Mechanisms of Hypoxia-mediated Immune Escape in Cancer

Ivraym B. Barsoum, Madhuri Koti, D. Robert Siemens and Charles H. Graham
DOI: 10.1158/0008-5472.CAN-14-2598 Published December 2014
Department of Biomedical and Molecular Sciences, Queen's University Kingston, Ontario, Canada.
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. Cancer Res; 74(24); 7185每90. ©2014 AACR.

Mechanisms of Hypoxia-Mediated Immune Escape in Cancer | Cancer Research

Hypoxic Stress-Induced Tumor and Immune Plasticity, Suppression, and Impact on Tumor Heterogeneity


imageSt谷phane Terry, imageSt谷phanie Buart and imageSalem Chouaib*
INSERM UMR 1186, Integrative Tumor Immunology and Genetic Oncology, Gustave Roussy, EPHE, Fac. de m谷decine 每 Univ. Paris-Sud, University Paris-Saclay, Villejuif, France
The microenvironment of a developing tumor is composed of proliferating cancer cells, blood vessels, stromal cells, infiltrating inflammatory cells, and a variety of associated tissue cells. The crosstalk between stromal cells and malignant cells within this environment crucially determines the fate of tumor progression, its hostility, and heterogeneity. It is widely accepted that hypoxic stresses occur in most solid tumors. Moreover, cancer cells found within hypoxic regions are presumed to represent the most aggressive and therapy-resistant fractions of the tumor. Here, we review evidence that hypoxia regulates cell plasticity, resistance to cell-mediated cytotoxicity, and immune suppression. Exposure to hypoxia occurs as a consequence of insufficient blood supply. Hypoxic cells activate a number of adaptive responses coordinated by various cellular pathways. Accumulating data also suggest that hypoxic stress in the tumor microenvironment promotes tumor escape mechanisms through the emergence of immune-resistant tumor variants and immune suppression. Thus, solid tumors seem to build up a hostile hypoxic microenvironment that hampers cell-mediated immunity and dampen the efficacy of the immune response.

The impact of hypoxia on tumor-associated macrophages

the secretion of immunosuppressive molecules (like lactic acid,TGF-beta, Il-10, VEGF, galectins etc)  by tumor cells under conditions of hypoxia provides a survival advantage, 

Hypoxic regulation of TAMs in cancer progression and therapy. (i) Hypoxia-induced release of chemoattractants results in enhanced TAM recruitment, which further amplifies the protumoral response. (ii) TAMs release survival factors for cancer cells, which protect them from chemotherapeutics. (iii) The hypoxic tumor environment is immunosuppressive and prevents an antitumor response.
Source publication

The role of tumor-associated macrophages (TAMs) in cancer is often correlated with poor prognosis, even though this statement should be interpreted with care, as the effects of macrophages primarily depend on their localization within the tumor. This versatile cell type orchestrates a broad spectrum of biological functions and exerts very complex and even opposing functions on cell death, immune stimulation or suppression, and angiogenesis, resulting in an overall pro- or antitumoral effect. We are only beginning to understand the environmental cues that contribute to transient retention of macrophages in a specific phenotype. It has become clear that hypoxia shapes and induces specific macrophage phenotypes that serve tumor malignancy, as hypoxia promotes immune evasion, angiogenesis, tumor cell survival, and metastatic dissemination. Additionally, TAMs in the hypoxic niches within the tumor are known to mediate resistance to several anticancer treatments and to promote cancer relapse. Thus, a careful characterization and understanding of this macrophage differentiation state is needed in order to efficiently tailor cancer therapy.

(PDF) The impact of hypoxia on tumor-associated macrophages

Targeting the renin-angiotensin system to improve cancer treatment: Implications for immunotherapy
Matthias Pinter1,2 and Rakesh K. Jain1,*
1Edwin L. Steele Laboratories for Tumor Biology, Department of Radiation Oncology, Harvard Medical School and Massachusetts General Hospital, Boston, MA 02114, USA.
2Division of Gastroenterology and Hepatology, Department of Internal Medicine III, Medical University of Vienna, Vienna, A-1090, Austria.

Renin-angiotensin system (RAS) inhibitors (RASi)〞widely prescribed for the treatment of cardiovascular diseases〞have considerable potential in oncology. The RAS plays a crucial role in cancer biology and affects tumor growth and dissemination directly and indirectly by remodeling the tumor microenvironment. We review clinical data on the benefit of RASi in primary and metastatic tumors and propose that, by activating immunostimulatory pathways, these inhibitors can enhance immunotherapy of cancer.

