Hypoxia-mimicking Conditions Research Articles

Heterogeneous porous hypoxia-mimicking scaffolds propel urethral reconstruction by promoting angiogenesis and regulating inflammation

The nasty urine microenvironment (UME) impedes neourethral regeneration by inhibiting angiogenesis and inducing an excessive inflammatory response. Cellular adaptation to hypoxia improves regeneration in numerous tissues. In this study, heterogeneous porous hypoxia-mimicking scaffolds were fabricated for urethral reconstruction via promoting angiogenesis and modulating the inflammatory response based on sustained release of dimethyloxalylglycine (DMOG) to promote HIF-1α stabilization. Such scaffolds exhibit a two-layered structure: a dense layer composed of electrospun poly (l-lactic acid) (PLLA) nanofibrous mats and a loose layer composed of a porous gelatin matrix incorporated with DMOG-loaded mesoporous silica nanoparticles (DMSNs) and coated with poly(glycerol sebacate) (PGS). The modification of PGS could significantly increase rupture elongation, making the composite scaffolds more suitable for urethral tissue regeneration. Additionally, sustained release of DMOG from the scaffold facilitates proliferation, migration, tube formation, and angiogenetic gene expression in human umbilical vein endothelial cells (HUVECs), as well as stimulates M2 macrophage polarization and its regulation of HUVECs migration and smooth muscle cell (SMCs) contractile phenotype. These effects were downstream of the stabilization of HIF-1α in HUVECs and macrophages under hypoxia-mimicking conditions. Furthermore, the scaffold achieved better urethral reconstruction in a rabbit urethral stricture model, including an unobstructed urethra with a larger urethral diameter, increased regeneration of urothelial cells, SMCs, and neovascularization. Our results indicate that heterogeneous porous hypoxia-mimicking scaffolds could promote urethral reconstruction via facilitating angiogenesis and modulating inflammatory response.

Abstract 5413: In vitro modeling of the migration ability of lung adenocarcinoma cell lines under hypoxic conditions

Abstract Hypoxia is a common condition in the tumor microenvironment that contributes to tumor cell migration, invasion, and metastasis, partly through inducing epithelial-mesenchymal transition (EMT). One of the key proteins in this process is hypoxia-inducible factor 1 (HIF-1), which regulates the expression of various genes as transcription factor and is vital for the cellular response in tissue hypoxia. Several studies have investigated the causation between hypoxic conditions and increased in vitro tumor cell motility and the role of potential regulatory signaling pathways. Understanding the precise molecular mechanisms underlying hypoxia-induced responses is crucial for better targeting tumor progression. However, establishing sound and predictive models is urgently needed to achieve this.In this study, we investigated the migration ability of four non-small cell lung cancer cell lines carrying different mutations of the growth factor pathway (H1975: EGFR T790M, PF901: BRAF V600E, PF139: KRAS G12C mutant, and H838: triple WT) under normoxic, hypoxic, and hypoxia-mimicking CoCl2-treated conditions. The motility of the cells was investigated by 48 h video microscopy (single cell random migration) and 72 h scratch assay. We analyzed the expression of crucial genes and proteins, including HIF-1α, as well as molecules associated with epithelial-mesenchymal transition (vimentin, n-cadherin, and e-cadherin), proliferation (PCNA), and apoptosis (PARP), using Western blot and qPCR techniques. Our work showed no change in cellular motility between any of the cell lines following treatment with CoCl2 compared to normoxic conditions. However, PF139 and PF901 cell lines showed a significant decrease in single-cell motility upon hypoxia. Interestingly, immunoblot analysis revealed stabilization of HIF-1α in response to both CoCl2 and hypoxia in the meantime. Additionally, we observed cell line-dependent changes in EMT marker expression upon CoCl2 treatment and hypoxia. Our work complements previous results published by our group and others on the tumor-promoting role of hypoxia, demonstrating that short-term exposure to hypoxia may act as an acute stressor for tumor cell lines. Our work suggests that the tumor progression-promoting effects are tissue-specific and should be investigated using long-term hypoxia models. Acknowledgments: This work was supported by the Hungarian Thematic Excellence Program (TKP2021-EGA-44) and the National Laboratories Program - National Laboratory for Tumor Biology (NLP-17). Sára Eszter Surguta is a grantee of the ITM-NKFIA funded Cooperative Doctoral Program (1018567). Citation Format: Sára Eszter Surguta, Marcell Baranyi, Laura Svajda, Ivan Ranđelović, Mihály Cserepes, József Tóvári. In vitro modeling of the migration ability of lung adenocarcinoma cell lines under hypoxic conditions [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 5413.

