Ivraym B. Barsoum

A mechanism of hypoxia-mediated escape from adaptive immunity in cancer cells

Immune escape is a fundamental trait of cancer in which mechanistic knowledge is incomplete. Here, we describe a novel mechanism by which hypoxia contributes to tumoral immune escape from cytotoxic T lymphocytes (CTL). Exposure of human or murine cancer cells to hypoxia for 24 hours led to upregulation of the immune inhibitory molecule programmed cell death ligand-1 (PD-L1; also known as B7-H1), in a manner dependent on the transcription factor hypoxia-inducible factor-1α (HIF-1α). In vivo studies also demonstrated cellular colocalization of HIF-1α and PD-L1 in tumors. Hypoxia-induced expression of PD-L1 in cancer cells increased their resistance to CTL-mediated lysis. Using glyceryl trinitrate (GTN), an agonist of nitric oxide (NO) signaling known to block HIF-1α accumulation in hypoxic cells, we prevented hypoxia-induced PD-L1 expression and diminished resistance to CTL-mediated lysis. Moreover, transdermal administration of GTN attenuated tumor growth in mice. We found that higher expression of PD-L1 induced in tumor cells by exposure to hypoxia led to increased apoptosis of cocultured CTLs and Jurkat leukemia T cells. This increase in apoptosis was prevented by blocking the interaction of PD-L1 with PD-1, the PD-L1 receptor on T cells, or by addition of GTN. Our findings point to a role for hypoxia/HIF-1 in driving immune escape from CTL, and they suggest a novel cancer immunotherapy to block PD-L1 expression in hypoxic-tumor cells by administering NO mimetics.

Hypoxia induces escape from innate immunity in cancer cells via increased expression of ADAM10: role of nitric oxide

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 hypoxia-inducible 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 α-subunit of HIF-1 (HIF-1α), 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 HIF-1α 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.

Mechanisms of hypoxia-mediated immune escape in cancer

An important aspect of malignant progression is the acquired ability of tumor cells to avoid recognition and destruction by the immune system (immune escape). Clinical cancer progression is also associated with the development of tumor hypoxia, which is mechanistically linked to the acquisition of malignant phenotypes in cancer cells. Despite the well-established role of hypoxia in tumor cell invasion and metastasis, and resistance to therapy, relatively few studies have examined the contribution of hypoxia to cancer immune escape. Accumulating evidence reveals that hypoxia can impair anticancer immunity by altering the function of innate and adaptive immune cells and/or by increasing the intrinsic resistance of tumor cells to the cytolytic activity of immune effectors. Here, we discuss certain aspects of the contribution of hypoxia to tumor immune escape and provide evidence for a novel role of cyclic guanosine monophosphate (cGMP) signaling in the regulation of hypoxia-induced immune escape. Thus, we propose that activation of cGMP signaling in cancer cells may have important immunotherapeutic applications.

Activation of the PD-1/PD-L1 immune checkpoint confers tumor cell chemoresistance associated with increased metastasis

The ability of tumor cells to avoid immune destruction (immune escape) as well as their acquired resistance to anti-cancer drugs constitute important barriers to the successful management of cancer. Interaction between the Programmed Death Ligand 1 (PD-L1) on the surface of tumor cells with the Programmed Death-1 (PD-1) receptor on cytotoxic T lymphocytes leads to inactivation of these immune effectors and, consequently, immune escape. Here we show that the PD-1/PD-L1 axis also leads to tumor cell resistance to conventional chemotherapeutic agents. Using a panel of PD-L1-expressing human and mouse breast and prostate cancer cell lines, we found that incubation of breast and prostate cancer cells in the presence of purified recombinant PD-1 resulted in resistance to doxorubicin and docetaxel as determined using clonogenic survival assays. Co-culture with PD-1-expressing Jurkat T cells also promoted chemoresistance and this was prevented by antibody blockade of either PD-L1 or PD-1 or by silencing of the PD-L1 gene. Moreover, inhibition of the PD-1/PD-L1 axis using anti-PD-1 antibody enhanced doxorubicin chemotherapy to inhibit metastasis in a syngeneic mammary orthotopic mouse model of metastatic breast cancer. To further investigate the mechanism of tumor cell survival advantage upon PD-L1 ligation, we show that exposure to rPD-1 promoted ERK and mTOR growth and survival pathways leading to increased cell proliferation. Overall, the findings of this study indicate that combinations of chemotherapy and immune checkpoint blockade may limit chemoresistance and progression to metastatic disease.

Glyceryl Trinitrate Inhibits Hypoxia-Induced Release of Soluble fms-Like Tyrosine Kinase-1 and Endoglin from Placental Tissues

Preeclampsia is associated with increased circulating levels of proinflammatory molecules, such as soluble fms-like tyrosine kinase 1 (sFlt-1) and soluble endoglin (sEng). On release by an inadequately perfused placenta into the maternal circulation, these molecules cause systemic endothelial dysfunction and the associated hypertension and proteinuria that characterize preeclampsia. We previously showed that glyceryl trinitrate (GTN) inhibits hypoxia/reoxygenation-induced apoptosis in the syncytiotrophoblast of term chorionic villi explants. Herein, we demonstrate that GTN inhibits the release of sFlt-1 and sEng from term chorionic villi explants exposed to hypoxia. Although transcript levels and secretion of sFlt-1 and sEng increased in explants exposed to hypoxia, low concentrations of GTN significantly inhibited the hypoxia-induced expression of these molecules at the mRNA and protein levels. Treatment of explants with GTN also prevented the hypoxia-induced accumulation of hypoxia-inducible factor-1α, a key mediator of cellular adaptations to hypoxia. Furthermore, knockdown of hypoxia-inducible factor-1α inhibited the hypoxia-induced secretion of sFlt-1 and sEng. This study provides evidence that hypoxia induces the release of sFlt-1 and sEng in the placenta via a mechanism that is inhibited by low concentrations of GTN. Our findings indicate that GTN may have potential applications in the treatment and/or prevention of preeclampsia.