The circulating renin-angiotensin system (RAS) is mainly known for its pivotal role in maintaining cardiovascular homeostasis and fluid and electrolyte balance. In addition, a local RAS is expressed in many tissues and mainly acts at the cellular level, where it mediates cell proliferation, growth, and metabolism. The local RAS works synergistically and independently of the systemic RAS. Angiotensin II (AngII) is the main effector and maintains tissue homeostasis by exerting regulatory and counterregulatory effects through its different receptors. Alternative peptide-receptor axes also assist in maintaining this balance (1每7). Figure 1 provides an overview of the main components of the RAS. Dysregulation of the RAS, for example, by overexpression of certain RAS components [such as renin, Ang-converting enzyme (ACE), or AngII type 1 receptor (AT1R)], can be involved in the pathophysiology and progression of a broad range of diseases, such as arterial hypertension, kidney disease, and other cardiovascular conditions (5, 8, 9).

Targeting the renin-angiotensin system to improve cancer treatment: Implications for immunotherapy | Science Translational Medicine

Intratumoral Immune Landscape: Immunogenicity to Tolerogenicity
Abir K Panda, Sayantan Bose, Sreeparna Chakraborty, Kirti Kajal and Gaurisankar Sa*

Division of Molecular Medicine, Bose Institute, India

*Corresponding author: Gaurisankar Sa, Division of Molecular Medicine, Bose Institute, P-1/12, CIT Scheme VII M, Kolkata-700054, India

Received: April 13, 2015; Accepted: September 05, 2015; Published: September 10, 2015

Immune system possesses distinct innate (less specific) and adaptive (more specific) branches which act in a collaborative way to eliminate cancer from the host. In spite of the presence of immune response, tumors develop in the body spontaneously through different immune escape strategies. During the progression of cancer, immune cells become paralyzed and altered. In tumor microenvironment both innate (macrophage and NK cells) and adaptive (CTLs and effector T cells) immune cells are unable to recognize and induce specific effector response against cancer to eradicate it. Tumor cells release different types of chemokines, cytokines, growth factors that can modulate immune cells to become tolerogenic and allow tumor cells to grow rapidly without any restriction. Immune cells also cannot discriminate the tumor antigens as they are concealed in stroma and are also less immunogenic. The immune cells thus become dormant and effective immune responses against tumors could not be elicited. Tumor cells exploit the plethora of immunosuppressive mechanisms which include abnormalities of antigen processing and presentation, induction of negative co-stimulatory signals that helps to establish tumor immune evasion. In addition, infiltration of T-regulatory cells, immature and tolerogenic Dendritic Cells (DCs), tumor-associated macrophages, and myeloid-derived stromal cells foster suppressive, tolerogenic condition. The understanding of different immune evasion mechanisms will help to design effective immunotherapies to overcome tolerogenic condition and elicit tumor regression.

Keywords: Immune cell dysfunction; Immunogenicity; Tolerogenicity; Tumor immune evasion; Tumor micro-environment

Figure 1: Major strategies adopted by tumor cells for immune evasion.

Figure 2: Negative Co-stimulatory signals between APC and Tcells in tumor microenvironment.

Figure 3: Different immunosuppressive factors and their interaction with immune cells in the tumor microenvironment.

Figure 4: 
Modulation of different immune cells in the tumor microenvironment.

Intratumoral Immune Landscape: Immunogenicity to Tolerogenicity

Tumor escape mechanisms.

The immune system exerts selective pressure on tumors through a variety of processes, including the destruction of antigen-positive tumor cells by CD8 + T cells. As a result, immunogenic tumor cells are eliminated, leaving behind tumor cell variants more adept at evading immune-mediated destruction (i.e., immunoselection). Over time, tumors evolve mechanisms to elude or inhibit immunity by both intrinsic and extrinsic means. Intrinsic alterations within tumor cells evade immunity by downregulating antigen presentation (MHC), upregulating inhibitors of apoptosis (Bcl- XL, FLIP), or expressing inhibitory cell surface molecules that directly kill cytotoxic T cells (PD-L1, FasL). In addition, tumor cells secrete factors that inhibit effector immune cell functions (TGF-汕, IL-10, VEGF, LXR-L, IDO, gangliosides, or soluble MICA) or recruit regulatory cells to generate an immunosuppressive microenvironment (IL-4, IL-13, GM-CSF, IL-1汕, VEGF, or PGE2). Once recruited, regulatory cells attenuate antitumor immunity through the liberation of immunosuppressive cytokines and alterations in the nutrient content of the microenvironment. Specifically, secretion of IL-4 and IL-13 leads to recruitment and polarization of M2 macrophages (M2 M朴) from myeloid precursors, which express TGF-汕, IL-10, and PDGF that inhibit T cells. The release of colony-stimulating factors, IL-1汕, VEGF, or PGE2 by tumor cells results in the accumulation of MDSCs that can block T cell function by expressing TGF-汕, ARG1, and iNOS. Regulatory T cells (Tregs) can also inhibit effector T cells through multiple mechanisms, including expression of CTLA-4. (Abbreviations: ARG1, arginase 1; Bcl-XL, B cell lymphoma extra-long; CTLA-4, cytotoxic T lymphocyte associated protein-4; DC, dendritic cell; FasL, Fas ligand; FLIP, apoptosis-stimulating fragment-associated protein with death domain-like interleukin-1 converting enzyme-like inhibitory protein; GM-CSF, granulocyte macrophage colony每stimulating factor; IDO, indoleamine 2,3-deoxygenase; IL, interleukin; iNOS, inducible nitric oxide synthase; LXR-L, liver X receptor ligand; MDSC, myeloid-derived suppressor cells; MHC, major histocompatibility complex; MICA, MHC class I polypeptide-related sequence A; PDGF, platelet-derived growth factor; PD-L1, programmed cell death 1 ligand 1; PGE2, prostaglandin-E2; TGF-汕, transforming growth factor-汕; Treg, regulatory T cell; VEGF, vascular endothelial growth factor) (Vesely et al. 2011).

