Hypoxia-induced Resistance Research Articles

Overcoming hypoxia-induced resistance of pancreatic and lung tumor cells by disrupting the PERK-NRF2-HIF-axis

Hypoxia-induced resistance of tumor cells to therapeutic treatment is an unresolved limitation due to poor vascular accessibility and protective cell adaptations provided by a network, including PERK, NRF2, and HIF signaling. All three pathways have been shown to influence each other, but a detailed picture remains elusive. To explore this crosstalk in the context of tumor therapy, we generated human cancer cell lines of pancreatic and lung origin carrying an inducible shRNA against NRF2 and PERK. We report that PERK-related phosphorylation of NRF2 is only critical in Keap1 wildtype cells to escape its degradation, but shows no direct effect on nuclear import or transcriptional activity of NRF2. We could further show that NRF2 is paramount for proliferation, ROS elimination, and radioprotection under constant hypoxia (1% O2), but is dispensable under normoxic conditions or after reoxygenation. Depletion of NRF2 does not affect apoptosis, cell cycle progression and proliferation factors AKT and c-Myc, but eliminates cellular HIF-1α signaling. Co-IP experiments revealed a protein interaction between NRF2 and HIF-1α and strongly suggest NRF2 as one of the cellular key factor for the HIF pathway. Together these data provide new insights on the complex role of the PERK-NRF2-HIF-axis for cancer growth.

IL-6 promotes nuclear translocation of HIF-1α to aggravate chemoresistance of ovarian cancer cells

The inflammatory milieu in tumor modulates the resistance to the conventional antitumoral therapies. Interleukin-6 (IL-6), a pleiotropic pro-inflammatory cytokine and a crucial mediator of tumor development, has been targeted as a therapeutic strategy to overcome chemoresistance in the treatment of tumors. The protein levels and nuclear translocation of HIFs (hypoxia-inducible factors), such as HIF-1α, are linked to the drug resistance of tumor cells. However, whether IL-6 promotes the nuclear translocation of HIF-1α and the related mechanism remain to be investigated. We applied two ovarian cancer (OvCa) cell lines, A2780 cells and SKOV3 cells for the in vivo and in vitro studies. We found that IL-6 up-regulates the HIF-1α expression via the signal transducer and activator of transcription 3 (STAT3) signaling under hypoxia in either endogenous or exogenous way, and then we proved that IL-6 enhances the transcriptional activity of HIF-1α via the STAT3 signaling. Further mechanism research revealed that IL-6 promotes the nuclear translocation of HIF-1α through the STAT3 signaling under hypoxia. Proliferation assay and apoptosis assay were applied and proved that IL-6 enhances the chemoresistance of OvCa cells against cisplatin through the upregulation of HIF-1α via the STAT3 signaling in vitro. The In vivo studies confirmed the effect of IL-6 in increasing the chemoresistance of OvCa cells against cisplatin through the IL-6/STAT3/HIF-1α loop in the animal models. Our data elucidates the explicit mechanism of IL-6/STAT3/HIF-1α loop in OvCa and also provides new insights into the development of different approaches for the inflammation-induced and hypoxia-induced resistance in tumor therapies.

Abstract PR09: Developing metabolic intervention strategies to reprogram neuroblastoma epigenome and overcome tumor resistance to differentiation therapy

