Reduction Of HIF-1α Expression Research Articles

ACY-241, a histone deacetylase 6 inhibitor, suppresses the epithelial-mesenchymal transition in lung cancer cells by downregulating hypoxia-inducible factor-1 alpha.

Hypoxia-inducible factor-1 alpha (HIF-1α) is a transcription factor activated under hypoxic conditions, and it plays a crucial role in cellular stress regulation. While HIF-1α activity is essential in normal tissues, its presence in the tumor microenvironment represents a significant risk factor as it can induce angiogenesis and confer resistance to anti-cancer drugs, thereby contributing to poor prognoses. Typically, HIF-1α undergoes rapid degradation in normoxic conditions via oxygen-dependent degradation mechanisms. However, certain cancer cells can express HIF-1α even under normoxia. In this study, we observed an inclination toward increased normoxic HIF-1α expression in cancer cell lines exhibiting increased HDAC6 expression, which prompted the hypothesis that HDAC6 may modulate HIF-1α stability in normoxic conditions. To prove this hypothesis, several cancer cells with relatively higher HIF-1α levels under normoxic conditions were treated with ACY-241, a selective HDAC6 inhibitor, and small interfering RNAs for HDAC6 knockdown. Our data revealed a significant reduction in HIF-1α expression upon HDAC6 inhibition. Moreover, the downregulation of HIF-1α under normoxic conditions decreased zinc finger E-box-binding homeobox 1 expression and increased E-cadherin levels in lung cancer H1975 cells, consequently suppressing cell invasion and migration. ACY-241 treatment also demonstrated an inhibitory effect on cell invasion and migration by reducing HIF-1α level. This study confirms that HDAC6 knockdown and ACY-241 treatment effectively decrease HIF-1α expression under normoxia, thereby suppressing the epithelial-mesenchymal transition. These findings highlight the potential of selective HDAC6 inhibition as an innovative therapeutic strategy for lung cancer.

Hypoxic Signaling Is Modulated by Calcium Channel, CaV1.3, in Androgen-Resistant Prostate Cancer.

Androgen deprivation therapy (ADT) remains a key treatment for advance prostate cancer (PCa), but resistance leads to terminal castrate-resistant prostate cancer (CRPC). Its development is linked to the emergence of a hypoxic tumor microenvironment and associated hypoxia inducible factor (HIF) signaling, which is known to be modulated by intracellular calcium. ADT is also known to upregulate store-operated calcium entry (SOCE) through voltage-gated calcium channel, CaV1.3. Consequently, the role of CaV1.3 in supporting hypoxic signaling and CRPC biology was explored. Androgen-sensitive PCa LNCaP cells were cultured with and without ADT bicalutamide, alongside ADT-resistant CRPC cells (LNCaP-ABL), either in normal or low oxygen (O2) (1%) conditions. HIF-1α, CaV1.3, and androgen receptor (AR) gene expression was measured by qPCR and protein expression with Western blot in the presence or absence of siCaV1.3. SOCE was determined through Fura-2AM fluorescence measurement. Cell proliferation was quantified by WST-1 assay and survival by colony formation. CaV1.3 expression was increased during ADT but not hypoxia, correlating with an associated increase in SOCE. HIF-1α expression was upregulated by ADT under normal O2 conditions and increased during hypoxia across all cells but with a higher fold change in early ADT-resistant and CRPC cells. Under hypoxic conditions CaV1.3 small interfering RNA resulted in a significant reduction in HIF-1α expression for ADT-sensitive cells but increased in CRPC. A similar pattern was also observed for AR expression. Cell survival was found significantly reduced by siCaV1.3 under hypoxic conditions for all cells, with and without ADT. Whereas cell proliferation under the same conditions was reduced in CRPC only. This study highlights that CaV1.3 can modulated HIF signaling and impact on PCa tumor biology under hypoxia, but further investigation is required to ascertain if this mediated through SOCE or a noncanonical mechanism.

Hif-1α Inhibitors Could Successfully Inhibit the Progression of Differentiated Thyroid Cancer in Vitro.

Hypoxia-inducible factor (HIF)-1α plays an important role in cancer progression. In various cancers, including thyroid cancer, overexpression of HIF-1α is related to poor prognosis or treatment response. However, few studies have investigated the role of HIF-1α inhibition in thyroid cancer progression. We evaluated the utility of the HIF-1α inhibitor IDF-11774 in vitro utilizing two thyroid cancer cell lines, K1 and BCPAP. Both cell lines were tested to elucidate the effects of IDF-11774 on cell proliferation and migration using soft agar and invasion assays. Here, we found that a reduction of HIF-1α expression in BCPAP cells was observed after treatment with IDF-11774 in a dose-dependent manner. Moreover, cell proliferation, migration, and anchorage-independent growth were effectively inhibited by IDF-11774 in BCPAP cells but not in K1 cells. Additionally, invasion of BCPAP but not K1 cells was controlled with IDF-11774 in a dose-dependent manner. Our findings suggest that promoting the degradation of HIF-1α could be a strategy to manage progression and that HIF-1α inhibitors are potent drugs for thyroid cancer treatment.