Blocking cholesterol formation and turnover improves cellular and mitochondria function in murine heart microvascular endothelial cells and cardiomyocytes.

Background: Statins and proprotein convertase subtilisin/kexin type 9 inhibitors (PCSK9i) are cornerstones of therapy to prevent cardiovascular disease, acting by lowering lipid concentrations and only partially identified pleiotropic effects. This study aimed to analyze impacts of atorvastatin and synthetic peptide PCSK9i on bioenergetics and function of microvascular endothelial cells and cardiomyocytes. Methods: Mitochondrial function and abundance as well as intracellular nucleotides, membrane potential, cytoskeleton structure, and cell proliferation rate were evaluated in mouse heart microvascular endothelial cells (H5V) and cardiomyocytes (HL-1) under normal and hypoxia-mimicking conditions (CoCl2 exposure). Results: In normal conditions PCSK9i, unlike atorvastatin, enhanced mitochondrial respiratory parameters, increased nucleotide levels, prevented actin cytoskeleton disturbances and stimulated endothelial cell proliferation. Under hypoxia-mimicking conditions both atorvastatin and PCSK9i improved the mitochondrial respiration and membrane potential in both cell types. Conclusion: This study demonstrated that both treatments benefited the endothelial cell and cardiomyocyte bioenergetics, but the effects of PCSK9i were superior.

Plumbagin Suppresses Breast Cancer Progression by Downregulating HIF-1α Expression via a PI3K/Akt/mTOR Independent Pathway under Hypoxic Condition.

Hypoxia-inducible factor-1α (HIF-1α) is a major transcriptional regulator that plays a crucial role in the hypoxic response of rapidly growing tumors. Overexpression of HIF-1α has been associated with breast cancer metastasis and poor clinical prognosis. Plumbagin, the main phytochemical from Plumbago indica, exerts anticancer effects via multiple mechanisms. However, its precise mechanisms on breast cancer cells under hypoxic conditions has never been investigated. This study aims to examine the anticancer effect of plumbagin on MCF-7 cell viability, transcriptional activity, and protein expression of HIF-1α under normoxia and hypoxia-mimicking conditions, as well as reveal the underlying signaling pathways. The results demonstrate that plumbagin decreased MCF-7 cell viability under normoxic conditions, and a greater extent of reduction was observed upon exposure to hypoxic conditions induced by cobalt chloride (CoCl2). Mechanistically, MCF-7 cells upregulated the expression of HIF-1α protein, mRNA, and the VEGF target gene under CoCl2-induced hypoxia, which were abolished by plumbagin treatment. In addition, inhibition of HIF-1α and its downstream targets did not affect the signaling transduction of the PI3K/Akt/mTOR pathway under hypoxic state. This study provides mechanistic insight into the anticancer activity of plumbagin in breast cancer cells under hypoxic conditions by abolishing HIF-1α at transcription and post-translational modifications.

Low-Dose-Rate Irradiation Suppresses the Expression of Cell Cycle-Related Genes, Resulting in Modification of Sensitivity to Anti-Cancer Drugs.

The biological effects of low-dose-rate (LDR) radiation exposure in nuclear power plant accidents and medical uses of ionizing radiation (IR), although being a social concern, remain unclear. In this study, we evaluated the effects of LDR-IR on global gene expression in human cells and aimed to clarify the mechanisms. RNA-seq analyses demonstrated that relatively low dose rates of IR modify gene expression levels in TIG-3 cells under normoxic conditions, but those effects were attenuated under hypoxia-mimicking conditions. Gene set enrichment analysis demonstrated that LDR-IR significantly decreased gene expression related to cell division, cell cycle, mitosis, and the Aurora kinase B and FOXM1 pathways. Quantitative RT-PCR confirmed the down-regulation of AURKB and FOXM1 genes in TIG-3 cells with LDR-IR or hypoxia-mimicking treatments without any dose-rate effect. Knock-down experiments suggested that HIF-1α and HIF-2α, as well as DEC1, participated in down-regulation of AURKB and FOXM1 under DFOM treatments, but to a lesser extent under LDR-IR treatment. FACS and microscopic analyses demonstrated that LDR-IR induced G0/G1 arrest and increased micronucleus or chromosome condensation. Finally, MTT assays demonstrated that LDR-IR decreased sensitivity to paclitaxel or barasertib in TIG-3 cells but not in A549 cells. In conclusion, LDR-IR modifies global gene expression and cell cycle control, resulting in a reduction of sensitivity to anti-cancer chemotherapy in non-cancer cells and thus a reduction in untoward effects (GA).