Abstract Patients with solid tumor usually do not respond to retinoic acid (RA)-based differentiation therapy because RA-resistance often develops. The goal of this study is to develop metabolic intervention strategies that target the cancer epigenome and overcome tumor resistance to differentiation therapy. Experimental procedures: We classified our neuroblastoma (NB) cell line collection into two categories: RA-sensitive cell lines and RA-resistant cell lines. Metabolic intervention agents were combined with RA to treat the RA-resistance cells. Chromatin markers including histone acetylations and methylations were determined using immunoblot. Global DNA methylation were accessed using dot blot. NB cell differentiation were quantified with neurite counting and immunofluorescence staining for NB differentiation marker β-tubulin III. Retinoic acid receptor (RAR) and neuron differentiation marker expression were measured using q-PCR. Summary of findings: Unlike the RA-sensitive NB cells, the RA-resistant cells did not show morphological change or proliferation arrest upon RA treatment. Previously we discovered that glycerol triacetate (GTA) supplementation restored histone acetylation and chromatin accessibility to reactivate the expression of differentiation markers and the RA-induced neuron differentiation program under hypoxia. However, the combination of GTA and RA did not induce proliferation arrest or morphologic change in RA-resistant cells. Interestingly, the combination of RA and the substrate of DNA demethylase, α-ketoglutarate (αKG), significantly induced neuronal differentiation in RA-resistant cells. RAR and differentiation markers expression were also restored by RA+αKG combination. αKG treatment rapidly reduced global 5-methylcytosine (5mC) levels within 1 hour. Although GTA alone or RA+GTA did not induce differentiation, adding GTA in combination with RA and αKG further enhanced the differentiation morphology change in resistant cells. In addition, αKG and GTA supplementation had a synergistic effect in reducing the clonogenic expansion of RA-resistant cells when combined with RA, suggesting that the RA+αKG+GTA combination inhibits self-renewal of RA-resistance NB cells. However, in RA-sensitive cells, αKG could not restore RAR repression caused by hypoxia, probably due to the elevated NADH/NAD+ ratio that favors the conversion of αKG to 2-hydroxyglutarate (2HG), which causes DNA hypermethylation. Conclusions: (1) Unlike hypoxia-induced resistance, which was mainly due to histone deacetylation, DNA methylation is the major cause of resistance in RA-resistant NB cells. (2) αKG+GTA combination effectively reduces DNA methylation and increases histone acetylation to promote RA-sensitivity in RA-resistant NB cells. Citation Format: Haowen Jiang, Yang Li, Michela Yip, Joshua Gruber, Albert Li, Jiangbin Ye. Developing metabolic intervention strategies to reprogram neuroblastoma epigenome and overcome tumor resistance to differentiation therapy [abstract]. In: Abstracts: AACR Special Virtual Conference on Epigenetics and Metabolism; October 15-16, 2020; 2020 Oct 15-16. Philadelphia (PA): AACR; Cancer Res 2020;80(23 Suppl):Abstract nr PR09.

The regulation of bcr-abl in hypoxia is through the mTOR pathway

Chronic myeloid leukemia (CML) is usually characterized by the formation of the fusion onco-protein bcr-abl. Therefore, the majority of CML treatments are bcr-abl specific tyrosine kinase inhibitors (TKIs). TKI resistance in CML treatment is becoming a major obstacle in managing this disease. One well-studied form of drug resistance is hypoxia-induced drug resistance, a phenomenon observed in many other cancers. This study aimed to determine the efficacy of TKIs in CML cells cultured in hypoxia. It was observed that bcr-abl translation was severely halted in hypoxia, rendering TKIs ineffective. We found that the mechanism by which bcr-abl protein levels were being suppressed in hypoxia was through the mTOR pathway, specifically via ribosomal protein S6 (RPS6). This information is vital to the improvement of CML treatments, as it can be used to determine how to best combat hypoxia-induced drug resistance in CML and subsequently to identify new targets for treatment.

Activatable nanomedicine for overcoming hypoxia-induced resistance to chemotherapy and inhibiting tumor growth by inducing collaborative apoptosis and ferroptosis in solid tumors

Hypoxia has been firmly correlated to the drug resistance of solid tumors. Alleviation of hypoxia by tumor reoxygenation is expected to sensitize the chemotherapy toward solid tumors. Alternatively, ferroptosis provides a therapeutic strategy to overcome apoptotic resistance and multidrug resistance of solid tumors, collaboratively strengthening the chemotherapy toward hypoxic tumors. Herein, an ultrasound (US)-activatable nanomedicine was developed for overcoming hypoxia-induced resistance to chemotherapy and efficiently inhibiting tumor growth by inducing sensitized apoptosis and collaborative ferroptosis of tumor cells. This nanomedicine was constructed by integrating ferrate and doxorubicin into biocompatible hollow mesoporous silica nanoplatforms, followed by assembling a solid-liquid phase-change material of n-heneicosane. The US-induced mild hyperthermia initiates the phase change of n-heneicosane, enabling US-activated co-release of ferrate and doxorubicin. Results reveal that the released ferrate effectively reacts with water as well as the over-expressed hydrogen peroxide and glutathione in tumor cells, achieving tumor-microenvironment-independent reoxygenation and glutathione-depletion in tumors. The reoxygenation down-regulates expressions of hypoxia-inducible factor 1α and multidrug resistance gene/transporter P-glycoprotein in tumor cells, sensitizing the apoptosis-based doxorubicin chemotherapy. More importantly, exogenous iron metabolism from the nanomedicine initiates intracellular Fenton reactions, leading to reactive oxygen species overproduction and iron-dependent ferroptotic death of tumor cells. Furthermore, the glutathione-depletion inactivates the glutathione peroxidase 4 (GPX4, a critical regulatory target in ferroptosis), inhibiting the reduction of lipid peroxides and reinforcing the ferroptotic cell death. The sensitized chemotherapy together with the iron-dependent ferroptosis of tumor cells play a synergistic role in boosting the growth suppression of hypoxic osteosarcoma in vivo. Additionally, the nanomedicine acts as a nanoprobe for in vivo photoacoustic imaging and glutathione tracking, showing great potential as theranostic agents for hypoxic solid tumors treatment.

