This study is of particular interest, as most prostate cancers have elevated AC8
, thus we may speculate that high levels of AC in those cell lines drives upregulation and secretion of cathepsin B, as descried in this study. Extracellular cathepsin B not only exhibits an invasive phenotype in vitro, as in this study, but also in animal models. In a metastatic model of colon cancer, cathepsin B activity was detected at the cell membrane, and treatment with an inhibitor of extracellular cathepsin B inhibited formation of liver metastases26
, hightlighting the importance of cathepsin B, particularly its extracellular form, in invasion and metastasis. Interestingly, a recent study demonstrated that invasion of prostate cancer cells in response to hepatocyte growth factor was incumbent upon anterograde lysosome trafficking and was accompanied by secretion of cathepsin B27
. The present study elucidates a novel mechanism by which cathepsin B is upregulated and secreted, and therefore adds knowledge of how prostate cancer may progress from localized to invasive.
Our findings that AC induces important changes in the regulation of cathepsin B are made relevant to the actual disease process by the finding that AC and cathepsin B are co-upregulated in prostate cancer tissues. Our studies lead us to propose that a contributing mechanism of cathepsin B upregulation in these patients is due to AC upregulation promoting local S1P secretion and transcriptional upregulation of cathepsin B through Ets1. While localized prostate cancer is slow to progress and frequently cured, our study points to AC upregulation as an important mediator of prostate cancer invasion and perhaps metastasis, the defining features of deadly prostate cancer. As such, AC and cathepsin B may be important contributors to the pathogenesis and progression of deadly prostate cancer, and each represents an opportunity for further study and possibly intervention.