A core-shell nanoplatform as a nonviral vector for targeted delivery of genes to the retina

Retinal diseases, including age-related macular degeneration (AMD), are a major cause of blindness. Efficient delivery of therapeutic genes to retinal cells to treat retinal disease is a formidable challenge. In this study, we developed a core-shell nanoplatform composed of a core and two external layers for targeted delivery of the gene to the retina. The inner core was composed of amino acid-functionalized dendrimers and a nuclear localization signal (NLS) for DNA complexation, nuclear transport and efficient transfection. The inner core was coated in a lipid bilayer that comprised pH-sensitive lipids as the inner shell layer. Hyaluronic acid (HA)-1,2-dioleoylphosphatidylethanolamine (DOPE) as the outermost shell layer was used for retinal cell targeting. This core-shell nanoplatform was developed so that the mobility in the vitreous body of these negatively charged carriers would not be affected by their surface charge, allowing diffusion into the retina, uptake into the retinal cells via CD44-mediated internalization, and finally transport into the nucleus by the NLS. The designed nanoparticles showed safety both in vitro and in vivo and inhibited the expression of VEGF under hypoxia-mimicking conditions. In vitro angiogenesis assays exhibited significant inhibitory effects on cell migration and tube formation. The in vivo assays indicated that this nanoplatform could be delivered to the retina. Taken together, this nanoplatform has the potential to transfer gene material into the retina for the treatment of retinal diseases, including AMD. Statement of significanceIt remains a challenge to develop an efficient nonviral vector for gene therapy, especially retinal gene therapy. Various barriers exist in gene delivery and the unique ocular environment, making gene delivery to the retina difficult. In this study, we designed a negatively charged core-shell nanoplatform (HD-NPPND) for the targeted delivery of gene to the retina. The developed nanoplatform possessed excellent transfection efficiency and safety both in vitro and in vivo. It efficiently delivered a gene to the retina. The results of this study suggested that this core-shell nanoplatform has the potential to deliver genes to the retina to treat retinal diseases, including age-related macular degeneration (AMD).

Distinct Roles of VEGFA and ANGPT2 in Lung Adenocarcinoma and Squamous Cell Carcinoma.

Background: Vascular endothelial growth factor A (VEGFA) and angiopoietin 2 (ANGPT2) are key mediators in angiogenesis. The expression and clinical significance of VEGFA and ANGPT2 have been investigated in lung cancer, but the results are controversial. The specific roles of VEGFA and ANGPT2 in adenocarcinoma (ADC) and squamous cell carcinoma (SQC) are still not fully understood. To characterize it, we conducted the current study.Materials and Methods: The relationships between clinic-pathological characteristics and the protein expressions of VEGFA and ANGPT2 were analyzed on tissue microarrays by immunohistochemistry (IHC) staining. Then public databases were used to evaluate the association of VEGFA and ANGPT2 mRNA expressions with clinic-pathological parameters and prognosis. Cobalt chloride (CoCl2) was adopted to mimic a hypoxic microenvironment and western blot was used to detect the expression of hypoxia inducible factor-1α (HIF-1α), VEGFA and ANGPT2 in lung cancer cell lines.Results: IHC staining revealed that the expressions of VEGFA and ANGPT2 were enriched in lung cancer tissues compared with normal tissues. Additionally, both VEGFA and ANGPT2 protein levels were significantly associated with the tumor size and lymph node metastasis only in ADC, not SQC. More importantly, increased VEGFA and ANGPT2 protein levels were negatively correlated with overall survival (OS) of ADC individuals. Meta-analyses of 22 gene expression omnibus (GEO) databases of lung cancer implicated that patients with higher VEGFA and ANGPT2 mRNA expressions tended to have advanced stage in ADC rather than SQC. Kaplan-Meier plot analyses further verified that high levels of VEGFA and ANGPT2 mRNA were associated with poor survival only in ADC. Moreover, the combination of VEGFA and ANGPT2 could more precisely predict prognosis in ADC. In hypoxia-mimicking conditions, induced expression of HIF-1α unregulated VEGFA and ANGPT2 proteins abundance.Conclusion: Our results showed hypoxia upregulated the protein levels of VEGFA and ANGPT2 in lung cancer cell lines, and the roles of VEGFA and ANGPT2 were distinct in ADC and SQC. Combined detections of VEGFA and ANGPT2 may be valuable prognostic biomarkers for ADC and double block of VEGFA and ANGPT2 may improve therapeutic outcome.