Inhibition of HIF-1a By PX-478 Normalizes Blood Vessels, Improves Drug Delivery and Suppresses Progression and Dissemination in Multiple Myeloma

Introduction: Multiple myeloma (MM) is a lymphoplasmacytic malignancy localized in the bone marrow (BM) characterized by the continuous metastasis. Despite the introduction of novel therapies, MM patients relapse due to the development of drug resistance that is, at least in part, promoted by hypoxia (insufficient oxygen). MM cells develop a hypoxic phenotype, leading to cellular adaptations that cause metastasis, angiogenesis, stemness and resistance to drugs, such as carfilzomib, promoted by a hypoxia-inducible factor-1α (HIF-1α) transcription factor. Herein, we explored the mechanisms underlying HIF pathway inhibition using for the first time in MM a HIF-1α selective small molecule inhibitor, PX-478, both in vitro and in vivo. Methods: In vitro, to test the effect of PX-478 (0 - 50 µM) in combination with carfilzomib on MM cell survival exposed to normoxia (21% O2) or hypoxia (1% O2) was assessed using MTT assay. Cell adhesion to endothelial cells (HUVECs), and cell migration to stromal cells of prelabeled MM cells treated with PX-478 was measured by fluorescent spectrophotometer and flow cytometry, respectively. Tube-like formation of HUVECs as well as survival was tested in the presence of PX-478. For in vivo study, MM.1S-Luc-GFP cells were injected intravenously (i.v.) into 40 SCID mice; 3 weeks post injection the mice were divided into 4 groups and treated with vehicle (PBS), carfilzomib, PX-478, and a combination of PX-478 and carfilzomib. Tumor progression and weight was monitored weekly by bioluminescent imaging, and survival was monitored daily. At day 28, 3 mice from each group were randomly taken: (i) to test the number of circulating tumor cells (MM-GFP+) in the peripheral blood counted by flow cytometry; (ii) to test the MM apoptosis in the femurs by TUNEL staining; and (iii) to test extramedullar metastasis of MM in the kidney, spleen and the liver using immunohistochemistry. Additionally, tumor vasculature was demonstrated in the skull using photoacoustic imaging as well as tumor involvement using fluorescent microscopy. Moreover, we tested the drug delivery by injecting fluorescent large molecule (Dextran-AF405 Mw=70,000) i.v. in MM-bearing mice treated with and without PX-478. Lastly, we tested the effect of PX-478 on prelabeled MM cell retention in the blood and homing to the BM one hour post-MM injection in naïve mice. Results: We found that PX-478 reversed the hypoxia-induced resistance of MM cells to carfilzomib, inhibited metastasis-related cell processes such as adhesion and migration, and reduced MM-mediated tube-like formation of HUVECs in vitro. In vivo, in MM-bearing mice PX-478 decreased the number of MM circulating cells, suppressed tumor metastasis, improved vascularization of the tumor thus delivery of chemotherapy, and as a result re-sensitized MM cells to carfilzomib by increasing tumor apoptosis thus completely abrogating tumor growth and significantly extending mice survival. Conclusions: This is the first study to show the efficacy of PX-478 in MM demonstrating that PX-478 is acting as a pleiotropic molecule in which it inhibited many different hypoxia-induced biological processes - migration, angiogenesis and drug resistance. By overcoming these cancer adaptations, PX-478 has a clear advantage over using agents that carry an effect against one of these processes. This data provides a preclinical basis for future clinical trials testing efficacy of PX-478 in MM. Disclosures No relevant conflicts of interest to declare.

Hypoxia Induces Resistance to EGFR Inhibitors in Lung Cancer Cells via Upregulation of FGFR1 and the MAPK Pathway.