α-Mangostin protects against myocardial ischemia reperfusion injury by suppressing the activation of HIF-1α

Purpose: To investigate the cytoprotective effect of α-mangostin on myocardial tissues in ischemic rats, and the underlying mechanism.Methods: Histopathological changes in myocardial tissues were determined using inverted microscope. Protein expressions were measured by western blotting, while enzyme-linked immunosorbent assay (ELISA) was used to assay the expression levels of caspase-3, caspase-9 and caspase-8.Results: Treatment with α-mangostin (20 mg/kg) suppressed production of reactive oxygen species (ROS) and lipid peroxides in myocardial tissues of MI/R rats, and significantly alleviated MI/R injurymediated reduction in ATP levels in cardiac tissues (p < 0.05). α-Mangostin treatment of MI/R injury rats suppressed HIF-1α activation, and markedly elevated BNIP3 levels, relative to model group. Moreover, MI/R-induced cardiomyocyte apoptosis was significantly alleviated by α-mangostin treatment (p < 0.05). Treatment with α-mangostin also suppressed I/R-induced increases in caspase-8 and caspase-3 activation in myocardial tissues, improved Nrf-2 activation, and promoted HO-1 and GST levels in MI/R injury rats (p < 0.05).Conclusion: These results suggest that α-mangostin protects rat cardiac tissues from MI/R-induced oxidative damage via reduction of HIF-1α expression, inhibition of ROS generation and suppression of apoptosis. Therefore, α-mangostin may be of therapeutic importance for the management of myocardial ischemia in humans.
 Keywords: α-Mangostin, Hypoxia, Inflammation, Nrf-2, Oxidative stress, Reperfusion

Hypoxia inducible factor-1α/B-cell lymphoma 2 signaling impacts radiosensitivity of H1299 non-small cell lung cancer cells in a normoxic environment.

Hypoxia inducible factor-1α (HIF-1α) is a critical transcriptional factor for the response of cells to hypoxic microenvironment and its expression induces resistance of hypoxic non-small-cell lung cancer (NSCLC) cells to radiotherapy. This study investigated how the activation of HIF-1α/B-cell lymphoma 2 (BCL-2) signaling under normoxic conditions impacted radiosensitivity of NSCLC cells. The recombinant pcDNA3.0-EGFP plasmids with wild-type or mutant HIF-1α complementary DNA (cDNA) were transfected into H1299 cells, an NSCLC cell line, establishing two H1299 sublines with high expression of HIF-1α. Compared with the levels of HIF-1α and BCL-2 proteins in non-transfected cells, increased levels of both proteins were found in transfected cells. Moreover, the expression of HIF-1α in non-transfected cells induced by chloride cobalt (CoCl2), a commonly used mimetic hypoxia reagent, was concomitant with the enhancement of BCL-2 expression. Conversely, reduction of HIF-1α expression by an inhibitor decreased the levels of BCL-2 proteins. The results revealed that the stabilization and expression of HIF-1α promoted the accumulation of BCL-2 proteins in H1299 cells. Subsequent experiments showed that intracellular HIF-1α/BCL-2 signaling was triggered in a normoxic environment after H1299 cells were exposed to irradiation, causing an elevated radioresistance. In contrast, blockage of HIF-1α/BCL-2 signaling leads to an elevated radiosensitivity. Proliferation of cells assay showed that, under normoxic conditions, population doubling times (PDTs) of irradiated cells were prolonged by suppression of HIF-1α/BCL-2 signaling. It is, therefore, indicated that HIF-1α/BCL-2 signaling activated by ionizing radiation reduces the radiosensitivity of H1299 cells independent of the hypoxic environment.

HGF regulate HTR-8/SVneo trophoblastic cells migration/invasion under hypoxic conditions through increased HIF-1α expression via MAPK and PI3K pathways.

Hepatocyte growth factor (HGF) is reported to be down-regulated in pregnancy complications like intrauterine growth retardation and preeclampsia, which are associated with abnormal trophoblast migration/invasion. In this study, role of HGF and associated signaling pathways has been investigated in HTR-8/SVneo trophoblastic cells migration/invasion under normoxia (20% O2) and hypoxia (2% O2). HTR-8/SVneo cells exposed to hypoxia showed increase in migration and invasion as compared to cells incubated under normoxic conditions. The migration/invasion under both normoxic and hypoxic conditions was further enhanced after treatment with HGF. Subsequent to treatment with HGF, a significant increase in expression of MMP2 & MMP3 under normoxia and MMP1 & MMP9 under hypoxia was observed. Treatment of HTR-8/SVneo cells with HGF under hypoxia also led to decrease in TIMP1. Treatment of the cells with HGF led to activation of mitogen activated protein kinases (MAPK) and phosphatidylinositol 3-kinase (PI3K) signaling pathways. Inhibition of MAPK by U0126 and PI3K by LY294002 led to concomitant decrease in the HGF-mediated migration/invasion of HTR-8/SVneo cells. HGF treatment under hypoxia also led to a significant increase in hypoxia inducible factor (HIF-1α) expression. Additionally, inhibition of HIF-1α by siRNA led to decrease in HGF-mediated migration of HTR-8/SVneo cells under hypoxic conditions. Inhibition of HGF activated MAPK and PI3K signaling led to reduction in HIF-1α expression under hypoxia. In conclusion, HGF facilitates HTR-8/SVneo cell migration/invasion by activation of MAPK/PI3K signaling pathways and increased expression of MMPs. HIF-1α has a role in HGF-mediated increase in migration under hypoxic conditions.