Mechanisms Of Hypoxia-Induced Immune Escape In Cancer And Their Regulation By Nitric Oxide

The acquired ability of tumour cells to avoid destruction by immune effector mechanisms (immune escape) is important for malignant progression. Also associated with malignant progression is tumour hypoxia, which induces aggressive phenotypes such as invasion, metastasis and drug resistance in cancer cells. Our studies revealed that hypoxia contributes to escape from innate immunity by increasing tumour cell expression of the metalloproteinase ADAM10 in a manner dependent on accumulation of the alpha subunit of the transcription factor hypoxia-inducible factor-1 (HIF-1α). Increased ADAM10 expression leads to shedding of the NK cell-activating ligand, MICA, from the surface of tumour cells, thereby resulting in resistance to NK cell-mediated lysis. Our more recent studies demonstrated that hypoxia, also via HIF-1α accumulation, increases the expression of the inhibitory co-stimulatory ligand PD-L1 on tumour cells. Elevated PD-L1 expression leads to escape from adaptive immunity via increased apoptosis of CD8+ cytotoxic T lymphocytes. Accumulating evidence indicates that hypoxia-induced acquisition of malignant phenotypes, including immune escape, is in part due to impaired nitric oxide (NO)-mediated activation of cGMP signalling and that restoration of cGMP signalling prevents such hypoxic responses. We have shown that NO/cGMP signalling inhibits hypoxia-induced malignant phenotypes likely in part by interfering with HIF-1α accumulation via a mechanism involving calpain. These findings indicate that activation of NO/cGMP signalling may have useful applications in cancer therapy.

Abstract 447: Hypoxia induces tumor cell escape from T cell-mediated immunity via up-regulation of B7-H1.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC In the current research we describe a novel mechanism by which hypoxia contributes to tumour cell escape from lysis mediated by cytotoxic T lymphocytes (CTLs). Specifically, we show that when exposed to hypoxia for 24 h, human DU145 prostate and MDA-MB-231 breast cancer cells, as well as mouse B16-OVA melanoma cells, increased their expression of the immune inhibitory ligand B7-H1 at both the mRNA and protein levels in a manner dependent on hypoxia-inducible factor-1α (HIF-1α). Moreover, our findings revealed that the hypoxia-induced up-regulation of B7-H1 expression in tumour cells resulted in increased tumour cell resistance to lysis mediated by primed CTLs. We previously demonstrated that activation of nitric oxide (NO) signalling blocks HIF-1α accumulation in tumour cells exposed to hypoxia. Here we show that the NO mimetic glyceryl trinitrate (GTN; 10 nM) prevented the hypoxia-induced up-regulation of tumour cell B7-H1 expression as well as the increased resistance to CTL-mediated lysis. Furthermore, co-culture studies revealed that the increased expression of B7-H1 following exposure of tumour cells to hypoxia led to the apoptotic death of CTLs as well as Jurkat leukemia cells in a manner that could be prevented by blocking B7-H1, programmed cell death-1 receptor (B7-H1 receptor expressed by T cells) or by incubation with low concentrations of GTN. These results indicate that a potential immunotherapy of cancer could involve interference with B7-H1 function in hypoxic tumour cells via administration of NO mimetics. Citation Format: Ivraym B. Barsoum, Chelsea A. Smallwood, D. Robert Siemens, Charles H. Graham. Hypoxia induces tumor cell escape from T cell-mediated immunity via up-regulation of B7-H1. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 447. doi:10.1158/1538-7445.AM2013-447

Abstract 4844: A novel mechanism of immune escape in cancer and its regulation by nitric oxide

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL 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 < 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

Abstract 447: A mechanism of hypoxia-induced immune escape in cancer cells

We previously showed that hypoxia induces resistance to natural killer (NK) cell-mediated lysis in tumor cells through a mechanism that involves the shedding of NK cell-activating ligands (MICA) from the cell surface. Furthermore, we were able to block this hypoxia-induced shedding of MICA, as well as resistance to NK cell-mediated lysis, by activating NO signaling in the cancer cells. The objective of the present study was to fully elucidate this mechanism of hypoxia-induced immune escape. We used siRNA knock-down to determine the role of hypoxia-inducible factor-1 (HIF-1), a transcriptional activator of hypoxia-inducible genes, and ADAM10, a metalloproteinase known to be involved in the shedding of MICA, in the hypoxia-induced immune escape in DU145 prostate cancer cells and MDA-MB-231 breast cancer cells. Our findings revealed that in vitro exposure to hypoxia increases ADAM10 expression in a HIF-1-dependent manner, and that knock-down of ADAM10 abolishes the hypoxia-induced MICA release and resistance to NK cell-mediated lysis. Treatment of the tumor cells with low concentrations of nitroglycerin inhibited the hypoxia-induced accumulation of HIF-1α, ADAM10 up-regulation, the shedding of the NK-activating ligands, and resistance to NK cell-mediated lysis. More importantly, continuous transdermal administration of nitroglycerin (1.8 µg/h) to nude mice attenuated the growth of transplanted human DU145 prostate tumors; however, this effect of nitroglycerin was lost in mice made deficient in NK cells by treatment with anti-asialo GM1 antibody. This latter finding indicates that nitroglycerin attenuates tumor growth by sensitizing tumor cells to innate immunity. These findings 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 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 447. doi:10.1158/1538-7445.AM2011-447