Development of resistance remains the key obstacle to the clinical efficacy of EGFR tyrosine kinase inhibitors (TKI). Hypoxia is a key microenvironmental stress in solid tumors associated with acquired resistance to conventional therapy. Consistent with our previous studies, we show here that long-term, moderate hypoxia promotes resistance to the EGFR TKI osimertinib (AZD9291) in the non-small cell lung cancer (NSCLC) cell line H1975, which harbors two EGFR mutations including T790M. Hypoxia-induced resistance was associated with development of epithelial-mesenchymal transition (EMT) coordinated by increased expression of ZEB-1, an EMT activator. Hypoxia induced increased fibroblast growth factor receptor 1 (FGFR1) expression in NSCLC cell lines H1975, HCC827, and YLR086, and knockdown of FGFR1 attenuated hypoxia-induced EGFR TKI resistance in each line. Upregulated expression of FGFR1 by hypoxia was mediated through the MAPK pathway and attenuated induction of the proapoptotic factor BIM. Consistent with this, inhibition of FGFR1 function by the selective small-molecule inhibitor BGJ398 enhanced EGFR TKI sensitivity and promoted upregulation of BIM levels. Furthermore, inhibition of MEK activity by trametinib showed similar effects. In tumor xenografts in mice, treatment with either BGJ398 or trametinib enhanced response to AZD9291 and improved survival. These results suggest that hypoxia is a driving force for acquired resistance to EGFR TKIs through increased expression of FGFR1. The combination of EGFR TKI and FGFR1 or MEK inhibitors may offer an attractive therapeutic strategy for NSCLC. SIGNIFICANCE: Hypoxia-induced resistance to EGFR TKI is driven by overexpression of FGFR1 to sustain ERK signaling, where a subsequent combination of EGFR TKI with FGFR1 inhibitors or MEK inhibitors reverses this resistance. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/21/4655/F1.large.jpg.

Hypoxia induces sorafenib resistance mediated by autophagy via activating FOXO3a in hepatocellular carcinoma

Sorafenib, a multikinase inhibitor, is considered as the only approved drug to cure the advanced hepatocellular carcinoma (HCC); however, the acquired chemoresistance caused by intratumoral hypoxia through sorafenib long term therapy induces sorafenib inefficacy. We demonstrated here that hypoxia significantly attenuated sensitivity of HCC cells to sorafenib treatment and reduced its proliferation. Autophagy was observed in sorafenib-treated HCC cells in hypoxia, and inhibition of autophagy by 3-MA eliminated hypoxia-induced sorafenib resistance. Further study revealed hypoxia-activated FOXO3a, an important cellular stress transcriptional factor, via inducing its dephosphorylation and nuclear location; and FOXO3a-dependent transcriptive activation of beclin-1 was responsible for hypoxia-induced autophagy in HCC cells. Knockout of FOXO3a inhibited the autophagy induced by sorafenib itself in normoxia and significantly enhanced the cytotoxicity of sorafenib in HCC cells; and it also inhibited the hypoxia-induced autophagy and achieved the same effect in sorafenib sensitivity-enhancement in HCC cells as it in normoxia. Finally, knockout of intratumoral FOXO3a significantly enhanced curative efficacy of sorafenib via inhibition of autophagy in xenograft tumors in nude mice. Collectively, our study suggests that FOXO3a plays a key role in regulating hypoxia-induced autophagy in sorafenib-treated HCC, and FOXO3-targeted therapy may serve as a promising approach to improve clinical prognosis of patients suffering from HCC.

Oroxylin A reverses hypoxia-induced cisplatin resistance through inhibiting HIF-1α mediated XPC transcription.

Hypoxia is a key concern during the treatment of non-small cell lung cancer (NSCLC), and hypoxia-inducible factor 1 alpha (HIF-1α) has been associated with increased tumor resistance to therapeutic modalities such as cisplatin. Compensatory activation of nucleotide excision repair (NER) pathway is the major mechanism that accounts for cisplatin resistance. In the present study, we suggest a novel strategy to improve the treatment of NSCLC and overcome the hypoxia-induced cisplatin resistance by cotreatment with Oroxylin A, one of the main bioactive flavonoids of Scutellariae radix. Based on the preliminary screening, we found that xeroderma pigmentosum group C (XPC), an important DNA damage recognition protein involved in NER, dramatically increased in hypoxic condition and contributed to hypoxia-induced cisplatin resistance. Further data suggested that Oroxylin A significantly reversed the hypoxia-induced cisplatin resistance through directly binding to HIF-1α bHLH-PAS domain and blocking its binding to HRE3 transcription factor binding sites on XPC promoter which is important to hypoxia-induced XPC transcription. Taken together, our findings not only demonstrate a crucial role of XPC dependent NER in hypoxia-induced cisplatin resistance, but also suggest a previously unrecognized tumor suppressive mechanism of Oroxylin A in NSCLC which through sensitization of cisplatin-mediated growth inhibition and apoptosis under hypoxia.

Self-Delivery Photodynamic Nanoinhibitors for Tumor Targeted Therapy and Metastasis Inhibition.

