Nitric oxide synthase (NOS) has been shown to be overexpressed in a number of human tumors compared to normal tissues and therefore potentially represents an exploitable target in future anticancer ...therapies. To achieve this, there will be a need to profile tumors to identify those expressing high levels of NOS; alternatively, endogenous (low) levels of NOS could be modulated by induction or through gene therapy approaches. NOS consists of a reductase domain which shares a high degree of sequence homology with P450 reductase and this domain supplies reducing equivalents to a haem containing oxygenase domain that is responsible for the production of nitric oxide. Thus, there are a number of routes of exploitation. Firstly, to take advantage of the reductase domain to activate bioreductive drugs as has been exemplified with tirapazamine and now extended to AQ4N (1,4-bis{2-(dimethylamino-
N-oxide)ethylamino}5,8-dihydroxy-anthracene-9,10-dione). Secondly, to take advantage of nitric oxide production for its ability to increase the sensitivity of resistant hypoxic cells to radiation. Lastly, to utilize inhibition of HIF-1 to amplify NO based therapies. In this review we provide examples/evidence of how these objectives can be achieved.
Tumor-associated macrophages (TAMs) are found in many solid tumors and have often been shown to accumulate in the hypoxic
regions surrounding areas of necrosis. TAMs are the major site of expression ...of nitric oxide synthase (NOS), a heme-containing
homodimeric enzyme consisting of oxygenase and reductase domains. The latter has a high degree of sequence homology to cytochrome
P 450 reductase and a functional consequence of this is the ability of NOS, under hypoxic conditions, to activate the bioreductive
drugs tirapazamine and RSU1069. Banoxantrone (AQ4N) is a bioreductive prodrug activated in hypoxia by an oxygen-dependent
two-electron reductive process to yield the topoisomerase II inhibitor AQ4. A feature of this process is that the final product
could potentially show bystander cell killing. Thus, in this study, we investigated the ability of inducible NOS (iNOS)-expressing
TAMs to activate AQ4N and elicit toxicity in cocultured human tumor cells. Murine macrophages were induced to overexpress
iNOS by treatment with a combination of cytokines, mixed with HT1080 and HCT116 human tumor cells, and the toxicity of AQ4N
was determined under aerobic or hypoxic conditions. The aerobic toxicity of AQ4N toward tumor cells was not affected through
coculturing with macrophages. However, under hypoxic conditions, the induction of iNOS activity in the macrophages was associated
with an increase in AQ4N metabolism and a substantial increase in tumor cell toxicity, which was dependent on the proportion
of macrophages in the culture. This study is the first demonstration of TAM-mediated prodrug activation to result in bystander
killing of human tumor cells.Mol Cancer Ther 2009;8(5):1261–9
The development of delivery systems capable of tumor targeting represents a promising strategy to overcome issues related to nonspecific effects of conventional anticancer therapies. Currently, one ...of the most investigated agents for cancer targeting is hyaluronic acid (HA), since its receptor, CD44, is overexpressed in many cancers. However, most of the studies on CD44/HA interaction have been so far performed in cell-free or genetically modified systems, thus leaving some uncertainty regarding which cell-related factors influence HA binding and internalization (collectively called “uptake”) into CD44-expressing cells. To address this, the expression of CD44 (both standard and variants, designated CD44s and CD44v, respectively) was evaluated in human dermal fibroblasts (HDFs) and a large panel of cancer cell lines, including breast, prostate, head and neck, pancreatic, ovarian, colorectal, thyroid, and endometrial cancers. Results showed that CD44 isoform profiles and expression levels vary across the cancer cell lines and HDF and are not consistent within the cell origin. Using composite information of CD44 expression, HA binding, and internalization, we found that the expression of CD44v can negatively influence the uptake of HA, and, instead, when cells primarily expressed CD44s, a positive correlation was observed between expression and uptake. In other words, CD44shigh cells bound and internalized more HA compared to CD44slow cells. Moreover, CD44shigh HDFs were less efficient in uptaking HA compared to CD44shigh cancer cells. The experiments described here are the first step toward understanding the interplay between CD44 expression, its functionality, and the underlying mechanism(s) for HA uptake. The results show that factors other than the amount of CD44 receptor can play a role in the interaction with HA, and this represents an important advance with respect to the design of HA-based carriers and the selection of tumors to treat according to their CD44 expression profile.
Hypoxia, a hallmark feature of the tumor microenvironment, causes resistance to conventional chemotherapy, but was recently reported to synergize with poly(ADP-ribose) polymerase inhibitors (PARPis) ...in homologous recombination-proficient (HR-proficient) cells through suppression of HR. While this synergistic killing occurs under severe hypoxia (<0.5% oxygen), our study shows that moderate hypoxia (2% oxygen) instead promotes PARPi resistance in both HR-proficient and -deficient cancer cells. Mechanistically, we identify reduced ROS-induced DNA damage as the cause for the observed resistance. To determine the contribution of hypoxia to PARPi resistance in tumors, we used the hypoxic cytotoxin tirapazamine to selectively kill hypoxic tumor cells. We found that the selective elimination of hypoxic tumor cells led to a substantial antitumor response when used with PARPi compared with that in tumors treated with PARPi alone, without enhancing normal tissue toxicity. Since human breast cancers with BRAC1/2 mutations have an increased hypoxia signature and hypoxia reduces the efficacy of PARPi, then eliminating hypoxic tumor cells should enhance the efficacy of PARPi therapy.
