Hypoxia-induced Resistance Research Articles

Fluorocarbon-Functionalized Polymerization-Induced Self-Assembly Nanoparticles Alleviate Hypoxia to Enhance Sonodynamic Cancer Therapy.

Sonodynamic therapy (SDT) is an ultrasound-based, noninvasive cancer treatment that targets tumor cells by triggering reactive oxygen species production. However, the limited accumulation of sonosensitizers and the insufficient supply of O2 to the hypoxic environment at the tumor site greatly limit the effectiveness of SDT. To address these issues, positively charged porphyrin-containing nanoparticles (NPs) from self-assembling of fluorocarbon/polyethylene glycol amphiphilic block copolymer, which is synthesized through reversible addition-fragmentation chain transfer polymerization, are constructed. The NPs with fluorocarbon core and positively charged hydrophilic shells not only stabilize the sonosensitizer and improve its cellular uptake, but also act as an O2 carrier alleviating the hypoxic tumor environment. In vitro and in vivo experiments demonstrate that the NPs effectively deliver O2 to the tumor and supply sufficient O2 to Renca cells after ultrasound treatment. Consequently, the NPs inhibit hypoxia-induced resistance to SDT and significantly produce reactive oxygen species by activated porphyrin moieties, inducing apoptosis in cancer cells. These oxygen-enhanced sonosensitizer NPs hold promise for cancer therapies such as photodynamic therapy, radiotherapy, and chemotherapy by overcoming hypoxia-induced resistance.

Local Regulation of Mitochondrial Respiratory for Enhancing Photodynamic Therapy and Breaking Chemoresistance in Hypoxia.

The hypoxic condition in solid tumors induces therapy resistance, limited therapeutic efficacy, and tumor recurrence, especially for chemotherapy and aerobic photodynamic therapy (PDT). To address this matter, an O2 regulator (SNP@Ato) is designed for breaking chemoresistance and enhancing PDT, which is constructed by loading Atovaquone (Ato) through self-assembly and host-guest interaction between β-cyclodextrin functionalized tetraphenylporphyrin (TPP-CD4) and thioketal-linked camptothecin/azobenzene (Azo-TK-CPT). Specifically, the porphyrin units in SNP@Ato are in "Off state" due to the photoinduced electron transfer (PET) effect between the porphyrin units and azobenzene. After encountering the hypoxic condition in solid tumors, SNP@Ato is dissociated by the cleaved azobenzene on account of over-expressed azo-reductase. Then the mitochondrial respiratory of cancer cells would be suppressed with the participation of Ato, generating a local hypoxia relief for sensitized chemotherapy and enhanced PDT. Accompanied by efficient PDT, the TK linker is broken by ROS, and the CPT is released from the prodrugs. Compared with the SNP group without oxygen-regulator, SNP@Ato exhibits a remarkable improvement of the therapeutic effect against hypoxic tumors in vitro and in vivo. This work proposes a novel paradigm for overcoming hypoxia-induced therapeutic resistance.

Priming of cancer-immunity cycle by alleviating hypoxia-induced ferroptosis resistance and immunosuppression

Stimulating a robust cancer-immunity cycle (CIC) holds promising potential for eliciting potent and enduring immune responses for cancer immunotherapy. However, designing a therapeutic nanomaterial capable of both enhancing tumor immunogenicity and mitigating immunosuppression is challenging and often associated with complicated design paradigms and immune-related adverse effects. Herein, a multienzyme-mimetic alloy nanosheet incorporating palladium (Pd) and iron (Fe) is developed, which can prime effective CIC by overcoming ferroptosis resistance for enhancing tumor immunogenicity and reprograming the tumor microenvironment for enhanced second near-infrared (NIR-II) photoimmunotherapy. The nanosheets accumulate in tumors when administered intravenously and counteract hypoxia through catalase-like oxygen production and subsequent reduction of hypoxia-inducible factor-1α, M2-like macrophages, regulatory T-cell, and programmed death-ligand 1 (PD-L1) expression. The surface plasmon resonance of the nanosheets enables NIR-II phototherapy and photoacoustic imaging, coupling with its ferroptosis and tumor microenvironment reprogram properties to synergize with anti-PD-L1 checkpoint blockade therapy to achieve satisfactory antitumor outcome. This study offers a strategy for localized tumor treatment and boosting the CIC through a straightforward and inexpensive nanomaterial design.