Simultaneous inhibitions of primary tumor growth and distant metastasis are very critical for cancer patients to improve their survival and cure rates. Although photodynamic therapy (PDT) shows great potential for primary tumor treatment, it often exacerbates hypoxia with a reduced therapeutic efficacy and subsequently contributes to carcinoma progression and metastatic dissemination. To solve these issues, self-delivery photodynamic nanoinhibitors (PNI) are developed for tumor targeted therapy and metastasis inhibition. PNI are composed of a carbonic anhydrase inhibitor (CAi), a hydrophilic poly(ethylene glycol) (PEG) linker, and a hydrophobic photosensitizer protoporphyrin IX (PpIX). Such self-delivery design of PNI avoids the premature release and heterogeneous distribution of CAi and PpIX to enhance the availability and synergism. Briefly, the CAi-based nanoinhibitors improve the selectivity of CAi for specific recognition and inhibition of tumor-associated isoform carbonic anhydrase (CA) IX, which would not only facilitate the targeted drug delivery of PNI but also regulate the hypoxia-induced signaling cascade and PDT resistance. Benefiting from the CA IX inhibition and targeted PDT, PNI exhibit a robust inhibitory effect on primary tumor growth and distant metastasis. This targeted self-delivery strategy sheds light on the photosensitizer-based molecular design to overcome the defect of traditional PDT.

Abstract 1877: Hypoxia induces EGFR inhibitor resistance in lung cancer cells by upregulation of fibroblast growth factor receptor 1 (FGFR1) via MAPK pathway

Abstract The development of small molecule tyrosine kinase inhibitors (TKIs) specific for epidermal growth factor receptors (EGFRs) with activating mutations has led to a new paradigm in the treatment of non-small cell lung cancer (NSCLC) patients. However, most patients eventually develop resistance. Hypoxia is a key micro-environmental stress in solid tumors that is associated with poor prognosis due in part to acquired resistance to conventional therapy. In our previous study, we showed that long-term, moderate hypoxia promotes resistance to the EGFR TKI, gefitinib, in the NSCLC cell line, HCC827, which harbors an activating EGFR mutation. In this study, we found that hypoxia also induces third generation EGFR TKI, osimertinib (AZD9291), resistance in the NSCLC cell line, H1975, which had developed resistance to first and second generation of EGFR TKIs by harboring second EGFR mutation, T790M. Following growth in hypoxia, hypoxia-induced gefitinib resistant HCC827 clones and hypoxic H1975 cells show increased N-cadherin expression and decreased E-cadherin expression, characteristics of an epithelial-mesenchymal transition (EMT), coordinated by an increased expression of ZEB1, an EMT activator. Mechanistically, we show that hypoxia induces increased fibroblast growth factor receptor 1 (FGFR1) expression in HCC827 and H1975 cells, and that knockdown of the FGFR1 by shRNA can attenuate hypoxia induced EGFR TKI resistance in both HCC827 and H1975 cells. We also found the upregulated expression of FGFR1 by hypoxia is mediated through the MAPK pathway to lead to EGFR TKI resistance. In keeping with this, inhibition of FGFR1 function by the selective small molecular inhibitor, BGJ398, attenuates hypoxia-induced EGFR TKI resistance and promotes upregulation of BIM levels. Similarly, inhibition of MEK activity by the inhibitor, trametinib, shows similar effects. In vivo in tumor xenografts in mice, BGJ398 treatment or trametinib treatment suppresses tumor growth and enhances AZD9291 response. These results suggest that hypoxia is a driving force for acquired resistance to EGFR TKIs through increased FGFR1 expression and coordination of EMT in NSCLC. The combination of EGFR TKIs and FGFR1 or MEK inhibitors may offer an attractive therapeutic strategy for NSCLCs. Citation Format: Yuhong Lu, Yanfeng Liu, Sebastian Oeck, Gary J. Zhang, Peter M. Glazer. Hypoxia induces EGFR inhibitor resistance in lung cancer cells by upregulation of fibroblast growth factor receptor 1 (FGFR1) via MAPK pathway [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1877.

Modulation of Tumor Hypoxia by pH-Responsive Liposomes to Inhibit Mitochondrial Respiration for Enhancing Sonodynamic Therapy.