Hypoxia inhibits TNF-α-induced TSLP expression in keratinocytes.

The expression of thymic stromal lymphopoietin (TSLP), a cytokine which greatly contributes to the induction of type I allergy, is upregulated in chronic inflammation such as atopic dermatitis and psoriasis. As hypoxia in the epidermis is important for maintaining skin homeostasis, we examined the regulation of TSLP expression by hypoxic conditions in normal skin epithelial tissues. TNF-α-induced expression of TSLP in human keratinocyte HaCaT and in mouse keratinocyte PAM212 cell lines were inhibited under hypoxic condition (1% O2), although the mRNA expressions of TNF-α, IL-6, IL-8, MCP-1, and VEGF-A were not inhibited. Hypoxia-mimicking conditions, which include NiCl2, CoCl2, and DMOG, an inhibitor of 2-oxoglutarate-dependent enzymes, also selectively inhibited TNF-α-induced TSLP expression. These results suggested that inactivation of prolyl hydroxylase by hypoxia and hypoxia-mimicking conditions is involved in the repression of TNF-α-induced TSLP expression. Interestingly, the inhibition of TSLP production by hypoxic treatment was significantly reversed by treatment with the HIF-2α antagonist but not with the HIF-1α inhibitor. DMOG-induced inhibition of TSLP promoter activity was dependent on the -71 to +185 bp promoter region, suggesting that the binding of HIF-2 to hypoxia response element (HRE) in this region repressed the TSLP expression. These results indicated that hypoxia and hypoxia-mimicking conditions inhibited TSLP expression via HIF-2 and HRE-dependent mechanisms. Therefore, PHD and HIF-2α could be a new strategy for treatment of atopic dermatitis and psoriasis.

Anticancer effect of Andrographis paniculata by suppression of tumor altered hypoxia signaling cascade in mouse melanoma cells

Background: Intratumor hypoxia, the main factor responsible for the angiogenic switch, represents one of the major events leading to tumor progression. Tumor hypoxia leads to the stabilization of hypoxia-inducible factor-1α (HIF-1α) which influences tumor angiogenesis. The new blood vessels formed by the HIF-1α-vascular endothelial growth factor (VEGF) signaling axis create the tumor microenvironment, which inhibits drug delivery to solid tumors. The present study aimed to investigate the effect of Andrographis paniculata leaf extract as a powerful anticancer agent targeting the HIF-1α-VEGF signaling axis in mouse melanoma cell. Materials and Methods: We induced hypoxia-mimicking conditions in mouse B16 melanoma cell with cobalt chloride. Total RNA was isolated followed by reverse transcriptase polymerase chain reaction to study the transcriptional expressions of HIF-1α and VEGF. Further confirmation of the transcriptional profiling was done at the protein level with Western blot analysis. Expression profiling of transcriptional factors involved in the hypoxia signaling cascade was done. An immunofluorescence study was also used to confirm the results obtained from transcriptional and translational analyses. Results: A. paniculata leaf extract significantly downregulated the expressions of HIF-1α and VEGF both at the transcriptional and translational level. Sp1, p300, CBP expressions were also downregulated, whereas the expression of Sp3 was significantly upregulated by A. paniculata leaf extract in B16 melanoma cells. Conclusion: In the present study, A. paniculata-treated cells demonstrated lower expressions of VEGF and HIF-1α both at the transcriptional and translational level. The mechanism of the downregulation of HIF-1α was probably through the altered expressions of transcriptional factors involved in the hypoxia-signaling cascade.

Contribution of the clock gene DEC2 to VEGF mRNA upregulation by modulation of HIF1α protein levels in hypoxic MIO-M1 cells, a human cell line of retinal glial (Müller) cells.

