Hypoxia inducible factor-1 influences sensitivity to paclitaxel of human lung cancer cell lines under normoxic conditions

Paclitaxel (PTX) is an anticancer drug that is effective against a wide range of solid tumors. The effect of PTX on two human lung cancer cell lines, PC14PE6 and NCI-H441 cells, was examined in an orthotopically transplanted animal model with an in vivo imaging devise. Although PTX effectively suppressed tumor growth and improved survival rate in NCI-H441, it did not influence these in PC14PE6. In vitro experiments confirmed that PC14PE6 cells are resistant to PTX under normoxic conditions and that both cell lines were resistant to PTX under hypoxic conditions. It was found that the expression level of endogenous hypoxia inducible factor (HIF)-1alpha in PC14PE6 is much higher than that in NCI-H441 cells under normoxic conditions. Furthermore, sensitivity to PTX in these cell lines was reversed when HIF-1alpha expression was decreased by siRNA specific to HIF-1alpha in PC14PE6 and increased by overexpression of the exogenous HIF-1alpha gene in NCI-H441. These results suggest that HIF-1 influences the PTX sensitivity of these cells. The authors further examined beta-tubulin, a target molecule of PTX, with western blotting and immunohistochemical analysis in these cells. The expression level of beta-tubulin was comparable in these cells under both normoxic and hypoxic conditions while the distribution of beta-tubulin and cell morphology were changed according to HIF-1alpha expression levels, suggesting that HIF-1 influences the conformation and dynamics of microtubules. These data support the potential development of HIF-1 targeted approaches in combination with PTX, where drug resistance tends to contribute to treatment failure.     

Augmenting tumor sensitivity to topotecan by transient hypoxia

We examined how the effect of topotecan is modulated by transient hypoxia in three different tumor lines, Lewis lung carcinoma (LLC), U87 human glioblastoma and DMS273 human small cell lung cancer. Four groups of tumor bearing mice were treated with saline or a single dose of topotecan, immediately followed by 6-h or 72-h exposure to a hypoxic environment (10% O₂) or normal air. Topotecan + hypoxia resulted in significantly greater suppression of tumor growth than normoxic topotecan or hypoxia alone. Correspondingly, the sensitivity of LLC cells to topotecan in a clonogenic survival assay was significantly higher under hypoxia. This effect of hypoxia was not a general phenomenon, since the tumor growth inhibitory effect of the alkylating agent cisplatin was not changed by hypoxic environment. In a parallel series of in vitro experiments, cell cultures were exposed to hypoxia (0.1% or 0.7% O₂) in a hypoxic chamber or normoxia for 24 h. We found a dose-dependent downregulation of HIF-1 by topotecan (30–270 nM). The hypoxic upregulation of Glucose transporter-1 and VEGF secretion to the culture medium was inhibited by the addition of topotecan, while doses up to 270 nM had no effect on VEGF under normoxia. VEGF protein levels in tumors were also reduced by topotecan. These data show that the effect of topotecan is increased by transient hypoxia, and this may be a direct effect on the ability of cells to survive under hypoxia as well as an antiangiogenic effect, mediated through the HIF-1 inhibitory effect of topotecan.     

Fluctuating and diffusion-limited hypoxia in hypoxia-induced metastasis.