A multifunctional mesoporous silica drug delivery nanosystem that ameliorates tumor hypoxia and increases radiotherapy efficacy

Radiotherapy (RT) is a widely used treatment with strong therapeutic effects, but overcoming challenges related to hypoxia-induced tumor resistance and ineffective antitumor immune responses is crucial for optimal outcomes. In this study, we developed a versatile nanosystem using mesoporous silica nanoparticles (MSNs), R837, and a small quantity of manganese peroxide (Mn/ZnO2). The synthesized MSN@R837-Mn/ZnO2 nanoparticles exhibited precise tumor targeting and accumulation, controlled drug release under acidic conditions, and increased sensitivity in magnetic resonance imaging. These attributes collectively augmented the therapeutic efficacy of RT by alleviating hypoxia and immunosuppression. Tumor cells treated with RT combined with these nanoparticles displayed reduced oxidative stress, alleviated hypoxia, and normalized blood vessel formation. Notably, all mice in the RT + PD-1 + MSN@R837-Mn/ZnO2 group achieved complete tumor regression with extended survival. Safety assessments confirmed the absence of MSN@R837-Mn/ZnO2 toxicity, highlighting its potential as a promising approach with dual functionality for the diagnostic imaging and treatment of cancer.

Hemoglobin nanoclusters-mediated regulation of KPNA4 in hypoxic tumor microenvironment enhances photodynamic therapy in hepatocellular carcinoma

BackgroundHepatocellular carcinoma (HCC) is a highly malignant tumor known for its hypoxic environment, which contributes to resistance against the anticancer drug Sorafenib (SF). Addressing SF resistance in HCC requires innovative strategies to improve tumor oxygenation and effectively deliver therapeutics.ResultsIn our study, we explored the role of KPNA4 in mediating hypoxia-induced SF resistance in HCC. We developed hemoglobin nanoclusters (Hb-NCs) capable of carrying oxygen, loaded with indocyanine green (ICG) and SF, named HPRG@SF. In vitro, HPRG@SF targeted HCC cells, alleviated hypoxia, suppressed KPNA4 expression, and enhanced the cytotoxicity of PDT against hypoxic, SF-resistant HCC cells. In vivo experiments supported these findings, showing that HPRG@SF effectively improved the oxygenation within the tumor microenvironment and countered SF resistance through combined photodynamic therapy (PDT).ConclusionThe combination of Hb-NCs with ICG and SF, forming HPRG@SF, presents a potent strategy to overcome drug resistance in hepatocellular carcinoma by improving hypoxia and employing PDT. This approach not only targets the hypoxic conditions that underlie resistance but also provides a synergistic anticancer effect, highlighting its potential for clinical applications in treating resistant HCC.

Identification of Hypoxia-ALCAMhigh Macrophage- Exhausted T Cell Axis in Tumor Microenvironment Remodeling for Immunotherapy Resistance.

Although hypoxia is known to be associated with immune resistance, the adaptability to hypoxia by different cell populations in the tumor microenvironment and the underlying mechanisms remain elusive. This knowledge gap has hindered the development of therapeutic strategies to overcome tumor immune resistance induced by hypoxia. Here, bulk, single-cell, and spatial transcriptomics are integrated to characterize hypoxia associated with immune escape during carcinogenesis and reveal a hypoxia-based intercellular communication hub consisting of malignant cells, ALCAMhigh macrophages, and exhausted CD8+ T cells around the tumor boundary. A hypoxic microenvironment promotes binding of HIF-1α complex is demonstrated to the ALCAM promoter therefore increasing its expression in macrophages, and the ALCAMhigh macrophages co-localize with exhausted CD8+ T cells in the tumor spatial microenvironment and promote T cell exhaustion. Preclinically, HIF-1ɑ inhibition reduces ALCAM expression in macrophages and exhausted CD8+ T cells and potentiates T cell antitumor function to enhance immunotherapy efficacy. This study reveals the systematic landscape of hypoxia at single-cell resolution and spatial architecture and highlights the effect of hypoxia on immunotherapy resistance through the ALCAMhigh macrophage-exhausted T cell axis, providing a novel immunotherapeutic strategy to overcome hypoxia-induced resistance in cancers.