Background and PurposeSonodynamic therapy (SDT) has been widely used for the noninvasive treatment of solid tumors, but the hypoxic tumor microenvironment limits its therapeutic effect. The current methods of reoxygenation to enhance SDT have limitations, prompting reconsideration of the design of therapeutic approaches. Here, we developed a tumor microenvironment-responsive nanoplatform by reducing oxygen consumption to overcome hypoxia-induced resistance to cancer therapy.MethodsA pH-responsive drug-loaded liposome (MI-PEOz-lip) was prepared and used to reduce oxygen consumption, attenuating hypoxia-induced resistance to SDT and thereby improving therapeutic efficiency. Photoacoustic imaging (PAI) and fluorescence imaging (FI) of MI-PEOz-lip were evaluated in vitro and in breast xenograft tumor models. The pH-sensitive functionality of MI-PEOz-lip was applied for pH-triggered cargo release, and its capacity was evaluated. The MI-PEOz-lip-mediated SDT effect was compared with other treatments in vivo.ResultsMI-PEOz-lip was demonstrated to specifically accumulate in tumors. Metformin molecules in liposomes selectively accumulate in tumors by pH-responsive drug release to inhibit the mitochondrial respiratory chain while releasing IR780 to the tumor area. These pH-responsive liposomes demonstrated PAI and FI imaging capabilities in vitro and in vivo, providing potential for treatment guidance and monitoring. In particular, the prepared MI-PEOz-lip combined with ultrasound irradiation effectively inhibited breast tumors by producing toxic reactive singlet oxygen species (ROS), while the introduction of metformin inhibited mitochondrial respiration and reduced tumor oxygen consumption, resulting in excellent sonodynamic therapy performance compared with other treatments.ConclusionIn this study, we present a novel strategy to achieve high therapeutic efficacy of SDT by the rational design of multifunctional nanoplatforms. This work provides a new strategy that can solve the current problems of inefficient oxygen delivery strategies and weaken resistance to various oxygen-dependent therapies.

Spatiotemporally Targeted Nanomedicine Overcomes Hypoxia-Induced Drug Resistance of Tumor Cells after Disrupting Neovasculature

Vascular disrupting agents (VDAs) are emerging anticancer agents, which show rising demand for combination with cytostatic drugs (CSDs), owing to inadequate tumor inhibition when applied singly. Nevertheless, the combination remains a challenge due to the different working sites of VDAs and CSDs and hypoxia-induced drug resistance after disrupting neovasculature by VDAs. Herein, we developed a shell-stacked nanoparticle (SNP) for coencapsulation of a VDA combretastatin A-4 phosphate (CA4P) and a proteasome inhibitor bortezomib (BTZ). The SNP could spatiotemporally deliver CA4P to tumor neovasculature and BTZ to tumor cells mediated by the site-specific stimuli-activated drug release. Moreover, the SNP also reversed the drug resistance caused by the overexpressed ABCG2 under CA4P-induced hypoxic conditions. The spatiotemporally targeted combination therapy significantly inhibited the growth of both the human A549 pulmonary adenocarcinoma xenograft model and patient-derived xenograft (PDX) model of colon cancer in mice, providing a promising strategy for treating advanced cancers.

Hypoxia-induced downregulation of B-cell translocation gene 3 confers resistance to radiation therapy of colorectal cancer.

Colorectal cancer (CRC) is now a major human cancer, and B-cell translocation gene 3 (BTG3) has been reported as a tumor-suppressor in CRC, but its upstream regulator has not been identified. Endogenous expression levels of BTG3 were compared between normal colorectal cell line CCD-18Co and two CRC cell lines SW480 and HT29, as well as between CRC patient tumor and adjacent normal tissues. Analysis of BTG3 genomic region was performed which identified a putative hypoxia response element (HRE). Effects of hypoxia condition, BTG3 overexpression, and their combination on the radiation sensitivity of CRC cell lines were assessed. BTG3 was downregulated in CRC cell lines and patient tumor samples, via the HRE in its promoter region. Hypoxia and BTG3 overexpression could both induce radiation resistance in CRC cells. Combining hypoxia with BTG3 overexpression effectively rendered the resistance of CRC cells to radiation to a level lower than hypoxia alone and higher than normoxia alone, indicating the essential role of BTG3 in hypoxia-induced radiation resistance of CRC cells. We therefore propose a novel signaling cascade involving hypoxia/BTG3 to be a potential risk factor for CRC patients undergoing radiation therapy, which could possibly serve as therapeutic targets among CRC patients with acquired radiotherapy resistance.

Targeted Myoglobin Delivery as a Strategy for Enhancing the Sensitivity of Hypoxic Cancer Cells to Radiation.