PURPOSE: Most tumors develop regions with hypoxic cells during growth, owing to permanent limitations in oxygen diffusion (chronic or diffusion-limited hypoxia) and/or transient limitations in blood perfusion (acute or fluctuating hypoxia). The aim of this study was to investigate the relative significance of chronic and acute hypoxia in the development of metastatic disease. EXPERIMENTAL DESIGN: D-12 and R-18 human melanoma xenografts were used as models of human cancer. D-12 tumors metastasize to the lungs, whereas R-18 tumors develop lymph node metastases. Fraction of radiobiologically hypoxic cells (HF(Rad)) was measured in individual primary tumors by using a radiobiological assay based on the paired survival curve method. Fraction of immunohistochemically hypoxic cells (HF(Imm)) was assessed in the same tumors by using a pimonidazole-based immunohistochemical assay optimized with respect to achieving selective staining of chronically hypoxic cells. HF(Imm) and the difference between HF(Rad) and HF(Imm), HF(Rad) - HF(Imm), were verified to be adequate variables for fraction of chronically hypoxic cells and fraction of acutely hypoxic cells, respectively. RESULTS: Chronic as well as acute hypoxia were found to promote spontaneous metastasis of D-12 and R-18 tumors. Acute hypoxia influenced metastasis to a greater extent than chronic hypoxia, partly because the fraction of acutely hypoxic cells was larger than the fraction of chronically hypoxic cells in most tumors and partly because acutely hypoxic cells showed a higher metastatic potential than chronically hypoxic cells. CONCLUSIONS: It may be beneficial to focus on fluctuating hypoxia rather than diffusion-limited hypoxia when searching for hypoxia-related prognostic variables and predictive assays.     

Acute hypoxia enhances spontaneous lymph node metastasis in an orthotopic murine model of human cervical carcinoma

An orthotopic mouse model of cervical carcinoma has been used to investigate the relationship between acute (cyclic) hypoxia and spontaneous lymph node metastasis in vivo. The human cervical carcinoma cell line ME-180 was stably transfected to express the fluorescent protein DsRed2, which allowed the in vivo optical monitoring of tumor growth and metastasis by fluorescent microscopy. The surgically implanted primary tumors metastasize initially to local lymph nodes and later to lung, a pattern consistent with the clinical course of the disease. The effect of acute hypoxia on the growth and spread of these tumors was examined by exposing tumor-bearing mice to treatment consisting of exposure to 12 cycles of 10 min 7% O(2) followed by 10 min air (total 4 h) daily during tumor growth. After 21 days, the tumors were excised, lymph node and lung metastases were quantified, and the hypoxic fraction and relative vascular area of the primary tumors were assessed by immunohistochemical staining for the hypoxic marker drug EF5 [2-(2-nitro-1H-imidazole-1-yl)-N-(2,2,3,3,3-pentafluoropropyl) acetamide] and the vascular marker CD31, respectively. In untreated mice, the primary tumor size was directly correlated with lymph node metastatic burden. The acute hypoxia treatment resulted in a significant decrease in the size of the primary tumors at the time of excision. However, the mice in the acute hypoxia group had an increased number of positive lymph nodes (2-4) as compared with control mice (1-3). Lung metastasis was not affected. The acute hypoxia treatment also decreased the relative vascular area in the primary tumors but did not affect the hypoxic fraction. These results suggest that fluctuating oxygenation in cervical carcinoma tumors may reduce tumor growth rate, but it may also enhance the ability of tumor cells to metastasize to local lymph nodes.     

Hsp90 inhibitor NVP-AUY922 enhances radiation sensitivity of tumor cell lines under hypoxia

NVP-AUY922, a novel inhibitor of Hsp90, was shown to enhance the effect of ionizing radiation (IR) on tumor cells under normoxic conditions. Since low oxygen tension is a common feature of solid tumors, we explore in the present study the impact of hypoxia on the combined treatment of lung carcinoma A549 and glioblastoma SNB19 cell lines with NVP-AUY922 and IR. Cellular analysis included the colony-forming ability, expression of CAIX, Hsp90, Hsp70, Raf-1, Akt, cell cycle progression and associated proteins, as well as DNA damage measured by histone γH2AX. The clonogenic assay revealed that in both cell lines NVP-AUY922 enhanced the radiotoxicity under hypoxic exposure to a level similar to that observed under oxic conditions. Irrespective of oxygen supply during drug treatment, NVP-AUY922 also reduced the expression of anti-apoptotic proteins Raf-1 and Akt. As judged by the levels of histone γH2AX, drug-treated hypoxic cells exhibited a lower repair rate of DNA double-strand breaks than normoxic cells. The drug-IR mediated changes in the cell cycle, i.e., S-phase depletion and G 2/M arrest, developed not directly during hypoxic exposure but first upon 24 h reoxygenation. Under both oxygen tensions, Hsp90 inhibition downregulated the cell cycle-associated proteins, Cdk1, Cdk4 and pRb. The finding that NVP-AUY922 can enhance the in vitro radiosensitivity of hypoxic tumor cells may have implications for the combined modality treatment of solid tumors.     