Mechanisms of Hypoxia-mediated Immune Escape in Cancer
Ivraym B. Barsoum, Madhuri Koti, D. Robert Siemens and Charles H. Graham
DOI: 10.1158/0008-5472.CAN-14-2598 Published December 2014
Department of Biomedical and Molecular Sciences, Queen's University Kingston, Ontario, Canada.
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. Cancer Res; 74(24); 7185每90. ©2014 AACR.

Hypoxia, a characteristic of many solid cancers, develops from an imbalance between oxygen consumption and oxygen supply. Although hypoxia is an important driver of tumor invasion and metastasis, as well as resistance to therapy (1), there is limited knowledge on the contribution of hypoxia to tumor cell escape from destruction by innate and adaptive immune effector mechanisms.

Immune escape in cancer is a multifaceted process resulting from the suppression of immune effector mechanisms and/or the acquisition of intrinsic tumor cell resistance to the cytotoxic activity of immune effectors. Hypoxia can influence these aspects of immune escape by modifying the intrinsic properties of tumor cells and of the stromal compartment. Here, we review some of the mechanisms by which hypoxia contributes to immune escape in cancer. Furthermore, we propose that activation of cyclic guanosine monophosphate (cGMP) signaling in cancer cells, via administration of low doses of nitric oxide (NO) mimetic drugs, may be a novel therapeutic approach to interfere with hypoxia-induced immune escape.

Hypoxia-Induced Release of Immunosuppressive Molecules by Tumor Cells
Upon exposure to hypoxia, tumor cells release a variety of immunosuppressive molecules. For example, in the severely hypoxic tumor microenvironment, dying cells release ATP that is metabolized to adenosine by the ectonucleotidases CD73 and CD39 (2). Soluble adenosine in the extracellular matrix binds specific receptors on T cells to increase their intracellular levels of cAMP, which, in turn, suppresses T-cell functions (3). Tumor-derived cytokines released under hypoxic conditions, such as IL10 and TGF汕, induce the differentiation of tumor-associated macrophages (TAM) into M2 macrophages with immune-suppressive activities (4). TGF汕 released by tumor cells also inhibits T-cell proliferation and effector function, promotes the generation of regulatory T cells (Treg), and blocks the expression of receptors required for the cytotoxic function of natural killer (NK) cells (5). In addition, TGF汕 negatively regulates the antigen presentation function of dendritic cells (DC), resulting in the inhibition of T-cell function and differentiation (5).

Interestingly, emerging evidence links hypoxia-induced angiogenesis with immune tolerance (6, 7). Hypoxia drives angiogenesis within the tumor microenvironment by inducing the secretion of vascular endothelial growth factor (VEGF) and other proangiogenic molecules by tumor cells. Tumor-derived VEGF suppresses the maturation of DCs and blocks the presentation of tumor-associated antigens to helper T cells, thereby promoting immune escape (6). Moreover, in response to tumor-derived VEGF, DCs increase their expression of the programmed death ligand 1 (PD-L1 or B7-H1), a negative regulator of T-cell function (7). VEGF promotes the accumulation of myeloid-derived suppressor cells (MDSC) in tumor tissues and secondary lymphoid organs (6). MDSCs are potent suppressors of anticancer T-cell responses and also contribute to tumor progression by releasing factors that promote angiogenesis and metastasis (for a review on MDSCs see refer. 8). Consequently, VEGF is a potential target for immune therapy. In support of this, anti-VEGF therapy was shown to be associated with increased numbers of activated DCs and heightened T-cell function in patients with cancer (9). However, targeting VEGF as an immunotherapeutic approach may lead to tumor hypoxia via inhibition of angiogenesis, thereby resulting in the activation of other hypoxia-induced immune escape pathways.

Tumor cells can secrete proteins such as CC-chemokine ligand 22 (CCL22) and various chemokines that inhibit effector T-cell responses and promote the generation and recruitment of immunosuppressive Tregs (10). In an ovarian cancer model, hypoxia was shown to promote the recruitment of Tregs via increased tumor cell expression of CCL28 (11). Tregs in turn can also secrete VEGF, thereby contributing to the VEGF pool in the tumor microenvironment that contributes to immune tolerance (11).