The SIRT3/GSK-3β/GLUT4 axis might be involved in maternal hypoxia-induced skeletal muscle insulin resistance in old male rat offspring

Maternal hypoxia is strongly linked to insulin resistance (IR) in adult offspring, and altered insulin signaling for muscle glucose uptake is thought to play a central role. However, whether the SIRT3/GSK-3β/GLUT4 axis is involved in maternal hypoxia-induced skeletal muscle IR in old male rat offspring has not been investigated. Maternal hypoxia was established from Days 5 to 21 of pregnancy by continuous infusion of nitrogen and air. The biochemical parameters and levels of key insulin signaling molecules of old male rat offspring were determined through a series of experiments. Compared to the control (Ctrl) old male rat offspring group, the hypoxic (HY) group exhibited elevated fasting blood glucose (FBG) (∼30%), fasting blood insulin (FBI) (∼35%), total triglycerides (TGs), and low-density lipoprotein cholesterol (LDL-C), as well as results showing impairment in the glucose tolerance test (GTT) and insulin tolerance test (ITT). In addition, hematoxylin–eosin (HE) staining and transmission electron microscopy (TEM) revealed impaired cellular structures and mitochondria in the longitudinal sections of skeletal muscle from HY group mice, which might be associated with decreased SIRT3 expression. Furthermore, the expression of insulin signaling molecules, such as GSK-3β and GLUT4, was also altered. In conclusion, the present results indicate that the SIRT3/GSK-3β/GLUT4 axis might be involved in maternal hypoxia-induced skeletal muscle IR in old male rat offspring.

Hypoxia-Induced Insulin Resistance Mediates the Elevated Cardiovascular Risk in Patients with Obstructive Sleep Apnea: A Comprehensive Review.

Patients with obstructive sleep apnea (OSA) experience insulin resistance and its clinical consequences, including hypertriglyceridemia, reduced high density lipoprotein-associated cholesterol (HDL-c), visceral adiposity, hepatic steatosis, increased epicardial fat thickness, essential hypertension, glucose intolerance, increased risk for type 2 diabetes, chronic kidney disease, subclinical vascular damage, and increased risk for cardiovascular events. Obesity is a major contributor to OSA. The prevalence of OSA is almost universal among patients with severe obesity undergoing bariatric surgery. However, insulin resistance and its clinical complications occur in OSA patients irrespective of general obesity (body mass index). In OSA patients, apnea episodes during sleep induce oxyhemoglobin desaturation and tissue hypoxia. Insulin resistance is an adaptive response to tissue hypoxia and develops in conditions with limited tissue oxygen supply, including healthy subjects exposed to hypobaric hypoxia (high altitude) and OSA patients. Indicators of oxyhemoglobin desaturation have been robustly and independently linked to insulin resistance and its clinical manifestations in patients with OSA. Insulin resistance mediates the elevated rate of type 2 diabetes, chronic kidney disease, and cardiovascular disease unexplained with traditional cardiovascular risk factors present in OSA patients. Pathophysiological processes underlying hypoxia-induced insulin resistance involve hypoxia inducible factor-1 upregulation and peroxisome proliferator-activated receptor-gamma (PPAR- ) downregulation. In human adipose tissue, PPAR- activity promotes glucose transport into adipocytes, lipid droplet biogenesis, and whole-body insulin sensitivity. Silencing of PPAR- in the adipose tissue reduces glucose uptake and fat accumulation into adipocytes and promotes insulin resistance. In conclusion, tissue hypoxia drives insulin resistance and its clinical consequences in patients with OSA, regardless of body mass index.

O8G Site-Specifically Modified tRF-1-AspGTC: A Novel Therapeutic Target and Biomarker for Pulmonary Hypertension.

