Here we report that the protease inhibitor PI-9 can protect prostate cancer cells from NK-92 cell- induced apoptosis by inhibiting Granzyme B (GrB). We also provide tantalizing preliminary data that this protective mechanism operates early in the progression of prostate cancer. In our experiments, PI-9 expressed by prostate cancer cells inhibited GrB. LNCaP cells that overexpressed PI-9 were resistant to apoptosis mediated by NK -92 cells, but LNCaP cells that lacked PI-9 were sensitive. This observation shows that PI-9 can protect prostate cells from NK cell-mediated apoptosis, one arm of immunosurveillance. Additionally, immunohistochemistry showed that PI-9 was present in HGPIN tumors, one of the earliest forms of prostate cancer, as well as in atrophic lesions. Our results suggest that PI-9 protects prostate tumors from immunosurveillance early in cancer progression by blocking the apoptotic response, while additional mechanisms protect tumors later in their progression through blocking recognition.
Why would PI-9 initially become upregulated in prostate tumors? As shown in , not only is PI-9 upregulated in early tumors, PI-9 is also abundantly expressed during atrophy, including prostatic inflammatory atrophy (PIA, Supplemental Figure 3
). Atrophy is a known hallmark of an inflamed prostate, and evidence has shown it can precede the development of PIN[27
]. A correlation between inflammation and prostate cancer has long been observed, and PI-9 could be the molecular mechanism that connects the two pathologies. PI-9 is often upregulated in response to inflammation to protect bystander cells from inadvertently introduced GrB[12
], and PI-9 expression can be induced by pro-inflammatory molecules like IL-1B and TNF-α[29
]. Therefore, prostatic inflammation may provide a trigger for PI-9 upregulation. In a subset of inflamed cells, this PI-9 upregulation may become permanent, creating a cancer-prone population of cells that are resistant to immunosurveillance.
While PI-9 allows cancer cells to block the apoptotic response of immunosurveillance, prostate cancer cells have also been shown to block recognition by CLs. through the process of MIC shedding. MICs, or MHC class I chain-related molecules, are expressed on the surface of cancerous cells and target these cells for destruction. MICs bind to the NKG2D receptor on NK cells[26
] which initiates granule exocytosis, killing the MIC-expressing cell[30
]. MIC is expressed in HGPIN and low grade prostate tumors, however, membrane bound MIC cleaved by a metalloprotease in high grade tumors. This allows cancerous cells to evade detection by NK cells[24
]. Early expression of PI-9 could allow prostate cancer cells to survive while MIC is present.
We hypothesize that PI-9 expression may protect prostate cancer tumors early in cancer progression, when MIC is still present on the surface of cells. We find that PI-9 is expressed in the early HGPIN stage, a stage in which MIC is also expressed, but stochastically expressed in advanced tumors (). These results imply that PI-9 is important early in cancer progression, when MIC is still present on the surface of cells. PI-9 may not be important in the later stages, after MIC is shed and cells are in less danger of NK cell-induced apoptosis. Therefore, PI-9 protects prostate cancer cells early in their progression, when surface MIC is expressed, and PI-9 is then dysregulated in later stages when MIC is shed. Future studies beyond cell lines could further elucidate the role of PI-9 and MIC early in prostate cancer progression, as most cell lines and patient samples are derived from late-stage tumors.
In summary, prostate cancer uses both PI-9 upregulation and MIC shedding at consecutive stages in its progression to evade immunosurveillance. Treatment for prostate cancer must take into account both mechanisms, as PI-9 has been shown to affect hormone therapy in breast cancer[18
] and immunotherapy in melanoma[19
]. MIC shedding could be counteracted by inhibiting matrix metalloprotease-14[32
]. PI-9 is a substrate of Granzyme M, so Granzyme M upregulation could alleviate the resistance to cell death caused by PI-9[33
]. Targeting both MIC shedding and PI-9 expression could lead to an effective treatment strategy that enhances the immune response to cancer cells.