Tumor cells can also produce galectin-1 and galectin-3 to induce apoptosis of activated lymphocytes (12, 13). In patients with melanoma, there was a strong correlation between expression of galectin-3 and apoptosis of tumor-infiltrating lymphocytes (TIL; ref. 12). In Wilms tumors and Schwannomas, galectin-3 was shown to colocalize with the transcription factor hypoxia-inducible factor-1汐 (HIF1汐; ref. 14). In addition, it was reported that galectin-1 expression is transcriptionally regulated by HIF1 in colorectal cancers (15) and head and neck squamous cell carcinomas (16).

Hypoxia was also shown to induce immunosuppression by upregulating COX-2 expression in tumor cells; and HIF1-mediated upregulation of COX-2 increased colorectal tumor cell survival and VEGF production (17). COX-2 is a proinflammatory enzyme that converts arachidonic acid into prostaglandin E2 (PGE2). The latter causes immunosuppression by increasing adenosine/cAMP signaling in effector T cells (18). PGE2 secreted by tumor cells can also inhibit antitumor immunity by inhibiting the maturation of DCs (19). Also, PGE2 enhances the suppressive activity of Tregs and supports the differentiation of Tregs (20). Finally, PGE2 can stimulate the immunosuppressive functions of MDSC by binding to EP-4 receptors on these cells (21). Hypoxia-induced immune suppression via COX-2 can explain why chronic administration of indomethacin, a COX-2 inhibitor, in the drinking water of mice led to significant reduction in the growth rate and metastasis of mammary tumors as well as restoration of splenic NK cell activity (22). A recent study revealed that use of NSAIDs reduced recurrence of breast cancer in overweight and obese women (23).

The above studies indicate that the secretion of immunosuppressive molecules by tumor cells under conditions of hypoxia provides a survival advantage, and therefore support the concept that hypoxia represents a selection pressure driving immune escape.

Direct Effects of Hypoxia on Immune Effectors
Hypoxia can also directly impair antitumor immune responses. For example, hypoxia in the tumor microenvironment can induce the release of VEGF by M2 macrophages (24). Furthermore, TAMs suppress T-cell function in a manner dependent on HIF1 (25), and TAMs in hypoxic regions of tumors exhibit increased expression of M2-promoting molecules, such as TGF汕 (26). Hypoxia inhibits the in vitro cytolytic activity of other immune effectors such as the NK cell每mediated killing of hepatocellular carcinoma cells and multiple myeloma cells (27, 28).

Hypoxia was shown to decrease T-cell survival (29), and incubation of naïve T cells under hypoxia decreases their secretion of the trophic cytokine IL2 in a HIF1-dependent manner (30). CD4+ and CD8+ T cells derived from HIF1汐-deficient mice exhibit increased proliferation, produce higher levels of interferon-污, and display increased antitumor responses (31). HIF1 was also shown to mediate Treg differentiation via increased expression of FoxP3 (32). Increased numbers of Tregs in the tumor stroma have been associated with poor survival of patients with various cancers (33, 34).

Another mechanism of tumor cell immune escape involves binding of the cytotoxic T lymphocyte antigen-4 (CTLA-4; an immune checkpoint regulator) to its natural receptors, CD80 (B7.1) and CD86 (B7.2). Interestingly, hypoxia was shown to increase the expression of CD86 by bone marrow每derived mouse DCs in a HIF1-independent manner (35). Studies revealed that CTLA-4 blockade attenuates the growth of several mouse tumors (36), reduces tumor-infiltrating Tregs, and promotes effector T-cell function in humans (37).

It is important to note that not all of the reported effects of hypoxia on T cells are detrimental to their function. Hypoxia was reported to upregulate CD137, a member of the TNF receptor family that is known for its costimulatory activity on T cells (38). Expression of CD137 on activated mouse T cells was shown to be stimulated by hypoxia (39), and tumors from HIF1汐-deficient mice exhibited undetectable numbers of CD137+ TILs (39). In tumor growth assays, hypoxia induced the activation of T cells via the upregulation of surface CD137 in a HIF1-dependent manner, which, in turn, resulted in improved immune response and slower tumor growth (39).

Hypoxia Induces Immune Tolerance via Regulation of Tumor Cell-Associated Immune Checkpoint Molecules
In addition to decreasing the cytolytic potential of immune effectors, hypoxia increases the intrinsic resistance of tumor cells to immune-mediated killing. One strategy that tumor cells use to avoid immune detection and destruction is to alter their expression of cell-surface immune checkpoint regulators. For example, tumor cells may shed stress-induced MHC class I chain-related proteins A and B (MICA/B) from their surface to avoid interaction with NKG2D receptors on NK cells, 污汛 T cells, and CD8+ 汐汕 T cells (40), thereby escaping cytolysis (41). We have shown that exposure of tumor cells to hypoxia leads to the shedding of surface MICA, which, in turn, results in increased resistance to lysis by innate immune effectors (42). We also showed that the hypoxia-induced release of MICA and resistance of tumor cells to lysis required HIF1-mediated expression of the metalloproteinase ADAM 10 in the tumor cells (Fig. 1; ref. 43).

