One key to malignant progression is the acquired ability of tumor cells to escape immune-mediated lysis. Whereas tumor hypoxia is known to play a causal role in cancer metastasis and resistance to therapy, the link between hypoxia and immune escape in cancer remains poorly understood. Here, we show that hypoxia induces tumor cell resistance to lysis mediated by immune effectors and that this resistance to lysis occurs via a hypoxiainducible factor-1 (HIF-1)-dependent pathway linked to increased expression of the metalloproteinase ADAM10. This enzyme is required for the hypoxia-induced shedding of MHC class I chain-related molecule A (MICA), a ligand that triggers the cytolytic action of immune effectors, from the surface of tumor cells. Indeed, our findings show a mechanistic link between hypoxia-induced accumulation of the a-subunit of HIF-1 (HIF-1a), increased expression of ADAM10, and decreased surface MICA levels leading to tumor cell resistance to lysis mediated by innate immune effectors. Nitric oxide mimetic agents interfered with the hypoxia-induced accumulation of HIF1a and with the hypoxia-induced upregulation of ADAM10 expression required for decreased surface MICA expression and resistance to lysis. Furthermore, treatment of tumor-bearing mice with nitroglycerin, a nitric oxide mimetic, attenuated tumor growth by a mechanism that relied upon innate immune effector cells. Together, these findings reveal a novel mechanism by which the hypoxic tumor microenvironment contributes to immune escape in cancer, lending support to potential immunotherapeutic strategies involving the use of nitric oxide mimetics. Cancer Res; 71(24); 7433-41. Ó2011 AACR.

These results suggest a rationale for future studies on the potential therapeutic applications of PDE inhibitors in men with prostate cancer.

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655 Background: Myeloid-derived suppressor cells (MDSC) are capable of inducing profound immune suppression of anti-tumor T cell responses. Here we examined the ability of intratumoral MDSC accumulation to alter the immune landscape of RCC and to identify the factors involved. Methods: Using flow cytometry, 41 clear cell RCC nephrectomy samples were assessed for their total MDSC infiltrate and the percentage of granulocytic (PMN-MDSC) or monocytic (M-MDSC) subsets. RNA isolated from these tumor samples and 4 non-tumor samples was also evaluated for expression levels of immune-related genes using a Nanostring PanCancer immune panel. A multiple linear regression model was used to identify which immune-related genes might be significantly associated with PMN-MDSC intratumoral accumulation. Log-Rank test was used for comparison of survival curves. Results: Genes encoding cytokines, cancer-testis antigens, interleukins, and T cell function were differentially expressed by clear cell RCC and the non-tumor samples. The transcription of 55 immune-related genes was significantly associated (FDR adjusted p < 0.05) with PMN-MDSC infiltration, including mediators of MDSC recruitment such as CXCL1, CXCL3, CXCL5, CXCR2, IL8, LIF, S100A8, CSF3R, and CCR3, and molecules governing MDSC expansion and function such as IL10, C/EBPB, CD44, COX2, and NFATC3. When a statistically significant 10 gene signature was analyzed in the context of patient survival using the TCGA database of clear cell RCC patients (n = 945), a strong association between the expression of these genes and reduced survival was observed. Moreover, this association seemed to be additive, with expression of 7-10 of these genes correlating with poorer survival when compared to patients expressing 3 or less (p < 0.0001). Conclusions: Consistent with previous studies defining the immunoregulatory role of MDSCs, these results suggest that MDSC accumulation can alter the inflammatory state of the tumor, and indicate that mediators associated with the function of PMN-MDSCs can have a negative impact on outcome. Mechanistic studies aimed at identifying the functionally relevant mediators are ongoing.

One key to malignant progression is the acquired ability of tumor cells to escape immune-mediated lysis. Whereas tumor hypoxia is known to play a causal role in cancer metastasis and resistance to therapy, the link between hypoxia and immune escape in cancer remains poorly understood. Previously, we showed that hypoxia induces resistance to natural killer (NK) cell-mediated lysis in tumour cells through a mechanism that involves the shedding of the NK cell-activating ligand, MHC Class I Chain Related molecule A (MICA), from the cell surface. Additionally, we reported that activation of NO signalling in the tumour cells was able to block this hypoxia-induced shedding of MICA, as well as resistance to NK-mediated lysis. Here, we show that hypoxia-induced tumor resistance to lysis is mediated by a HIF-1-dependent increased expression of the metalloproteinase ADAM10, which is required for the hypoxia-induced shedding of MICA. Our findings demonstrate that HIF-1α knock down attenuated the hypoxia-induced expression of ADAM10 and MICA in tumour cells. Also, siRNA-mediated knockdown of HIF-1α or ADAM10 prevented the hypoxia-induced resistance of DU145 cells to innate immune effector cell-mediated lysis. Treatment with nitric oxide mimetic agents interfered with the hypoxia-induced accumulation of HIF-1α, and with the hypoxia-induced up-regulation of ADAM10 expression required for decreased surface MICA expression and resistance to lysis. To validate the in vitro results, we adopted a model in which human DU145 cells were injected subcutaneously into Swiss nude mice (T and B cell-deficient). Continuous transdermal delivery of nitroglycerine (1.8 µg/h for up to 62 days) significantly attenuated attenuated the tumor growth (P &lt; 0.05). This tumor growth-inhibitory effect of nitroglycerine was absent in mice depleted of innate immune effector cells. Within the tumours, areas of hypoxia co-localized with the metalloproteinase ADAM10 expression. Our results demonstrate a novel mechanism by which hypoxia contributes to immune escape in tumor cells and provide evidence that activation of nitric oxide signalling interferes with this mechanism. The findings described here are important because they indicate that nitric oxide mimetics could potentially be used as immunosensitizers in the treatment and/or prevention of cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4844. doi:1538-7445.AM2012-4844