YC-1 induces S cell cycle arrest and apoptosis by activating checkpoint kinases

Hypoxia-inducible factor-1alpha (HIF-1alpha) seems central to tumor growth and progression because it up-regulates genes essential for angiogenesis and the hypoxic adaptation of cancer cells, which is why HIF-1alpha inhibition is viewed as a cancer therapy strategy. Paradoxically, HIF-1alpha also leads to cell cycle arrest or the apoptosis of cancer cells. Thus, the possibility cannot be ruled out that HIF-1alpha inhibitors unlock cell cycle arrest under hypoxic conditions and prevent cell death, which would limit the anticancer effect of HIF-1alpha inhibitors. Previously, we reported on the development of YC-1 as an anticancer agent that inhibits HIF-1alpha. In the present study, we evaluated the effects of YC-1 on hypoxia-induced cell cycle arrest and cell death. It was found that YC-1 does not reverse the antiproliferative effect of hypoxia, but rather that it induces S-phase arrest and apoptosis at therapeutic concentrations that inhibit HIF-1alpha and tumor growth; however, YC-1 did not stimulate cyclic guanosine 3',5'-monophosphate production in this concentration range. It was also found that YC-1 activates the checkpoint kinase-mediated intra-S-phase checkpoint, independently of ataxia-telangiectasia mutated kinase or ataxia-telangiectasia mutated and Rad3-related kinase. These results imply that YC-1 does not promote the regrowth of hypoxic tumors because of its cell cycle arrest effect. Furthermore, YC-1 may induce the combined anticancer effects of HIF-1alpha inhibition and cell growth inhibition.     

Influence of different hypoxia models on metastatic potential of SGC-7901 gastric cancer cells

Gastric cancers are one of the most common gastrointestinal tumors with proclivity to metastasis. Hypoxia is an important feature in solid tumor microenvironment; accumulating evidence suggests that chronic and repetitive exposure to hypoxia and reoxygenation seems to provide an advantage to tumor growth. However, mechanisms of intermittent hypoxia regulating gastric cancer metastatic potential remain to be elucidated. In the present study, we established the continuous and intermittent gastric cancer hypoxia models, and we compared the effects of these models on gastric cancer cell metastatic potential. Hypoxia-inducible factor-1α (HIF-1α) and hypoxia target protein/genes expression in response to different hypoxia models were analyzed by Western blot, immunofluorescence, and real-time PCR assays. Gastric cancer cell migration and invasion ability were analyzed by wound healing and Boyden chamber assay. Colony formation and tumor spheroid formation assays were used to assess the ability of self-renewal. Stem-related protein OCT4 and HIF-1α expression were also analyzed by confocal immunofluorescence in single tertiary generation spheroid. We demonstrated that the intermittent hypoxia model upregulated expression of both HIF-1α and hypoxia target protein/genes in a time-dependent manner compared with that of SGC-7901 cells cultured under normoxia. Remarkably, HIF-1α protein was more prone to being located in the nucleus of SGC-7901 cells under conditions of intermittent hypoxia. Gastric cancer cells’ migration and invasion ability were significantly enhanced after hypoxic culture; moreover, intermittent hypoxia-conditioned SGC-7901 cells exhibited higher invasiveness than the continuous hypoxia-conditioned SGC-7901 cells. Gastric cancer stem/progenitor cell subpopulation was also enlarged which indicated an enhanced self-renewal ability in hypoxic cultured SGC-7901 cells. Our study emphasizes the significance of hypoxia in regulating gastric cancer cell metastasis potential. Compared with continuous hypoxia, intermittent hypoxia is a more effective and potent hypoxic stimulus. These results provide a new insight to investigate the hypoxic microenvironment within solid tumors.     