Clock genes are components of the molecular clock. Their malfunction is thought to increase the risk of numerous diseases, including cancer. Vascular endothelial growth factor (VEGF) has a pivotal role in angiogenesis, and its expression levels are controlled by clock genes in tumor cells. Ophthalmic diseases such as age-related macular degeneration, proliferative diabetic retinopathy, and neovascular glaucoma are also associated with abnormal angiogenesis followed by upregulation of VEGF in the eye. In the present study, we aimed to uncover the relationship between clock genes and VEGF in the eye. Laboratory investigation METHODS: Oxygen-induced retinopathy (OIR) mice were prepared to mimic hypoxic conditions in the eye. Deferoxamine (DFO) was used to mimic hypoxic conditions in human Müller cell line MIO-M1 cells. Expression levels of mRNA and protein were quantified by quantitative reverse transcription polymerase chain reaction and Western blot analysis, respectively. In the retinas of OIR mice, the expression levels of Vegf and the clock gene Dec2 increased transiently, and their temporal profiles were correlated. Knockdown of DEC2 resulted in a significant (26.7%) reduction of VEGF expression in MIO-M1 cells under hypoxia-mimicking conditions induced by DFO (P < .05). Levels of HIF1α protein were also reduced significantly, by 60.2%, in MIO-M1 cells treated with siRNA against the DEC2 gene (P < .05). Moreover, HIF1α levels showed a significant (2.5-fold) increase in MIO-M1 cells overexpressing DEC2 (P < .05). DEC2 could upregulate retinal VEGF gene expression through modulation of HIF1α levels under hypoxic conditions.

Combination therapy of simvastatin and 5, 6-dimethylxanthenone-4-acetic acid synergistically suppresses the aggressiveness of B16.F10 melanoma cells

The major drawback of current anti-angiogenic therapies is drug resistance, mainly caused by overexpression of the transcription factor, hypoxia-inducible factor 1α (HIF-1α) as a result of treatment-induced hypoxia, which stimulates cancer cells to develop aggressive and immunosuppressive phenotypes. Moreover, the cancer cell resistance to anti-angiogenic therapies is deeply mediated by the communication between tumor cells and tumor-associated macrophages (TAMs)—the most important microenvironmental cells for the coordination of all supportive processes in tumor development. Thus, simultaneous targeting of TAMs and cancer cells could improve the outcome of the anti-angiogenic therapies. Since our previous studies proved that simvastatin (SIM) exerts strong antiproliferative actions on B16.F10 murine melanoma cells via reduction of TAMs-mediated oxidative stress and inhibition of intratumor production of HIF-1α, we investigated whether the antitumor efficacy of the anti-angiogenic agent—5,6-dimethylxanthenone-4-acetic acid (DMXAA) could be improved by its co-administration with the lipophilic statin. Our results provide confirmatory evidence for the ability of the combined treatment to suppress the aggressive phenotype of the B16.F10 melanoma cells co-cultured with TAMs under hypoxia-mimicking conditions in vitro. Thus, proliferation and migration capacity of the melanoma cells were strongly decelerated after the co-administration of SIM and DMXAA. Moreover, our data suggested that the anti-oxidant action of the combined treatment, as a result of melanogenesis stimulation, might be the principal cause for the simultaneous suppression of key molecules involved in melanoma cell aggressiveness, present in melanoma cells (HIF-1α) as well as in TAMs (arginase-1). Finally, the concomitant suppression of these proteins might have contributed to a very strong inhibition of the angiogenic capacity of the cell co-culture microenvironment.

Inhibition of P-glycoprotein stimulates cell death under Hypoxia-mimicking conditions.

The most common drug resistance mechanism in tumor cells is expression on their surface of the energy-dependent pump like P-glycoprotein (P-gp) that expels chemotherapeutic agents from the interior. An imitation of the hypoxic condition by the iron chelator deferoxamine caused Hypoxia-inducible factor 1-alpha (HIF-1α) stabilization and inhibition of doxorubicin-induced apoptosis in colon cancer НСТ116 cells. P-gp blocker verapamil suppressed doxorubicin accumulation leading to cell death induction. Considering these results, P-gp may be used as a potential target to stimulate chemotherapeutic drugs activity that will contribute to more efficient tumor cells elimination.

The Potent Inhibitory Effect of a Naproxen-Appended Cobalt(III)-Cyclam Complex on Cancer Stem Cells.