Hypoxia and oxidative stress contribute to the development of pulmonary hypertension (PH). tRNA-derived fragments play important roles in RNA interference and cell proliferation, but their epitranscriptional roles in PH development have not been investigated. We aimed to gain insight into the mechanistic contribution of oxidative stress-induced 8-oxoguanine in pulmonary vascular remodeling. Through small RNA modification array analysis and quantitative polymerase chain reaction, a significant upregulation of the 8-oxoguanine -modified tRF-1-AspGTC was found in the lung tissues and the serum of patients with PH. This modification occurs at the position 5 of the tRF-1-AspGTC (5o8G tRF). Inhibition of the 5o8G tRF reversed hypoxia-induced proliferation and apoptosis resistance in pulmonary artery smooth muscle cells. Further investigation unveiled that the 5o8G tRF retargeted mRNA of WNT5A (Wingless-type MMTV integration site family, member 5A) and CASP3 (Caspase3) and inhibited their expression. Ultimately, BMPR2 (Bone morphogenetic protein receptor 2) -reactive oxygen species/5o8G tRF/WNT5A signaling pathway exacerbated the progression of PH. Our study highlights the role of site-specific 8-oxoguanine-modified tRF in promoting the development of PH. Our findings present a promising therapeutic avenue for managing PH and propose 5o8G tRF as a potential innovative marker for diagnosing this disease.

Cisplatin-activated and hemoglobin-mediated injectable hydrogel system for antitumor chemodynamic and chemotherapy

Low specificity and hypoxia-induced drug resistance are significant challenges in traditional cancer treatment. To enhance the anticancer efficacy, an injectable hydrogel system is developed through the formation of dynamic covalent bonds in hyaluronic acid, allowing for localized controlled release of drugs. This system also utilizes double-stranded DNA sequences for the intercalation delivery of the chemotherapeutic drug, enabling a multifaceted approach to therapy. Cisplatin not only serves as a chemotherapy drug but also acts as a catalyst for chemodynamic therapy (CDT) to initiate CDT cascades by creating hydrogen peroxide for the Fenton reaction. Hemoglobin, enclosed in PLGA nanoparticles, provides ferrous ions that react with hydrogen peroxide in an acidic environment, yielding hydroxyl radicals that induce cancer cell death. Additionally, oxygen released from hemoglobin mitigates hypoxia-induced chemoresistance, bolstering overall anticancer efficacy. Results demonstrate the shear-thinning properties and injectability of the hydrogel. Cisplatin elevates intracellular hydrogen peroxide levels in tumor cells, while hemoglobin efficiently releases ferrous ions and generates reactive oxygen species (ROS) in the presence of hydrogen peroxide. In in vitro and in vivo study, the combinational use of chemo- and chemodynamic therapies achieves a synergistic anticancer effect on combating glioblastoma. In summary, our CDT-based hydrogel, activated by endogenous cues and mediated by chemo drugs, spontaneously produces ROS and ameliorates the adverse tumor microenvironment with rational and selective antitumor strategies.

Pioglitazone does not affect exogenous glucose oxidation or metabolic clearance rate during steady-state aerobic exercise at high altitude

Background: Hypoxia-induced insulin insensitivity decreases exogenous glucose oxidation and metabolic clearance rate (MCR, proxy for peripheral glucose uptake) in native lowlanders exercising during the initial hours of high-altitude (HA) exposure compared sea level. These data suggest that administering an insulin sensitizing drug before HA exposure may increase exogenous glucose oxidation rate by stimulating peripheral glucose uptake. Objective: To compare the effects of pioglitazone (PIO) and placebo (PLA) on exogenous glucose oxidation and MCR during steady-state aerobic exercise at HA. We hypothesized that exogenous glucose oxidation and MCR would be higher with PIO than PLA. Methods: Using a randomized, crossover design, native lowlanders (n = 7 males, mean ± SD, age: 23 ± 6 y, body mass: 84 ± 11 kg) consumed 145 g (1.8 g/min) of glucose while performing 80-min of metabolically-matched (1.43 ± 0.16 VȮ2 L/min) treadmill exercise at HA (460 mmHg) after being administered PIO (15 mg) or PLA (microcrystalline cellulose pill) daily for five days. Substrate oxidation and glucose turnover were determined using indirect calorimetry, oral 13C-glucose and [6,6-2H2]-glucose primed, continuous infusions. Results: Exogenous glucose oxidation rate was not different between PIO (0.31 ± 0.03 g/min) and PLA (0.32 ± 0.09 g/min). Total carbohydrate oxidation with PIO (1.65 ± 0.22 g/min) was the same as PLA (1.68 ± 0.32 g/min). There were no treatment effects on glucose rate of appearance (PIO: 2.46 ± 0.27, PLA: 2.43 ± 0.27 mg/kg/min) disappearance (PIO: 2.19 ± 0.17, PLA: 2.20 ± 0.22 mg/kg/min), and MCR (PIO: 1.63 ± 0.37, PLA: 1.73 ± 0.40 mL/kg/min). Conclusion: These data do not support our hypothesis that exogenous glucose oxidation during exercise at HA can be restored through the short-term administration of PIO. Findings from this study may suggest that hypoxia-induced insulin resistance may be a symptom and not the primary etiology of dysregulated glucose metabolism during acute HA exposure. Supported by MOMRP; authors’ views not offcial US Army or DoD policy. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Hypoxia treatment and resistance training alters microRNA profiling in rats skeletal muscle