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Background: PARP inhibitors (PARPi) have demonstrated activity in HR deficient tumors through synthetic lethality. However, their clinical efficacy has not been highly promising, ...with response rates ranging between 30-60% for Olaparib, Talazoparib and Veliparib (1). This highlights the necessity to understand the mechanisms of resistance and how these agents work in the context of the tumor microenvironment. In this study, we explore the effect of hypoxia on the efficacy of PARPi in a range of HR-deficient tumors. Hypoxia is known to downregulate genes of homologous recombination (2) but its role in HR deficiency in still unknown. Methods: HR deficient tumor cell lines were treated with PARPi under both aerobic and hypoxic conditions. DNA damage markers such as γH2AX, 53BP1 and RIF1 were detected by immunofluorescence. Tumor xenografts were grown to 100 mm
3
prior to treatment with vehicle, PARPi, hypoxia activated cytotoxin, or the combination and tumor volumes were measured. Co-localization of apoptosis and hypoxia was detected by IHC. Finally, hypoxic signature in breast PDX models was correlated with their sensitivity to PARPi. Results: We found that hypoxic cells are significantly more resistant to PARPi in cell culture models. Efficiency of inhibition of PARP activity by these agents was similar under both air and hypoxia. The DNA damage marker γH2AX increased in cells treated in air, but not under hypoxia. There was no loss of 53BP1 protein that regulate engagement of the c-NHEJ pathway, indicating that HR has not been restored in these cells (3). However, it’s binding partner RIF1 was lost in hypoxia upon treatment indicating deficient signaling downstream of 53BP1. RIF1 is known to inhibit 5’ resection and its loss leads to resistance to PARPi in BRCA-deficient cells (4). In vivo, combining PARPi with hypoxia activated prodrugs led to substantial tumor growth delay in three xenograft models. DNA damage and apoptosis in these tumors was only detected outside of hypoxic regions positive for pimonidazole. In the breast PDX models, there was a strong correlation between the level of hypoxia in these tumors and their sensitivity to Olaparib. Conclusions: The significant increase in anti-tumor effects with the combination of PARPi and hypoxia-targeting cytotoxins leads us to propose a new strategy for tumors which are not responsive to PARPi alone. 1. Brown JS et al. Br J cancer 2016, 114, 713-5 2. Chan N et al. Cancer Res 2010, 70,8045-54 3. Bunting SF et al. Cell 2010, 141, 243-54 4. Chapman JR et al. Mol Cell 2013, 49, 858-71.
Prognosis of HPV negative head and neck squamous cell carcinoma (HNSCC) patients remains poor despite surgical and medical advances and inadequacy of predictive and prognostic biomarkers in this type ...of cancer highlights one of the challenges to successful therapy. Statins, widely used for the treatment of hyperlipidaemia, have been shown to possess anti-tumour effects which were partly attributed to their ability to interfere with metabolic pathways essential in the survival of cancer cells. Here, we have investigated the effect of statins on the metabolic modulation of HNSCC cancers with a vision to predict a personalised anticancer therapy. Although, treatment of tumour-bearing mice with simvastatin did not affect tumour growth, pre-treatment for 2 weeks prior to tumour injection, inhibited tumour growth resulting in strongly increased survival. This was associated with increased expression of the monocarboxylate transporter 1 (MCT1) and a significant reduction in tumour lactate content, suggesting a possible reliance of these tumours on oxidative phosphorylation for survival. Since MCT1 is responsible for the uptake of mitochondrial fuels into the cells, we reasoned that inhibiting it would be beneficial. Interestingly, combination of simvastatin with AZD3965 (MCT1 inhibitor) led to further tumour growth delay as compared to monotherapies, without signs of toxicity. In clinical biopsies, prediagnostic statin therapy was associated with a significantly higher MCT1 expression and was not of prognostic value following conventional chemo-radiotherapy. These findings provide a rationale to investigate the clinical effectiveness of MCT1 inhibition in patients with HNSCC who have been taking lipophilic statins prior to diagnosis.
Abstract
Severe hypoxia causes resistance to conventional chemotherapy and has been reported to synergize with PARP inhibitors (PARPi) through suppression of homologous recombination (HR). While this ...synergistic killing is true at oxygen levels less than 0.5%, our study shows that less severe hypoxia (e.g. 2% oxygen) is instead associated with resistance to PARPi in HR proficient cells. Interestingly, we demonstrate that HR deficient hypoxic tumors are significantly less responsive to PARPi, due to limited ROS-induced intrinsic DNA damage. To determine the contribution of hypoxic cells to PARPi, we used the hypoxic cytotoxin Tirapazamine to target hypoxic tumor cells. We found that the elimination of hypoxic tumor cells by Tirapazamine led to a substantial antitumor response with PARPi compared to PARPi treated tumors alone, without enhancing normal tissue toxicity. These studies indicate that tumor hypoxia reduces the efficacy of PARPi to tumor cells and that eliminating hypoxic tumor cells will enhance the efficacy of PARPi therapy.