Cell cycle kinase inhibitor expression and hypoxia-induced cell cycle arrest in human cancer cell lines

Flow cytometric analysis of fibroblasts, normal breast epithelial cells and breast or other cancer cell lines identified variation in the abilities of cell lines to undergo cell cycle arrest as a response to hypoxia. Human mammary epithelial cells (HMEC), normal fibroblasts (Hs68 and WI38), HeLa cervical carcinoma and HTB-30 breast carcinoma cells arrest in G(1)/S in response to severe hypoxia. Hep3B hepatocellular carcinoma cells did not exhibit orderly G(1)/S arrest in response to severe hypoxia. We found a general decrease in p16(INK4a) (p16) mRNA levels, with an associated decrease in p16 protein levels in both normal cells and in cancer cells, regardless of their cell cycle response to hypoxia. p27 protein levels did not correlate with the cell line's ability to enter a hypoxic G(1)/S arrest. Furthermore, cell lines that underwent G(1)/S arrest showed decreased expression of hypoxia inducible factor 1 (HIF-1alpha) and at least one member of INK4 or Sdi cell cycle kinase inhibitors families after 12-24 h of hypoxia. Conversely, Hep3B, which did not exhibit orderly hypoxia-associated G(1)/S arrest, also did not show decreased HIF-1alpha, INK4 or Sdi protein levels in hypoxia. Furthermore, Hep3B showed constitutive activating phosphorylation of Akt and inhibitory phosphorylation of GSK3beta, which was the opposite pattern to that exhibited by the cell lines showing the G(1)/S arrest phenotype. Inhibition of GSK3beta by lithium chloride treatment of HeLa cells converted the HIF-1alpha, p16 and p27 loss to levels unchanged by hypoxic exposure. Our results suggest that regulation of the cell cycle during hypoxia in either normal or cancer cells is not simply due to up-regulation of cell cycle kinase inhibitors. Furthermore, decreased protein expression of HIF-1alpha, p16 and p27 was associated with both a hypoxia-induced G(1)/S arrest phenotype and increased GSK3beta activity.     

Modulation of cell death in the tumor microenvironment

The microenvironment of solid human tumors is characterized by heterogeneity in oxygenation. Hypoxia arises early in the process of tumor development because rapidly proliferating tumor cells outgrow the capacity of the host vasculature. Formation of solid tumors thus requires coordination of angiogenesis with continued tumor cell proliferation. However, despite such neovascularization, hypoxia is persistent and frequently found in tumors at the time of diagnosis. Tumors with low oxygenation have a poor prognosis, and strong evidence suggests this is because of the effects of hypoxia on malignant progression, angiogenesis, metastasis, and therapy resistance. The presence of viable hypoxic cells is likely a reflection of the development of hypoxia tolerance resulting from modulation of cell death in the microenvironment. This acquired feature has been explained on the basis of clonal selection-the hypoxic microenvironment selects cells capable of surviving in the absence of normal oxygen availability. However, the persistence and frequency of hypoxia in solid tumors raises a second potential explanation. We suggest that stable microregions of hypoxia may play a positive role in tumor growth. Although hypoxia inhibits cell proliferation and in tumor cells will eventually induce cell death, hypoxia also provides angiogenic and metastatic signals. The development of hypoxia tolerance will thus allow prolonged survival in the absence of oxygen and generation of a persistent angiogenic signal. We will discuss the concept of hypoxia tolerance and review mechanisms used by cancer cells to acquire this phenotype. The concept of hypoxia tolerance has important implications for current and future therapeutic approaches. Most therapeutic efforts to combat hypoxia have focused on targeting the presence of hypoxia itself. Our hypothesis predicts that targeting the biological responses to hypoxia and the pathways leading to hypoxia tolerance may also be attractive therapeutic strategies. Copyright 2003, Elsevier Science (USA). All rights reserved.     