MicroRNAs (miRNAs) may play a crucial regulatory role in the process of muscle atrophy induced by high-altitude hypoxia and its amelioration through resistance training. However, research in this aspect is still lacking. Therefore, this study aimed to employ miRNA microarray analysis to investigate the expression profile of miRNAs in skeletal muscle from an animal model of hypoxia-induced muscle atrophy and resistance training aimed at mitigating muscle atrophy. The study utilized a simulated hypoxic environment (oxygen concentration at 11.2%) to induce muscle atrophy and established a rat model of resistance training using ladder climbing, with a total intervention period of 4 weeks. The miRNA expression profile revealed 9 differentially expressed miRNAs influenced by hypoxia (e.g., miR-341, miR-32-5p, miR-465-5p) and 14 differentially expressed miRNAs influenced by resistance training under hypoxic conditions (e.g., miR-338-5p, miR-203a-3p, miR-92b-3p) (∣log2(FC)∣ ≥ 1.5, p < 0.05). The differentially expressed miRNAs were found to target genes involved in muscle protein synthesis and degradation (such as Utrn, mdm2, eIF4E), biological processes (such as negative regulation of transcription from RNA polymerase II promoter, regulation of transcription, DNA-dependent), and signaling pathways (such as Wnt signaling pathway, MAPK signaling pathway, ubiquitin-mediated proteolysis, mTOR signaling pathway). This study provides a foundation for understanding and further exploring the molecular mechanisms underlying hypoxia-induced rats muscle atrophy and the mitigation of atrophy through resistance training.

Abstract 374: Hypoxia-induced mitochondrial glutathione transporter expression causes drug resistance in colorectal cancer

Abstract Colorectal cancer (CRC) is the third most common type of cancer worldwide, with higher incidence and mortality rates. In order to adapt stress conditions during cancer progression, the cancer cells will be subject to metabolism reprogramming to increase energy production and remove hazardous substances. One of the important organelles, mitochondria is responsible for cellular ATP production, ion homeostasis, and several metabolic pathways. Therefore, mitochondria require a series of transporters called solute carrier family 25 (SLC25) to transport metabolites crossing the mitochondrial inner membrane, and dysregulation of those transporters causes abnormal cell morphology and diseases. In this study, members of the SLC25 family were analyzed in CRC via bioinformatics analysis, and a mitochondrial glutathione transporter was identified to overexpress in several public datasets of CRC and our own clinical specimens. In addition, its role has not been reported in any disease, including cancer. To systemically analyze the underlying mechanism causing overexpression of glutathione transporter during CRC progression, several stress conditions had been applied to colon cancer cells. However, only hypoxia was identified to induce its protein expression but not mRNA expression. Since a previous study reported that glutathione deprivation induced its protein expression, hypoxia didn’t alter the abundance of glutathione level suggesting that another glutathione-independent regulatory mechanism might be involve it. Interestingly, our findings revealed that hypoxia-induced glutathione transporter protein expression was mediated through the activation of internal ribosome binding site-mediated translation under hypoxia. To identify the potential role of glutathione transporter during colon cancer progression, its mitochondrial metabolite profiles from knockout cells of previous studies were further analyzed and identified that the changes of metabolites were associated with the urea cycle which had been reported to be associated with the sensitivity of oxaliplatin and 5-fluorouracil (5-FU) in cancer treatment. Importantly, our results showed that hypoxia-attenuated those chemotherapeutic effects were abolished by loss of glutathione transporter in colon cancer cells, indicating its crucial role in hypoxia-induced drug resistance. In conclusion, our study shows that glutathione transporter increased by hypoxia might lead to drug resistance of cancer therapy in colon cancer. Citation Format: Chin-Chun Lin. Hypoxia-induced mitochondrial glutathione transporter expression causes drug resistance in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 374.