Figure 1.
Proposed mechanisms of hypoxia-induced tumor cell escape from innate and adaptive immunity. Hypoxia increases the accumulation of HIF1汐 in tumor cells, which, in turn, leads to higher levels of ADAM 10 and PD-L1 on the surface of tumor cells. ADAM 10 cleaves MICA from the cell surface to limit binding to NKG2D receptors on NK cells, leading to escape from innate immunity, whereas interaction of PD-L1 with PD-1 or CD80 on activated CTLs causes apoptosis in the CTLs and escape from adaptive immunity. NO/cGMP signaling is proposed to block the effect of hypoxia on ADAM 10 and PD-L1 upregulation by inhibiting HIF1汐 accumulation.

As discussed earlier, there is evidence that tumor cells can suppress cytotoxic T lymphocyte (CTL) function through the interaction of inhibitory costimulatory molecules with their ligands. Certain members of the B7 family of costimulatory molecules expressed on the surface of tumor cells provide signals that suppress CTL responses. For example, binding of PD-L1 with PD-1 or with CD80 (B7.1) on activated CTLs leads to suppression of immune responses via mechanisms that include induction of apoptosis and anergy (nonresponsiveness to antigen) in the T cells (44). Recent clinical studies revealed that therapy with blocking anti-PD-1 antibody (nivolumab) produced objective responses in patients with non每small-cell lung cancer, melanoma, or renal-cell cancer (45). Also, reinduction therapy with anti-PD-1 antibody for late tumor recurrence showed durable remissions in patients with colorectal cancer, renal cell cancer, and melanoma (46). In another study, concurrent therapy with anti-CTLA-4 antibody (ipilimumab) and nivolumab resulted in tumor regression in a substantial proportion of patients with unresectable, stage III or IV melanoma (47). We recently provided evidence that, when exposed to hypoxia, human and mouse cancer cells increased their expression of PD-L1 and acquired resistance to CTL-mediated lysis in a manner dependent on HIF1汐 (Fig. 1; ref. 48). Furthermore, the hypoxia-induced expression of PD-L1 in tumor cells led to increased apoptosis of cocultured CTLs as well as Jurkat T cells (48).

In addition, hypoxia may induce immune escape in cancer cells via epigenetic mechanisms. For example, tumor cells can upregulate miR210 in lung cancer and melanoma (49). In turn, miR210 was shown to block the susceptibility of tumor cells to lysis by antigen-specific CTLs. This effect was mediated via increased expression of protein tyrosine phosphatase, nonreceptor type I (PTPN1), homeobox A1 (HOXA1), and tumor protein p53-inducible protein 11 (TP53I11; ref. 49). Further studies are required to elucidate the mechanisms used by these molecules to suppress CTL activity.

Exposure of tumor cells to hypoxia also resulted in resistance to autologous CTL-mediated lysis in a manner dependent on the signal transducer and activator of transcription (STAT) 3 (50). STAT3 modulates the cross-talk between tumor and immune cells (51). A small-molecule inhibitor of STAT3, WP1066, was reported to reverse immune tolerance in patients with malignant glioma (52). Another STAT3 inhibitor, sunitinib, reduced the immunosuppressive phenotype of renal cell carcinomas (53) and reversed MDSC-mediated immune suppression via increased recruitment of CD4+CD8+ cytotoxic T cells (54).

Regulation of Immune Tolerance via Hypoxia-Induced Autophagy
Cancer cells often rely on autophagy as a mechanism of survival under conditions of stress including hypoxia, nutrient starvation, growth factor withdrawal, and chemotherapy (55, 56). However, the mechanisms by which autophagy enables survival of normal or malignant cells are not well known.

Hypoxia-induced autophagy is partly dependent on the HIF1/BNIP3每BNIP3L每Beclin1 axis (57), and partly on HIF1/platelet-derived growth factor receptor signaling (58). Through the activating transcription factor 4 and C/EBP homologous protein (CHOP), hypoxia increases the expression of microtubule-associated protein 1 light chain 3 (LC3) and autophagy protein 5 (ATG5) involved in formation and maturation of autophagosomes (59).

Hypoxia-induced autophagy is known to promote tumor cell survival via several mechanisms, including the removal of damaged mitochondria that produce cytotoxic reactive oxygen species (57) and the degradation of harmful protein aggregates (59). Activation of autophagy in cancer cells during hypoxia or exposure to other microenvironmental stressors may also lead to inhibition of death signals such as those triggered by CTLs (60). Furthermore, stress-induced release of the molecular pattern molecule HMGB1 induces cytoprotective autophagy and leads to recruitment of Tregs (60).