Hypoxia-activated cytotoxicity of benznidazole against clonogenic tumor cells

Solid tumors contain numerous regions with insufficient oxygen concentrations, a condition termed hypoxia. Tumor hypoxia is significantly associated with metastasis, refractory to conventional cancer therapies, and poor patient survival. Therefore, eradication of hypoxic tumor cells will likely have significant impact on the overall progression-free patient survival. This article reports a new discovery that Benznidazole, a bioreductive drug currently used to treat Chagas disease caused by the parasitic protozoan Trypanosoma cruzi, is activated by hypoxia and can kill clonogenic tumor cells especially those under severe hypoxic conditions (≤0.1 % O₂). This type of hypoxia selectivity is important in that severely hypoxic tumor microenvironment is where tumor cells exhibit the strongest resistance to therapy. Mechanistically, activation of Benznidazole coincides with the stabilization of the Hypoxia-Inducible Factor 1α (HIF-1α), suggesting that Benznidazole is activated after tumor cells have entered into a fully hypoxic state. Under such hypoxic conditions, Benznidazole induces the formation of 53BP1 foci, a hallmark of DNA double-stranded breaks that can cause clonogenic inhibition or cell death. These results demonstrate that Benznidazole is a hypoxia-activated cytotoxin with the potential to specifically eliminate hypoxic tumor cells.     

Modulation of TRAIL-induced tumor cell apoptosis in a hypoxic environment

Hypoxia induces Hif-1alpha and selects for loss of wild-type p53 function, both of which can promote tumor cell survival. We evaluated the ability of TRAIL to induce apoptosis of human tumor cell lines exposed to hypoxia. H460 lung cancer cells express low levels of Hif-1alpha, stabilize wild-type p53 during hypoxia, and undergo TRAIL-induced apoptosis. In U2OS osteosarcoma or PA1 ovarian teratocarcinoma cells, high levels of Hif-1alpha and low levels of stable p53 are detected during hypoxia, and cells undergo low levels of TRAIL-induced apoptosis as compared to H460 cells. H460 cells are sensitized to TRAIL-induced apoptosis, whereas U2OS are protected, and little apoptosis is observed in relatively TRAIL-resistant PA1 during hypoxia. Forced expression of Hif-1alpha is also surprisingly a potent inducer of apoptosis in wild-type p53 expressing H460 cells and further promotes TRAIL-induced apoptosis. TRAIL-sensitive wild-type p53-expressing HCT116 colon carcinoma cells modestly elevate Hif-1alpha levels and are equally or slightly more sensitive to TRAIL during hypoxia. In contrast, p53-null HCT116 have higher levels of Hif-1alpha during normoxia and are extremely sensitive to TRAIL, but are protected from TRAIL-induced apoptosis during hypoxia. We hypothesize that a hypoxic tumor microenvironment may alter sensitivity to TRAIL, which may be impacted by Hif-1alpha levels and p53 status. These findings suggest that particular attention to hypoxic regions of tumors and sensitizers to hypoxia-induced cell death may be required to optimize therapeutic combinations using TRAIL.     

Systemic inflammation increases cancer cell adhesion to hepatic sinusoids by neutrophil mediated mechanisms

Interactions between endothelial selectins and selectin ligands expressed on tumor cells have been implicated in the binding of circulating metastatic cancer cells to the vascular endothelium during extravasation. Moreover, there is mounting evidence that inflammatory environments can accelerate the progression of metastasis by neutrophil mediated mechanisms. In this study, a physiologically relevant in vivo model of early metastasis coupled with intravital microscopy was used to visualize the trafficking of tumor cells within the liver vasculature in real time. Using GFP‐labeled Lewis lung carcinoma subline H‐59 cells, we show here that disrupting the interactions between endothelial selectins and tumor cell selectin ligands diminished tumor cell recruitment to the liver. Furthermore, systemic inflammation induced by intravenous injection of lipopolysaccharide significantly enhanced the metastatic potential of these lung carcinoma cells by increasing their propensity to adhere to the liver sinusoidal endothelium. Confocal microscopy revealed frequent colocalization of cancer cells with neutrophils and neutrophil depletion in vivo significantly attenuated the lipopolysaccharide‐induced increase in H‐59 cell adhesion. Although direct selectin–selectin ligand interactions contributed significantly to tumor cell adhesion to sinusoidal endothelial cells, we show here that in addition, interactions between adherent neutrophils within the inflamed sinusoids and circulating tumor cells may further increase tumor cell arrest in the liver. © 2009 UICC     