We report the potency against cancer stem cells (CSCs) of a new cobalt(III)-cyclam complex (1) that bears the nonsteroidal anti-inflammatory drug, naproxen. The complex displays selective potency for breast CSC-enriched HMLER-shEcad cells over breast CSC-depleted HMLER cells. Additionally, it inhibited the formation of three-dimensional tumour-like mammospheres, and reduced their viability to a greater extent than clinically used breast cancer drugs (vinorelbine, cisplatin and paclitaxel). The anti-mammosphere potency of 1 was enhanced under hypoxia-mimicking conditions. Detailed mechanistic studies revealed that DNA damage and cyclooxygenase-2 (COX-2) inhibition contribute to the cytotoxic mechanism of 1. To the best of our knowledge, 1 is the first cobalt-containing compound to show selective potency for CSCs over bulk cancer cells.

Abstract 1983: Aryl hydrocarbon receptor activates NDRG1 transcription under hypoxia in breast cancer cells

Abstract Hypoxia has been intensively investigated over past decades based on the observations that hypoxic tumors were more resistant to therapy and had a worse prognosis. Previously, we reported that N-myc downstream-regulated gene 1 (NDRG1) was strongly up-regulated under hypoxia and may play an important role in tumor adaptation to fluctuation of oxygen concentrations. However, the regulatory mechanism of NDRG1 under hypoxia remains elusive. Therefore, the purpose of this study is to identify the novel transcription factors that regulate NDRG1 and to investigate the functional roles of NDRG1 in hypoxia. In silico analysis predicted binding sites of aryl hydrocarbon receptor (AHR) existing in the NDRG1 promoter. Western blotting showed that nuclear AHR was up-regulated in the presence of cobalt and hypoxia. Immunofluorescence staining and chromatin immunoprecipitation assays showed that AHR translocated to nuclei and bound to NDRG1 promoter in hypoxia. Luciferase reporter assays further showed that the binding site of AHR at -412 ∼ -388 bp played a crucial role in regulating NDRG1 under hypoxia-mimicking conditions. Moreover, hypoxia-mimetic induction of NDRG1 by was attenuated by knockdown of AHR expression using short interfering RNA. Also, overexpression of AHR facilitated cell proliferation and migration via up-regulation of NDRG1, whereas shRNA knockdown of NDRG1 reduced cell growth and motility. In summary, these results showed for the first time that AHR positively regulates NDRG1 transcription through a AHR binding site by hypoxia-mimetic signaling, which may lead to development of a specific therapeutic regime to prevent tumor malignancy under hypoxia. Citation Format: En-Yu Li, Wei-Yung Huang, Mong-Hsun Tsai, Eric Y. Chuang, Liang-Chuan Lai. Aryl hydrocarbon receptor activates NDRG1 transcription under hypoxia in breast cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1983. doi:10.1158/1538-7445.AM2015-1983

Abstract P1-07-16: Galectin-7 increases resistance of breast cancer cells to drug-induced apoptosis and promotes tumor escape by killing T cells

Abstract Resistance to apoptosis induced by anti-cancer drugs is a major obstacle for the treatment of aggressive forms of breast cancer. Galectin-7 was recently shown to be specifically expressed in basal-like and HER-2 positive but not in luminal subtypes of human breast cancer. Galectin-7 also increases the metastatic behavior of breast cancer cells since overexpression of this protein in poorly metastatic breast cancer cells increases their ability to metastasize to the bone and to the lung. This pro-tumoral activity of galectin-7 in breast cancer is surprising since galectin-7 had been previously associated with activation of p53 and breast cancer cells often harbor a transcriptionally inactive form of p53. We have recently reconcile this apparent contradiction by showing that elevated levels of galectin-7 in breast cancer cells occurs via a gain-of-function mechanism of mutant p53. In p53-null breast cancer cells, we have shown that C/EBPβ-2 (also known as LAP2), the most transcriptionally active of the C/EBPβ isoforms, is responsible for the upregulation of galectin-7. How galectin-7 contributes to the progression of breast cancer remains unclear. Up to now, regulation of apoptosis by intracellular galectins has been largely attributed to their ability to translocate to mitochondria, possibly following their interaction with bcl-2. Here, we report that a mutant of galectin-7 that is unable to translocate to mitochondria induces resistance of human breast cancer cells to apoptosis induced by etoposide or by hypoxia-mimicking conditions. Surprisingly, this mutant and the wild-type form of galectin-7 bind equally well to bcl-2 in vitro and in vivo. Interestingly, both forms decreases the translocation of p53 to the nucleus and reduce the expression of p21 following treatment with doxorubicin. We also found that galectin-7 was released by breast cancer cells and that recombinant galectin-7 increased apoptosis of monocytes, T CD4+ and T CD8+ cells. This suggests that galectin-7 contributes to the establishment of an immunosuppressive tumor microenvironment by killing helper and effector T cells. Taken together, these results challenges the current paradigm that mitochondrial galectins are important for resistance to apoptosis and call for a greater focus on the role of galectin-7 in breast cancer. They also indicate that targeting both cytosolic and extracellular galectin-7 could improve the efficacy of anti-cancer drugs for the treatment of aggressive forms of breast cancer. Citation Format: Yves St-Pierre, Andrée-Anne Grosset, Marilyne Labrie, Donald Gagné, Maria-Claudia Vladoiu, Louis Gaboury, Nicolas Doucet. Galectin-7 increases resistance of breast cancer cells to drug-induced apoptosis and promotes tumor escape by killing T cells [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P1-07-16.