Citation Format: Manal Mehibel, Jimmy Xu, Grace Li, Jung Moon, Kaushik Thakkar, Anh Diep, Ryan Kim, Joshua Bloomstein, Siren Xiao, Julien Bacal, Joshua Saldivar, Quynh Le, Karlene Cimprich, Erinn Rankin, Amato Giaccia. Oxygen dependent resistance to PARP inhibitors abstract. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr IA-003.
Banoxantrone (AQ4N) is a prototype hypoxia selective cytotoxin that is activated by haem containing reductases such as inducible nitric oxide synthase (iNOS). In the present study, we evaluate ...whether elevated levels of iNOS in human tumour cells will improve their sensitivity to AQ4N. Further, we examine the potential of radiation to increase cellular toxicity of AQ4N under normoxic (aerobic) and hypoxic conditions. We employed an expression vector containing the cDNA for human iNOS to transfect human fibrosarcoma HT1080 tumour cells. Alternatively, parental cells were exposed to a cytokine cocktail to induce iNOS gene expression and enzymatic activity. The cells were then treated with AQ4N alone and in combination with radiation in the presence or absence of the iNOS inhibitor N-methyl-L-arginine. In parental cells, AQ4N showed little difference in toxicity under hypoxic verses normoxic conditions. Notably, cells with upregulated iNOS activity showed a significant increase in sensitivity to AQ4N, but only under conditions of reduced oxygenation. When these cells were exposed to the combination of AQ4N and radiation, there was much greater cell killing than that observed with either modality alone. In the clinical development of hypoxia selective cytotoxins it is likely they will be used in combination with radiotherapy. In the present study, we demonstrated that AQ4N can selectively kill hypoxic cells via an iNOS-dependent mechanism. This hypoxia-selective effect can be augmented by combining AQ4N with radiation without increasing cytotoxicity to well-oxygenated tissues. Collectively, these results suggest that targeting hypoxic tumours with high levels of iNOS with a combination of AQ4N and radiotherapy could be a useful clinical therapeutic strategy.
Murine macrophages were induced to over-express iNOS by treatment with a combination of cytokines, mixed with HT1080 and HCT116 human tumour cells and the toxicity of AQ4N was determined under ...normoxic or hypoxic conditions. The normoxic toxicity of AQ4N towards tumour cells was not affected through co-culturing with macrophages. However, under hypoxic conditions, the induction of iNOS activity in the macrophages was associated with an increase in AQ4N metabolism and a substantial increase in tumour cell toxicity, which was dependent upon the proportion of macrophages in the culture. This study is the first demonstration of tumour associated macrophage mediated pro-drug activation to result in bystander killing of human tumour cells. The oxygenase domain of the iNOS enzyme is responsible for the production of nitric oxide (NO), a potent biological mediator, whose complex role in tumour biology is still not fully understood as it seems to have both anti- and protumour effects. We were able to use the co-culture model of NO-producing macrophages and cancer cells to demonstrate that NO is also a potent tumour radiosensitiser under hypoxic conditions with a sparing effect on well-oxygenated tumour cells, also a characteristic of normal tissues. Additionally, NO produced by the macrophages resulted in a significant induction of hypoxia inducible factor-1 (HIF-1) driven transcriptional activity in the co-cultured tumour cells under both normoxic and hypoxic conditions. We therefore, suggest the use of HIF-1 inhibitors in combination with NO-based therapies and radiation to favour tumour regression.
Murine macrophages were induced to over-express iNOS by treatment with a combination of cytokines, mixed with HT1080 and HCT116 human tumour cells and the toxicity of AQ4N was determined under ...normoxic or hypoxic conditions. The normoxic toxicity of AQ4N towards tumour cells was not affected through co-culturing with macrophages. However, under hypoxic conditions, the induction of iNOS activity in the macrophages was associated with an increase in AQ4N metabolism and a substantial increase in tumour cell toxicity, which was dependent upon the proportion of macrophages in the culture. This study is the first demonstration of tumour associated macrophage mediated pro-drug activation to result in bystander killing of human tumour cells. The oxygenase domain of the iNOS enzyme is responsible for the production of nitric oxide (NO), a potent biological mediator, whose complex role in tumour biology is still not fully understood as it seems to have both anti- and protumour effects. We were able to use the co-culture model of NO-producing macrophages and cancer cells to demonstrate that NO is also a potent tumour radiosensitiser under hypoxic conditions with a sparing effect on well-oxygenated tumour cells, also a characteristic of normal tissues. Additionally, NO produced by the macrophages resulted in a significant induction of hypoxia inducible factor-1 (HIF-1) driven transcriptional activity in the co-cultured tumour cells under both normoxic and hypoxic conditions. We therefore, suggest the use of HIF-1 inhibitors in combination with NO-based therapies and radiation to favour tumour regression.
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