Autophagy can also promote activation of anticancer immunity. For example, autophagy has been shown to be crucial for proliferation of immune cells as well as for their effector functions such as antigen presentation and T cell每mediated tumor cell cytotoxicity (61). In T cells, autophagy is activated upon TCR engagement in both CD4+ and CD8+ T-cell subtypes (62). The knockdown of the essential autophagy-related genes, ATG5 or ATG7, during TCR stimulation leads to a significant decrease in cellular proliferation demonstrating the importance of autophagy during T-cell activation (62, 63). Furthermore, culture of DCs under low-oxygen results in the stabilization of HIF1汐, which initiates BNIP3 expression and promotes survival of mature DCs, possibly due to induction of autophagy (64). Hypoxia-induced autophagy in antigen-presenting cells infiltrating a tumor can occur via Toll-like receptor (TLR) signaling (65).

Together, the above findings indicate a dual role for autophagy in cancer immune escape. Therefore, immunotherapeutic strategies designed to target autophagy will need to consider its impact on the immune system.

Nitric Oxide/cGMP每Mediated Inhibition of Hypoxia-Induced Immune Escape
Our research over the last 15 years has revealed that classical NO signaling involving cGMP production functions as an O2-sensing mechanism playing a key role in tumor cell adaptations to hypoxia (42, 43, 48, 66每69). On the basis of our findings, we postulated that an important aspect of the mechanism by which cancer cells adapt to hypoxia involves inhibition of endogenous NO/cGMP signaling. Our research demonstrates that low concentrations of NO mimetics [e.g., glyceryl trinitrate (GTN), DETA/NO], known to selectively activate soluble guanylyl cyclase (sGC), inhibit malignant adaptations to hypoxia such as increased invasiveness, metastatic ability, and drug resistance (66每69). Moreover, because NO production is dependent on O2 availability, endogenous NO generation is severely limited in cells exposed to hypoxia (70, 71). This is despite the fact that hypoxia was shown to increase the expression of inducible NO synthase (iNOS) in the same cells (RAW 264.7 macrophages; ref. 71). We previously reported that cGMP levels are decreased in MDA-MB-231 breast tumor cells incubated for 6 hours in 0.5% O2 (68). This observation is consistent with the more recent findings of Hickok and colleagues (71), who reported decreased sGC activation in a murine macrophage line incubated under 5% O2. It is likely that our observed effects of NO/cGMP signaling on hypoxia-induced malignant phenotypes are at least partly mediated via inhibition of HIF1 transcriptional activity. This conclusion is based on evidence that NO mimetics, including the cGMP analogue 8-bromo-cGMP, inhibit the accumulation of HIF1汐 in cells exposed to hypoxia (43, 72). Our research has also revealed that NO mimetics interfere with the HIF1-mediated upregulation of ADAM10 expression involved in the shedding of MICA from the tumor cell surface and resistance to immune-mediated lysis (Fig. 1; ref. 43). In that same study, treatment of mice with GTN attenuated the growth of transplanted prostate tumors via a mechanism dependent on innate immune effectors. More recently, we demonstrated that low concentrations of GTN interfere with hypoxia-induced escape from T cell每mediated immunity in tumor cells by preventing the HIF1-dependent expression of PD-L1 (Fig. 1; ref. 48). Together, these studies indicate that activation of NO/cGMP signaling may have important applications in the prevention and/or treatment of cancer.

Although there is evidence that hypoxia can activate certain components of pathways involved in antitumor immunity, most studies indicate that hypoxia is a major contributor to cancer immune escape. Hypoxia-induced tumor cell escape from innate and adaptive immunity is likely a consequence of multiple mechanisms operating in a complementary, and sometimes redundant, manner. Thus, targeting individual mechanisms of hypoxia-induced immune escape will likely prove to be ineffective as a therapeutic strategy. However, it is clear that several mechanisms of such immune escape rely on the transcriptional activity of HIF1. This raises the possibility that interference with hypoxia response pathways involving HIF1 activity may be a fruitful immunotherapeutic approach. Interference with such pathways could be achieved through the use of molecules that directly inhibit HIF1 activity or block HIF1汐 accumulation in hypoxia. Our studies on the inhibitory effect of NO/cGMP signaling on HIF1汐 accumulation and malignant adaptations to hypoxia, including tumor cell escape from innate and adaptive immunity, support the therapeutic potential of NO mimetic agents. In this review, we highlighted some key mechanisms of hypoxia-mediated immune escape. However, because tumor cell avoidance of immune destruction is multifaceted, it is likely that hypoxia influences escape mechanisms not described herein. The role of the hypoxic tumor microenvironment on other key aspects of cancer immune surveillance, such as antigen presentation, additional immune checkpoints and effector mechanisms of tumor cell destruction, warrants investigation.

Mechanisms of Hypoxia-Mediated Immune Escape in Cancer | Cancer Research

Little bang for the Big Cancer? Nitroglycerin in the anti-cancer arsenal
James Bond on a lilo couldn*t match this relaxation agent
By Pan Pantziarka 27 Oct 2015 at 09:28 37 Reg comments SHARE
Big Bang
It's often the case that when people talk of wonder drugs in cancer they most often think of the latest exquisitely engineered molecules that closely target very specific biochemical pathways. Think high cost, think high science, and think high hopes.

And yet there's evidence mounting that one of our oldest and most widely used medical treatments is something of a wonder drug too.