Modeling hypoxia-induced radiation resistance and the impact of radiation sources

Hypoxia contributes significantly to resistance in radiotherapy. Our research rigorously examines the influence of microvascular morphology on radiotherapy outcome, specifically focusing on how microvasculature shapes hypoxia within the microenvironment and affects resistance to a standard treatment regimen (30×2GyRBE). Our computational modeling extends to the effects of different radiation sources. For photons and protons, our analysis establishes a clear correlation between hypoxic volume distribution and treatment effectiveness, with vascular density and regularity playing a crucial role in treatment success. On the contrary, carbon ions exhibit distinct effectiveness, even in areas of intense hypoxia and poor vascularization. This finding points to the potential of carbon-based hadron therapy in overcoming hypoxia-induced resistance to RT. Considering that the spatial scale analyzed in this study is closely aligned with that of imaging data voxels, we also address the implications of these findings in a clinical context envisioning the possibility of detecting subvoxel hypoxia.

Development of Anticancer Ferric Complex Based on Human Serum Albumin Nanoparticles That Generate Oxygen in Cells to Overcome Hypoxia-Induced Resistance in Metal Chemotherapy.

To achieve the remarkable therapeutic efficacy of a ferric (Fe) complex via a reactive oxygen species (ROS) mechanism in solid tumors, a therapeutic Fe-based Schiff-base complex (Fe1) was synthesized and encapsulated in human serum albumin (HSA) nanoparticles (NPs), which generated oxygen (O2) in cancer cells in situ. The HSA-Fe1-O2 NP (HSA-Fe1-O2NP) delivery system effectively overcame hypoxia-induced resistance in metal chemotherapy, alleviated the hypoxic condition of tumor tissues, and showed excellent tumor suppression by generating excess ROS and promoting the apoptosis of SK-N-MC tumor cells. The HSA-Fe1-O2NPs not only enhanced the ability of the Fe1 complex to target tumor cells but also decreased adverse effects in vivo.

A continuously efficient O2-supplying strategy for long-term modulation of hypoxic tumor microenvironment to enhance long-acting radionuclides internal therapy

Radionuclides internal radiotherapy (RIT) is a clinically powerful method for cancer treatment, but still poses unsatisfactory therapeutic outcomes due to the hypoxic characteristic of tumor microenvironment (TME). Catalase (CAT) or CAT-like nanomaterials can be used to enzymatically decompose TME endogenous H2O2 to boost TME oxygenation and thus alleviate the hypoxic level within tumors, but their effectiveness is still hindered by the short-lasting of hypoxia relief owing to their poor stability or degradability, thereby failing to match the long therapeutic duration of RIT. Herein, we proposed an innovative strategy of using facet-dependent CAT-like Pd-based two-dimensional (2D) nanoplatforms to continuously enhance RIT. Specifically, rationally designed 2D Pd@Au nanosheets (NSs) enable consistent enzymatic conversion of endogenous H2O2 into O2 to overcome hypoxia-induced RIT resistance. Furthermore, partially coated Au layer afford NIR-II responsiveness and moderate photothermal treatment that augmenting their enzymatic functionality. This approach with dual-effect paves the way for reshaping TME and consequently facilitating the brachytherapy ablation of cancer. Our work offers a significant advancement in the integration of catalytic nanomedicine and nuclear medicine, with the overarching goal of amplifying the clinical benefits of RIT-treated patients.Graphical abstract