The acquired radioresistance in HeLa cells under conditions mimicking hypoxia was attenuated by a decreased expression of HIF subunit genes induced by RNA interference

The cancer cells residing in the hypoxic layer are resistant to radiation and these are ones responsible for cancer recurrence after radiation therapy. One of the reasons why hypoxic cancer cells acquire radioresistance may be attributable to changes in the gene expression profile by the activation of hypoxia inducible factors (HIFs). However, the details underlying this process remain unknown. In this study, we investigated the effects of knockdown of HIF subunit genes to elucidate how HIF subunit genes may be involved in the radioresistance acquired by HeLa cells following exposure to a hypoxia mimic. Interestingly, HIF-1α and HIF-2α seemed mutually complementary for each other when either of them was suppressed. We thus suppressed the expression of both genes simultaneously. To do this, we developed a short hairpin RNA (shRNA) targeting a high homology region between HIF-1α and HIF-2α. It was shown that the expression of the shRNA effectively suppressed the acquisition of radioresistance following the hypoxia mimic. Moreover, it was confirmed that suppression of both subunits resulted in the downregulation of stem cell markers and the suppression of spheroid formation during the hypoxia mimicking-conditions. This shRNA-mediated knockdown method targeting a common region shared by a family of genes may offer a new candidate cancer treatment.

Abstract 1352: Cytoplasmic galectin-7 has an antiapoptotic function by decreasing p53 nuclear translocation in breast cancer cells

Abstract Resistance to apoptosis induced by anti-cancer drugs is a major obstacle for the treatment of aggressive forms of breast cancer. Galectin-7 was recently shown to be specifically expressed in basal-like and HER-2 positive but not in luminal subtypes of human breast cancer. Overexpression of galectin-7 in breast cancer cells also increases lung and bone metastases. However, the molecular mechanisms used by galectin-7 to increase the dissemination of breast cancer cells remain largely unknown. Although galectins are best known for their ability to bind cell surface receptors, there is increasing evidence that their intracellular forms have important functions in cancer cells. This is particularly true in the control of apoptosis. Up to now, regulation of apoptosis by intracellular galectins has been largely attributed to their ability to translocate to mitochondria, possibly following their interaction with bcl-2. Here, we report that a mutant of galectin-7 that is unable to translocate to mitochondria and nucleus induces resistance of human breast cancer cells to apoptosis induced by etoposide or by hypoxia-mimicking conditions. Surprisingly, this mutant and the wild-type form of galectin-7 bind equally well to bcl-2 in vitro and in vivo. Interestingly, both forms decreases the translocation of p53 to the nucleus and reduce the expression of p21 following treatment with doxorubicin. These results challenges the current paradigm that mitochondrial galectins are important for resistance to apoptosis and call for a greater focus on the role of cytosolic galectins in cancer cells. Our data also indicate that targeting cytosolic galectin-7 could improve the efficacy of anti-cancer drugs for the treatment of aggressive forms of breast cancer. Citation Format: Andrée-Anne Grosset, Donald Gagné, Marilyne Labrie, Maria Vladoiu, Louis Gaboury, Nicolas Doucet, Yves St-Pierre. Cytoplasmic galectin-7 has an antiapoptotic function by decreasing p53 nuclear translocation in breast cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1352. doi:10.1158/1538-7445.AM2014-1352

The anti-diabetic drug metformin inhibits vascular endothelial growth factor expression via the mammalian target of rapamycin complex 1/hypoxia-inducible factor-1α signaling pathway in ELT-3 cells