Glucose requirement for hypoxic accumulation of hypoxia-inducible factor-1alpha (HIF-1alpha)

Hypoxia-inducible factor-1alpha (HIF-1alpha) is both a potential endogenous marker of tumor hypoxia and therapeutic target, and elevated HIF-1alpha protein levels have been shown to be associated with increased hypoxic radiation resistance in FaDu human pharyngeal carcinoma cells in vitro. Here, we found that in FaDu xenografts, no significant HIF-1alpha protein accumulation was detectable by either flow cytometry or Western blot, despite the presence of hypoxic (pimonidazole-positive, radiation resistant) cells. To investigate the effect of different tumor microenvironment conditions on hypoxic HIF-1alpha accumulation, we performed in vitro hypoxia experiments (0.1% O2, 24 h) with manipulation of pH (7.4 vs. 6.7), glucose (0-5.5 mM) and serum (0 or 10%) availability in FaDu and HT 1080 human fibrosarcoma cells. Hypoxic induction of HIF-1alpha protein was strongly dependent on glucose availability and largely abolished at 0.55 mM glucose or less in both cell lines. This glucose effect was confirmed in a hypoxia-responsive-element (HRE)/enhanced-green-fluorescent-protein (EGFP) reporter assay in transfected HT 1080 cells and possibly explains a lack of HIF-1alpha protein in hypoxic tumor cells.     

Onconase induces caspase-independent cell death in chemoresistant neuroblastoma cells

Hypoxia-inducible factor-1alpha (HIF-1alpha) is believed to promote tumor growth, and thus, is viewed as one of the most compelling cancer therapy targets. YC-1 is widely used as a potent inhibitor of HIF-1alpha both in vitro and in vivo, and is also being developed as a novel anticancer drug. However, little is known about the effects of YC-1 on tumor invasion or metastasis. In the present study, we found that the Hep3B cell migration-stimulatory effect of hypoxia was abolished by HIF-1alpha siRNA or YC-1. YC-1 also significantly inhibited the migrations of other cancer cells. Furthermore, YC-1 effectively inhibited cell invasion through Matrigel. In nude mice, GFP-expressing stable cell-lines of Hep3B or H1299 were inoculated into spleens to induce liver metastasis or into the pleural cavity to induce lung invasion. In untreated mice, many tumor lesions emitting strong fluorescence were found in livers or lungs, and fluorescence intensities and tumor lesion numbers were markedly reduced in YC-1-treated mice. These results suggest that YC-1 effectively inhibits tumor invasion and metastasis, and imply that YC-1 is worth while to further develop as a multipurpose anticancer drug.     

Platelets and p-selectin control tumor cell metastasis in an organ-specific manner and independently of NK cells

The prometastatic role of platelets has long been recognized with proposed mechanisms of action including shielding tumor cells from natural killer (NK) cell destruction and aiding endothelial attachment and extravasation of tumor cells with platelet P-selectin being implicated in these processes. However, many aspects of the prometastatic function of platelets remain unclear. In this study, we used mouse models of metastatic breast cancer and melanoma to investigate the platelet effect, focusing on organ specificity, the relationship with NK cells and the relative importance of platelet-derived versus endothelial-derived P-selectin. We found that platelets promote lung metastasis in the absence of NK cells in both acute and spontaneous metastasis models. In addition, the prometastatic action of platelets was found to be organ specific, clearly enhancing lung metastasis but not affecting B16F1 liver metastasis, in fact, liver metastasis was enhanced in the absence of platelets. Furthermore, the profound antimetastatic activity of NK cells was equally effective in the presence or absence of platelets and chronologically distinct from the prometastatic role of platelets. Finally, it was shown that endothelial-derived P-selectin is just as important as platelet-derived P-selectin in promoting lung metastasis and also plays an important role in liver metastasis. Taken together, our findings help clarify the roles of platelets, NK cells and P-selectin in metastasis, and they identify P-selectin as an attractive therapeutic target for preventing metastasis in multiple organs. Cancer Res; 72(18); 4662-71. ?2012 AACR.     