Another paper published in the British Journal of Cancer (Aspirin as a neoadjuvant agent during preoperative chemoradiation for rectal cancer) is reporting on a clinical trial that showed that the humble aspirin had a positive effect on overall survival when used during the chemoradiation treatment of rectal cancer patients.

It is one of a number of studies which show that aspirin has positive effects as a cancer treatment, not just as a cancer prevention agent.

All this from a drug that has existed in its modern form for more than 115 years 每 I*d say that qualifies aspirin as something of a wonder drug.

But while the story of aspirin as an anticancer drug is gaining increasing public attention, less well known is the story of another venerable old drug 每 glyceryl trinitrate; better known as nitroglycerin.

Like aspirin, this is a drug with a long history 每 the original report of its medical application dates back to a paper in the Lancet by William Murrell in 1879, so it predates aspirin by about 20 years. A medical application for a compound better known as a potent explosive 每 most of us know of nitroglycerin as the active ingredient in dynamite 每 was something of a surprise.

But then there are a lot of surprising things about nitroglycerin, not the least of which is that it might enable us to tackle one of the most difficult problems in cancer: treatment resistance.

It is this possibility that attracted our attention at the Repurposing Drugs in Oncology project and explored in a recent paper (Repurposing Drugs in Oncology (ReDO) 每 nitroglycerin as an anti-cancer agent). Before looking at what we found we need to back up a little bit and take a look at what nitroglycerin does when it*s not blowing things up, and at what treatment failure means in cancer.

Nitroglycerin is used as a vasodilator 每 that is, it eases the muscle cells that surround blood vessels so they relax and open up. It*s this property which makes it useful as treatment for hypertension, angina and congestive heart failure. As an aside it*s this property that makes the related drug amyl nitrite (the recreational drug known as poppers) of interest in certain sexual subcultures.

In the case of nitroglycerin it can be used in an acute situation 每 usually delivered via a sublingual tablet or spray 每 or as a chronic treatment, where it is delivered via transdermal patches, which are worn during the day. It*s a safe, cheap and well-known drug with decades of use behind it.

You would think, therefore, that we would know all there is to know about how it works its magic. But that*s not case. The actual mechanism by which it works at the molecular level has still not been fully worked out. The question is how do we go from the drug to the action of vasodilation?

The drug was developed at a time when we had very little knowledge of fundamental biochemistry 每 for a long time drug development was a rather empirical process in comparison with the targeted molecular approach we now use. In fact, even now there are competing theories to explain how it works 每 but that it works is not in doubt.

What has all this got to do with cancer? Well, potentially rather a lot. Two of the mainstays of cancer treatment are high dose chemotherapy and radiotherapy. Even though the anti-cancer armoury has expanded and continues to expand, most cancer treatments will involve one or both of these.

Both of these treatments work by massively killing tumour cells 每 and we need to do this quickly before resistance evolves and before we kill the patient. If there*s one thing we know about cytotoxic chemotherapy in particular is that there*s a lot of collateral damage 每 to cells in the gastrointestinal tract (vomiting), immune system (neutropenia), hair follicles (hair loss) and so on.

Radiotherapy carries its own set of side effects, and there too we have the problem that some tumours can become radio-resistant. The treatment resistance is a major problem 每 because once the first-line treatments fail the second often fail too.

There are numerous factors involved in these forms of resistance, and some of them are architectural. Tumours need a supply of nutrients, they need oxygen, they need all the support systems that normal tissues need 每 and they need lots of them.

However, these tumours are very good are kicking off a process called angiogenesis 每 the sprouting of new blood vessels to keep themselves going.

Angiogenesis is a normal physiological function in wound healing, foetal growth, and general development; but what happens in cancer is that this process is hijacked. The result though is a highly disordered and chaotic vasculature around a tumour, with immature blood vessels, some areas starved of oxygen and nutrients and some areas well-supplied with both.

The problem is we need that blood supply to get the chemo drugs deep inside tumours. And we need oxygen to react with the radiotherapy to cause the cell damage to kill cancer cells. Therefore, if we can tackle this then we can get more bang for our buck 每 the chemotherapy drugs make it deeper into the tumours, the radiotherapy causes more reactive oxygen to kill resistant cells. And this is where nitroglycerin comes in.

By causing vasodilation, nitroglycerin causes the immature blood vessels to become more leaky 每 so the chemo drugs which are circulating in the blood seep out more readily into the tumour. While it doesn*t molecularly target cancer cells specifically, it does mean that you get more chemo where you want it and less where you don*t. Nitroglycerin also has the nice property that it increases the oxygenation of tissues which are relatively hypoxic (lacking oxygen).

Hypoxia is a factor in treatment resistance to both chemo and radiotherapy, and it*s also a selective pressure associated with the evolution of more aggressive and metastatic disease. It*s a major target of drug research at the moment, although from the evidence that we*ve uncovered in our paper it looks like nitroglycerin does a good job there too.