The aim of this study was to elucidate whether metformin can regulate the expression of vascular endothelial growth factor (VEGF) in rat-derived uterine leiomyoma cells (ELT-3 cells). In vitro studies were conducted using ELT-3 cells. Under normoxic conditions, metformin suppressed VEGF protein levels in the supernatant and cells in a dose-dependent manner. In hypoxia-mimicking conditions, VEGF and hypoxia-inducible factor-1α (HIF-1α) proteins were both highly expressed and were suppressed by the metformin treatment. Metformin did not affect HIF-1α mRNA levels, which indicated that its effects occurred at the post-translational level. Metformin inhibited mammalian target of rapamycin complex 1 (mTORC1) activity by phosphorylating the mTORC1 component raptor. This study revealed the anti-angiogenic activity of metformin in ELT-3 cells by suppressing the expression of VEGF via the mTORC1/HIF-1α pathway. These results indicate that metformin may represent an effective alternative in the future treatment of uterine leiomyomas.

Hypoxia inducible factor-1 is involved in growth factor, glucocorticoid and hypoxia mediated regulation of vascular endothelial growth factor-A in human meningiomas

In meningiomas, neovascularization through angiogenesis is essential for tumor expansion. As the vascular endothelial growth factor-A (VEGF-A) plays an outstanding role in this process, we have studied basal VEGF-A release and some aspects of its regulation in 46 meningiomas and in Ben-Men-1 cells in vitro. Among two putative VEGF-A stimulating growth factors tested, TGF-1β was more potent than TGF-α in enhancing VEGF-A secretion. Hypoxia-mimicking conditions induced by CoCl2 treatment also strongly increased VEGF-A secretion. The synthetic glucocorticoid dexamethasone (DEX) potently suppressed both basal and growth factor or CoCl2-induced VEGF-A release. All these effects were also seen in the Ben-Men-1 cell line in which studies on the role of HIF-1 in the regulation of VEGF-A showed that not only hypoxia but also the growth factors induced HIF-1α and DEX suppressed HIF-1α induction. Therefore, in Ben-Men-1 cells with HIF-1α knock-down the effects of hypoxia, growth factors and DEX on VEGF-A production were strongly impaired. This clearly indicates that HIF-1 not only regulates hypoxia-induced VEGF-A production but also mediates at least in part the effects of growth factors and DEX on VEGF-A synthesis and release. Our findings show the complexity of VEGF-A regulation in meningiomas and point to new options for the pharmacological treatment of these tumors.

Acetylation mediated by the p300/CBP-associated factor determines cellular energy metabolic pathways in cancer

Normal cells produce energy either through OXPHOS in the presence of oxygen or glycolysis in its absence. Cancer cells produce energy preferably through glycolysis even in the presence of oxygen, thereby, acquiring survival and proliferative advantages. Oncogenes and tumour suppressors control these metabolic pathways by regulating the expression of their target genes involved in these processes. During hypoxia, HIF-1 favours high glycolytic flux by upregulating glycolytic enzymes. Conversely, p53 inhibits glycolysis and increases OXPHOS expression through TIGAR and SCO2 gene expression, respectively. We hypothesise that the p300/CBP-associated factor (PCAF) as a common co-factor shared between p53 and HIF-1 plays an important role in the regulation of energy production by modulating SCO2 and TIGAR gene expression mediated by these two transcription factors. The possible involvement of HIF-1 in the regulation of SCO2 and TIGAR gene expression was investigated in cells with different p53 status in normoxia- and hypoxia-mimicking conditions. Putative hypoxia response elements (HREs) were identified in the regulatory region of SCO2 and TIGAR gene promoters. Chromatin immunoprecipitation experiments suggested that HIF-1 was recruited to the putative HREs present in the SCO2 and TIGAR promoters in a cell type-dependent manner. Transcriptional assays endorsed the notion that PCAF may be involved in the determination of the SCO2 and TIGAR cellular levels, thereby, regulating cellular energy metabolism, a view supported by assays measuring lactic acid production and oxygen consumption in cells ectopically expressing PCAF. The present study identified HIF-1 as a potential regulator of SCO2 and TIGAR gene expression. Furthermore, evidence to suggest that PCAF is involved in the regulation of cellular energy production pathways in hypoxia-mimicking conditions is presented. This effect of PCAF is exerted by orchestrating differential recruitment of HIF-1α and p53 to the promoter of TIGAR and/or SCO2 genes, thereby, tailoring physiological needs and environmental conditions to SCO2 and TIGAR gene expression.