Acute (cyclic) hypoxia enhances spontaneous metastasis of KHT murine tumors.

Hypoxia exists in most human and rodent solid tumors and has been shown to correlate with poor survival in carcinoma of the cervix, carcinoma of the head and neck, and soft tissue sarcoma. It exists both chronically, due to the poorly organized vasculature of solid tumors, and acutely, due to fluctuations in blood flow. It has been found that tumors that are more hypoxic are more likely to metastasize in humans and in rodent models, and it has been demonstrated that exposure of tumor cells to hypoxia in vitro can transiently enhance their metastatic potential when they are reinjected i.v. into mice. The purpose of the present study was to determine whether experimentally imposed hypoxic stress in vivo, either chronic or acute, affects the process of spontaneous metastasis in tumor-bearing mice. We exposed mice bearing KHT tumors to low oxygen conditions (5-7% O(2) breathing) daily during tumor growth in an attempt to induce additional chronic (2 h/day) and acute (12 x 10 min/day) hypoxia in their tumors. By monitoring tumor pO(2) levels over the course of treatment, we demonstrated that these treatments produce acute and chronic hypoxia within the tumor tissue. The acute but not the chronic hypoxia treatment significantly increased the number of spontaneous microscopic lung metastases in the mice by a factor of about 2, and the results suggest that this effect was due to the changes induced in the primary tumor. This study describes a novel method for studying the effects of hypoxia in solid tumors and demonstrates that acute and chronic hypoxia can have different effects on tumor cell behavior in vivo.     

Contextual synthetic lethality of cancer cell kill based on the tumor microenvironment

Acute and chronic hypoxia exists within the three-dimensional microenvironment of solid tumors and drives therapy resistance, genetic instability, and metastasis. Replicating cells exposed to either severe acute hypoxia (16 hours with 0.02% O(2)) followed by reoxygenation or moderate chronic hypoxia (72 hours with 0.2% O(2)) treatments have decreased homologous recombination (HR) protein expression and function. As HR defects are synthetically lethal with poly(ADP-ribose) polymerase 1 (PARP1) inhibition, we evaluated the sensitivity of repair-defective hypoxic cells to PARP inhibition. Although PARP inhibition itself did not affect HR expression or function, we observed increased clonogenic killing in HR-deficient hypoxic cells following chemical inhibition of PARP1. This effect was partially reversible by RAD51 overexpression. PARP1(-/-) murine embryonic fibroblasts (MEF) showed a proliferative disadvantage under hypoxic gassing when compared with PARP1(+/+) MEFs. PARP-inhibited hypoxic cells accumulated γH2AX and 53BP1 foci as a consequence of altered DNA replication firing during S phase-specific cell killing. In support of this proposed mode of action, PARP inhibitor-treated xenografts displayed increased γH2AX and cleaved caspase-3 expression in RAD51-deficient hypoxic subregions in vivo, which was associated with decreased ex vivo clonogenic survival following experimental radiotherapy. This is the first report of selective cell killing of HR-defective hypoxic cells in vivo as a consequence of microenvironment-mediated "contextual synthetic lethality." As all solid tumors contain aggressive hypoxic cells, this may broaden the clinical utility of PARP and DNA repair inhibition, either alone or in combination with radiotherapy and chemotherapy, even in tumor cells lacking synthetically lethal, genetic mutations.