Hypoxia-Inducible Transgelin-2 Confers Treatment Resistance through Activation of PI3K/Akt/GSK3β Pathway in Glioblastoma

Glioblastoma (GBM) patients with wild-type IDH experience worse survival response to the standard treatment of surgery followed by radiation therapy (RT) and temozolomide (TMZ) chemotherapy compared to their mutant IDH counterparts. This treatment has remained relatively ineffective partly due to the highly invasive phenotype of GBM leading to therapeutic resistance and tumor recurrence. Hypoxia is one of the key characteristic features of GBM which results in cancer metastasis and confers treatment resistance. Therefore, it is paramount to identify targets to help overcome hypoxia-induced treatment resistance in glioblastoma. Our lab has identified transgelin-2 (TAGLN2) to be significantly upregulated in IDH-wt GBM through multiple molecular profiling studies. This study aims to understand the mechanisms by which TAGLN2 confers treatment resistance for developing additional treatments for GBM. Additionally, active drug development efforts are also underway to target TAGLN2 for circumventing these therapeutic resistance mechanisms for effective GBM therapy. RNAi-mediated TAGLN2 knockdown (KD) approach was employed to assess the functions of TAGLN2 in GBM patient-derived xenograft (PDX) cell lines. Series of in vitro functional assays were performed to assess the role of TAGLN2 in these cell lines. Cell proliferation, invasion ± RT and/or TMZ were assessed by MTS and Trans-well invasion assays. Subsequently, WB analysis of oncogenic signaling pathways was performed following Transgelin-2 KD. Co-IP assays and Biacore/SPR analyses were performed to study the binding affinity and kinetics for the interaction of PTEN with TAGLN2. Further, cells were intracranially implanted in nude mice to assess the role of TAGLN2 on tumor growth in vivo. Conditional KD of TAGLN2 reduces cell proliferation, survival and invasive potential of GBM PDX cell lines. TAGLN2 KD also improved the sensitivity of these cells to both TMZ and radiation in vitro, as assessed by proliferation, survival, clonal expansion, and invasion. Histopathological studies of human GBM tumors and mouse xenograft tumors showed elevated expression of TAGLN2 in the peri-necrotic region of the tumors indicating that TAGLN2 protein level was upregulated by hypoxia. We also show that TAGLN2 is induced in hypoxic microenvironments with GBM PDX cell lines and its overexpression may enhance cellular resistance towards conventional therapy. Subsequently, we also show that hypoxia-induced TAGLN2 activates the PI3K/Akt oncogenic pathway through binding and inhibition of PTEN. Finally, in vivo data using an orthotopic xenograft mouse model shows reduction of tumor growth with knockdown of TAGLN2. Our in vitro and in vivo xenograft studies suggest that TAGLN2 confers treatment resistance to GBM contributing to tumor recurrence. Altogether, TAGLN2 may serve as a potential therapeutically vulnerable target in GBM specifically through its role in cell survival and invasion.

Targeting sphingolipid metabolism with the sphingosine kinase inhibitor SKI-II overcomes hypoxia-induced chemotherapy resistance in glioblastoma cells: effects on cell death, self-renewal, and invasion