The good news is that there is some clinical work already going on to prove that this works in practice. For example, we are supporting a clinical trial in the Netherlands in non-small cell lung cancer in which patients being treated with combined chemo and radiotherapy wear a transdermal nitroglycerin patch.

There are other trials too, including a Phase III trial in prostate cancer in which transdermal nitroglycerin is used as a standalone therapy in men who show biochemical signs of recurrence after treatment.

Our hope is that in the future patients undergoing standard cancer treatments will also be wearing transdermal nitroglycerin patches in those cancers in which we have proved that doing so increases the overall survival. The potential impact might be explosive. ®

Little bang for the Big C? Nitro in the anti-cancer arsenal • The Register

Glyceryl trinitrate‑induced cytotoxicity of docetaxel‑resistant prostatic cancer cells is associated with differential regulation of clusterin

Laboratoire d'Immunologie et Immunoth谷rapie des Cancers, EPHE, PSL Research University, F‑75000 Paris, France, Centre Georges‑François Leclerc, F‑21000 Dijon, France

Metastatic castration resistant prostate cancer (mCRPC) relapse due to acquired resistance to chemotherapy, such as docetaxel, remains a major threat to patient survival. Resistance of mCRPC to docetaxel can be associated with elevated levels of soluble clusterin (sCLU) and growth differentiation factor‑15 (GDF‑15). Any strategies aiming to modulate sCLU and/or GDF‑15 in docetaxel‑resistant prostate cancer cells present a therapeutic interest.

The present study reports the cytotoxic effect of a nitric oxide donor, glyceryl trinitrate (GTN), on docetaxel‑resistant mCRPC human cell lines and demonstrates that GTN displays greater inhibition of cell viability toward docetaxel‑resistant mCRPC cells than on mCRPC cells. It is also demonstrated that GTN modulates the level of expression of clusterin (CLU) which is dependent of GDF‑15, two markers associated with docetaxel resistance in prostate cancer. The results indicate that GTN represses the level of expression of the cytoprotective isoform of CLU (sCLU) and can increase the level of expression of the cytotoxic isoform (nuclear CLU) in docetaxel resistant cells. Furthermore, it was observed that GTN differentially regulates the level of the precursor form of GDF‑15 between resistant and parental cells, and that recombinant GDF‑15 can modulate the expression of CLU isoforms and counteract GTN‑induced cytotoxicity in resistant cells. A link was established between GDF‑15 and the expression of CLU isoforms. The present study thus revealed GTN as a potential therapeutic strategy to overcome docetaxel‑resistant mCRPC.

Glyceryl trinitrate‑induced cytotoxicity of docetaxel‑resistant prostatic cancer cells is associated with differential regulation of clusterin

Chemosensitization of Cancer In vitro and In vivo by Nitric Oxide Signaling

Department of Pathology and Molecular Medicine, Queen's University
Lisa J. Frederiksen, Richard Sullivan, Lori R. Maxwell, Shannyn K. Macdonald-Goodfellow, Michael A. Adams, Brian M. Bennett, D. Robert Siemens and Charles H. Graham
DOI: 10.1158/1078-0432.CCR-06-1807 Published April 2007
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Purpose: Hypoxia contributes to drug resistance in solid cancers, and studies have revealed that low concentrations of nitric oxide (NO) mimetics attenuate hypoxia-induced drug resistance in tumor cells in vitro. Classic NO signaling involves activation of soluble guanylyl cyclase, generation of cyclic GMP (cGMP), and activation of cGMP-dependent protein kinase. Here, we determined whether chemosensitization by NO mimetics requires cGMP-dependent signaling and whether low concentrations of NO mimetics can chemosensitize tumors in vivo.

Experimental Design: Survival of human prostate and breast cancer cells was assessed by clonogenic assays following exposure to chemotherapeutic agents. The effect of NO mimetics on tumor chemosensitivity in vivo was determined using a mouse xenograft model of human prostate cancer. Drug efflux in vitro was assessed by measuring intracellular doxorubicin-associated fluorescence.

Results: Low concentrations of the NO mimetics glyceryl trinitrate (GTN) and isosorbide dinitrate attenuated hypoxia-induced resistance to doxorubicin and paclitaxel. Similar to hypoxia-induced drug resistance, inhibition of various components of the NO signaling pathway increased resistance to doxorubicin, whereas activation of the pathway with 8-bromo-cGMP attenuated hypoxia-induced resistance. Drug efflux was unaffected by hypoxia and inhibitors of drug efflux did not significantly attenuate hypoxia-induced chemoresistance. Compared with mice treated with doxorubicin alone, tumor growth was decreased in mice treated with doxorubicin and a transdermal GTN patch. The presence of GTN and GTN metabolites in plasma samples was confirmed by gas chromatography.

Conclusion: Tumor hypoxia induces resistance to anticancer drugs by interfering with endogenous NO signaling and reactivation of NO signaling represents a novel approach to enhance chemotherapy.

Chemosensitization of Cancer In vitro and In vivo by Nitric Oxide Signaling | Clinical Cancer Research