SummaryThe effectiveness of cancer radiotherapy is frequently hindered by the hypoxia of the tumor microenvironment. Direct delivery of oxygen to hypoxic tumor tissues is an attractive strategy to overcome this hypoxia-associated radioresistance. Herein, we report the generation of submicron-sized particles comprising myoglobin fused to the crystal-forming domain of Cry3Aa protein for the targeted delivery of oxygen to cancer cells. We demonstrate that myoglobin-containing particles were successfully produced in Bacillus thuringiensis with the assistance of the Cry3Aa domain I. Furthermore, these particles could be genetically modified to incorporate the cell penetrating peptide TAT and cell targeting peptide A549.1, resulting in particles that exhibited improved cellular uptake and targeting toward A549 cells. Notably, these myoglobin-containing particles increased the intracellular oxygen levels of A549 cells and thereby sensitized them to radiation. These findings suggest that the targeted delivery of O2-bound myoglobin could be an effective approach to enhance the efficacy of radiotherapy.

β-Escin alleviates cobalt chloride-induced hypoxia-mediated apoptotic resistance and invasion via ROS-dependent HIF-1α/TGF-β/MMPs in A549 cells.

Hypoxia is contributed in various pathophysiological conditions including obesity, cardiovascular diseases, and cancer. In cancer, hypoxia is a salient phenomenon and has been correlated with tumor progression, metastasis, and provoke resistance to therapies in cancer patients, which exert with stabilization of main effector, hypoxia inducible factor-1 alpha (HIF-1α). Therefore, therapeutic targeting of hypoxic responses in cancer is the potential approach to improve the better treatment efficacy. In the present study, we evaluated the effect of β-Escin (β-Es) on hypoxia-induced resistance to apoptosis and metastasis in human non-small-cell lung cancer cells. The MTT assay revealed that β-Es treatment decreased the A549 cells viability under cobalt chloride-induced hypoxia. Apoptotic proteins were analyzed by western blot that showed cancer cells treated with β-Es induced cell death in hypoxia condition as proteins compared with normoxia. Moreover, we observed that cobalt chloride induced hypoxia through the generation of intracellular reactive oxygen species and stabilized the transcriptional factor HIF-1α, which leads to cancer metastasis. This notion was supported by the migration, invasion, and adhesion assays. Furthermore, hypoxia increased the expression of transforming growth factor-β, and the activation of matrix metalloproteinases were suppressed by the treatment of β-Es as well as pretreatment with N-acetylcysteine (NAC). Therefore, we demonstrate that a concurrent activation of HIF-1α, transforming growth factor-β, and matrix metalloproteinases participate in hypoxia-induced metastasis and that β-Es prevent A549 cells metastasis by inhibition of reactive oxygen species.

Circular RNA circNRIP1 Sponges microRNA-138-5p to Maintain Hypoxia-Induced Resistance to 5-Fluorouracil Through HIF-1α-Dependent Glucose Metabolism in Gastric Carcinoma.

BackgroundHypoxia-induced chemoresistance is recognized as a major obstacle to the successful treatment of gastric cancer (GC). Circular RNAs (circRNAs) have been proposed to implicate in resistance to chemotherapeutic drugs. However, whether circNRIP1 is involved in the development of hypoxia-induced 5-fluorouracil (5-FU) resistance remains largely unknown.MethodsGene expression was evaluated using quantitative real-time polymerase chain reaction and Western blot. The impact of circNRIP1 on hypoxia-induced resistance to 5-FU was investigated by determining glucose consumption, lactate production and glucose-6-phosphate (G6P) levels. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolim bromide assay was performed to assess cell survival.ResultscircNRIP1 was upregulated in GC cells. Hypoxia induced the upregulation of circNRIP1 and reduced the sensitivity of GC cells to 5-FU, as evidenced by the increase in multidrug resistance 1 gene, P-glycoprotein, hypoxia-inducible factor-1α (HIF-1α) and G6P levels, glucose consumption, lactate production, as well as cell survival. Silencing of circNRIP1 enhanced the sensitivity of GC cells to 5-FU under a hypoxic condition. microRNA (miR)-138-5p was confirmed as a downstream target gene of circNRIP1, and upregulation of miR-138-5p could reverse the effect of circNRIP1 on hypoxia-induced 5-FU resistance. Additionally, HIF-1α was a target gene of miR-138-5p. More significantly, the effect of circNRIP1 on hypoxia-induced 5-FU resistance was markedly blocked by 2-DG treatment.ConclusioncircNRIP1 functioned as a miR-138-5p sponge to enhance hypoxia-induced resistance to 5-FU through modulation of HIF-1α-dependent glycolysis, which provides a novel potential approach to overcome hypoxia-induced 5-FU resistance in GC.