BackgroundGlioblastoma patients commonly develop resistance to temozolomide chemotherapy. Hypoxia, which supports chemotherapy resistance, favors the expansion of glioblastoma stem cells (GSC), contributing to tumor relapse. Because of a deregulated sphingolipid metabolism, glioblastoma tissues contain high levels of the pro-survival sphingosine-1-phosphate and low levels of the pro-apoptotic ceramide. The latter can be metabolized to sphingosine-1-phosphate by sphingosine kinase (SK) 1 that is overexpressed in glioblastoma. The small molecule SKI-II inhibits SK and dihydroceramide desaturase 1, which converts dihydroceramide to ceramide. We previously reported that SKI-II combined with temozolomide induces caspase-dependent cell death, preceded by dihydrosphingolipids accumulation and autophagy in normoxia. In the present study, we investigated the effects of a low-dose combination of temozolomide and SKI-II under normoxia and hypoxia in glioblastoma cells and patient-derived GCSs.MethodsDrug synergism was analyzed with the Chou-Talalay Combination Index method. Dose–effect curves of each drug were determined with the Sulforhodamine B colorimetric assay. Cell death mechanisms and autophagy were analyzed by immunofluorescence, flow cytometry and western blot; sphingolipid metabolism alterations by mass spectrometry and gene expression analysis. GSCs self-renewal capacity was determined using extreme limiting dilution assays and invasion of glioblastoma cells using a 3D spheroid model.ResultsTemozolomide resistance of glioblastoma cells was increased under hypoxia. However, combination of temozolomide (48 µM) with SKI-II (2.66 µM) synergistically inhibited glioblastoma cell growth and potentiated glioblastoma cell death relative to single treatments under hypoxia. This low-dose combination did not induce dihydrosphingolipids accumulation, but a decrease in ceramide and its metabolites. It induced oxidative and endoplasmic reticulum stress and triggered caspase-independent cell death. It impaired the self-renewal capacity of temozolomide-resistant GSCs, especially under hypoxia. Furthermore, it decreased invasion of glioblastoma cell spheroids.ConclusionsThis in vitro study provides novel insights on the links between sphingolipid metabolism and invasion, a hallmark of cancer, and cancer stem cells, key drivers of cancer. It demonstrates the therapeutic potential of approaches that combine modulation of sphingolipid metabolism with first-line agent temozolomide in overcoming tumor growth and relapse by reducing hypoxia-induced resistance to chemotherapy and by targeting both differentiated and stem glioblastoma cells.

Vav1-dependent Rac1 activation mediates hypoxia-induced gemcitabine resistance in pancreatic ductal adenocarcinoma cells through upregulation of HIF-1α expression.

Hypoxia has been shown to induce gemcitabine (GEM) resistance in pancreatic ductal adenocarcinoma (PDAC) cells, however, the underlying mechanisms remain to be clarified. In the present study, we investigated whether activation of Vav1/Rac1/HIF-1α axis is responsible for hypoxia-induced GEM resistance in PDAC cells. Our results showed that Rac1 activation contributed to hypoxia-induced GEM resistance in PANC-1 cells. Hypoxia treatment led to an increased expression level of Vav1, which was responsible for Rac1 activation and GEM resistance in PDAC cells. Furthermore, Rac1 mediated hypoxia-induced GEM resistance by upregulating HIF-1α in PDAC cells. Taken together, these findings suggest that hypoxia induces GEM resistance in PDAC cells by activating the Vav1/Rac1/HIF-1α signaling pathway.

Enhancing chemotherapy for pancreatic cancer through efficient and sustained tumor microenvironment remodeling with a fibroblast-targeted nanosystem

Pancreatic cancer (PC) carries a poor prognosis among all malignancies and poses great challenges to clinical drug accessibility due to the severely fibrotic and hypoxic tumor microenvironment (TME). Therein, cancer-associated fibroblasts (CAFs), which are extremely abundant in PC, play a key role in forming the complex PC microenvironment. Therefore, a highly efficient TME reprogramming therapeutic paradigm that can specifically inhibit CAF function is urgently needed. Herein, we successfully developed a novel CAF-tailored nanosystem (Dex-GP-DOCA, DPD) loaded with a potent anti-fibrosis flavonoid compound (Quercetin, QUE), which possesses biological responsiveness to fibroblast activation protein alpha (FAP-α), prolonged TME remodeling and enhancement of clinical chemotherapeutics. Specifically, DPD/QUE allowed for extracellular matrix (ECM) reduction, vessel normalization, hypoxia-induced drug resistance reversal, and blockade of Wnt16 paracrine in CAFs. More importantly, this chemotherapy conducive microenvironment persisted for at least 8 days following treatment with DPD/QUE. It should also be noted that the effective and prolonged microenvironment modulation induced by DPD/QUE significantly improved the chemotherapy sensitivity of Abraxane and gemcitabine, the first-line chemotherapeutic drugs for PC, with inhibition rates increasing from 37.5% and 40.0% to 87.5% and 85.2%, respectively. Overall, our CAFs-targeted nanosystem showed promising prospects for remodeling the TME and facilitating chemotherapy for refractory pancreatic cancer.