Hypoxia-induced up-regulation of miR-27a promotes paclitaxel resistance in ovarian cancer.

Ovarian cancer (OC) is a malignant tumor with high mortality in women. Although cancer patients initially respond to paclitaxel chemotherapy following surgery, most patients will relapse after 12–24 months and gradually die from chemotherapy resistance. In OC, cancer cells become resistant to paclitaxel chemotherapy under hypoxic environment. The miR-27a has been identified as an oncogenic molecular in ovarian cancer, prostate cancer, liver cancer etc. In addition, the miR-27a is involved in hypoxia-induced chemoresistance in various cancers. However, the role of miR-27a in hypoxia-induced OC resistance remains unclear. The aim of the present study was to investigate the regulatory mechanism of miR-27a in hypoxia-induced OC resistance. The expression of HIF-1α induced Hypoxia overtly up-regulated. At the same time, hypoxia increased viability of Skov3 cells and decreased cell apoptosis when treated with paclitaxel. The expression of the miR-27a was obviously up-regulated under hypoxia and involved in hypoxia-induced paclitaxel resistance. Follow-up experiments portray that miR-27a improved paclitaxel resistance by restraining the expression of APAF1 in OC. Finally, we further elucidated the important regulatory role of the miR-27a-APAF1 axis in OC through in vivo experiments. According to our knowledge, we first reported the regulation of miR-27a in hypoxia-induced chemoresistance in OC, providing a possible target for chemoresistance treatment of OC.

Overcoming the hypoxia-induced drug resistance in liver tumor by the concurrent use of apigenin and paclitaxel

The chemotherapeutic efficacy of paclitaxel against hypoxic tumors is usually unsatisfactory, which is partially due to the so-called hypoxia-induced drug resistance. The mechanism of hypoxia-induced resistance is primarily associated with hypoxia-inducible factor 1α (HIF-1α), which is an oxygen-sensitive transcriptional activator coordinating the cellular response to hypoxia. Apigenin is a natural occurring HIF-1α inhibitor that can suppress the expression of HIF-1α through multiple pathways and reverse the hypoxia-induced resistance found in cancer cells. Here we report that the use of apigenin can suppress the HIF-1α expression in hypoxic tumors through the simultaneous inhibition of the AKT/p-AKT pathway and HSP90, which is beneficial for enhancing the anticancer activity of the co-administered paclitaxel. The potential synergistic effect of apigenin and paclitaxel was further validated on HepG2 cell line and tumor-bearing mouse models.

HIF2A overexpression reduces cisplatin sensitivity in cervical cancer by inducing excessive autophagy.

BackgroundHypoxia-induced autophagy is a crucial factor that induces chemotherapy resistance in tumor cells. As a key regulator facilitating the adaptation of solid tumors to hypoxia, the role of hypoxia-inducible factors (HIFs) in regulating hypoxia-induced chemotherapy resistance and autophagy has been extensively studied. However, the majority of studies have mainly focused on HIF-1. Direct evidence concerning the role of HIF2A in cisplatin resistance is sparse, and its underlying mechanism is not yet known.MethodsAnimal models were constructed by subcutaneously injecting cervical cancer cells stably overexpressing HIF2A (LV-HIF2A) with or without intraperitoneal injection of cisplatin. Tumor size and weight were evaluated to determine tumor growth. Apoptosis was detected by TUNEL assay and protein expression by western blotting.ResultsNude mice injected with cells overexpressing HIF2A showed larger and heavier tumors than those in mice injected with negative control lentivirus (LV-NC)-infected cells, with or without cisplatin. Fewer apoptotic cells were noted in tumor tissues from the LV-HIF2A group than from the LV-NC group, with or without cisplatin. Additionally, expression of the anti-apoptotic protein, B-cell lymphoma 2 (BCL2), and autophagy-related proteins, beclin 1 and autophagy related 5 (ATG5), were found to be higher in the LV-HIF2A group than in the LV-NC group, regardless of cisplatin treatment. Moreover, expression of the pro-apoptotic protein, BCL2-associated X (BAX), was lower in tumor tissues from the LV-HIF2A group than from the LV-NC group. Effect of HIF2A overexpression on cisplatin sensitivity was found to be alleviated in vivo by the autophagy inhibitor, 3-methyladenine (3-MA).ConclusionsHIF2A overexpression promoted tumor growth and autophagy but suppressed apoptosis in vivo, with or without cisplatin. The HIF2A overexpression-affected cisplatin sensitivity was alleviated by 3-MA. Therefore, we suggest that HIF2A overexpression reduces cisplatin sensitivity in cervical cancer by inducing excessive autophagy.