Abstract 5055: Multiplatform metabolomics analysis of growth arrest in pancreatic tumor xenografts

Abstract R,S-4-Methoxy-1-naphthylfenoterol, MNF, inhibits the G protein-coupled receptor GPR55. In this study, we determine the effect of MNF on human PANC-1 pancreatic tumor growth in mice and apply multiplatform metabolomics analysis to identify pathways associated with growth arrest. Methods: Female Balb/c nude mice, 6-8 weeks old, 18-20g, were inoculated subcutaneously with 5 x 106 PANC-1 cells. On Day 8, mice were placed in groups of 10 using random block design based upon tumor volume. Mice received daily ip injections of vehicle or 40mgxkg-1 MNF 5 days/week for 3 treatment cycles. Mice were monitored daily and tumor volumes measured at beginning and end of each dosing cycle. On Day 33, mice were euthanized, and plasma samples and tumors collected. Tumor tissue was homogenized, extracted and analyzed using liquid chromatography-QTOF-MS, capillary electrophoresis-TOF-MS and gas chromatography-EI-Q-MS. Differences between groups were evaluated by unpaired t-test or Mann−Whitney test with post hoc Benjamini-Hochberg correction. Statistical significance was set at P≤0.05. Compound identification was accomplished using online databases and in-house standards Results Tumor volume increased in vehicle-treated mice by ~700%, 142 ± 8 mm3 to 957 ± 79 mm3 and only ~250% in MNF-treated mice, 143 ± 8 mm3 to 259 ± 27 mm3. On Day 33, MNF was not detected in plasma but accumulated in tumor tissues, 43.9 ± 32.7 ng/g. Plasma L-lactate levels were reduced from 3.29 ± 0.66 mmol/L το 2.81 ± 0.60 mmol/L, P≤0.001. Differences in tumor tissue metabolome were observed with MNF compared to vehicle, which was reflected by significant, P≤0.05, changes in relative metabolite signals. MNF treatment was accompanied by the disruption of pyrimidine nucleotide biosynthesis at uridine 5'-monophosphate, UMP, and increased UMP degradation. Moreover, the +237% increase in ophthalmic acid and +95% increase in its precursor 2-aminobutyrate indicate higher oxidative stress and the +51% increase in 2-hydroxyproline suggest greater HFI-1α proteolysis upon MNF treatment. Conclusions These results are consistent with our previous observations in PANC-1 cells showing that GPR55 inhibition attenuates activation of the EGFR-MEK-ERK, Wnt-β-catenin and PI3K/AKT pathways resulting in lower cyclin D1 expression and halting the cell cycle in G1. The reduction in HIF-1α expression and glycolytic flux in MNF-treated PANC-1 cells is in line with the decline in L-lactate plasma levels in MNF-treated mice. The data from this study indicate that MNF may be useful in the treatment of pancreatic cancer. Citation Format: Irving W. Wainer, Danuta Dudzik, Michel Bernier, Coral Barbas. Multiplatform metabolomics analysis of growth arrest in pancreatic tumor xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5055. doi:10.1158/1538-7445.AM2017-5055

P08.58 Impact of selected pentabromobenzylisothiourea (ZKK-3) combined with hyperbaric oxygen therapy on therapeutic response and oxygenation status of T98G glioblastoma cell line

Abstract Introduction: Glioblastoma (GBM), the primary brain tumour derived from astroglial cells, is the most frequently diagnosed intracranial malignancy in adults. Poor prognosis for GBM patients results from high invasiveness of neoplastic cells related with faulty angiogenesis and persistent hypoxia states. In such conditions glioma cells develop radio- and chemotherapy-resistant phenotype. It is postulated that application of hyperbaric oxygen (HBO) may improve therapeutic response via refinement of tumour tissue oxygenation. The more effective therapeutic strategies are also required. One of the novel compounds that exhibit anti-tumour properties are pentabromobenzylisothioureas. The aim of this study was to examine whether HBO may enhance anti-tumour efficacy of selected pentabromobenzylisothiourea when both agents were administered as combined ZKK-3/HBO therapy. Materials and Methods: Human glioblastoma T98G cell line was cultured in medium supplemented with novel isothiourea derivative - ZKK-3. The proliferation and viability of glioma cells cultured in hypoxia or hyperbaric oxygen conditions were examined. Cell proliferation was tested 24 hours after ZKK-3 addition using Nikon Inverted Microscope and Multisizer 3 Beckman Coulter. The viability assay was taken 24 and 48 hours post ZKK-3 supplementation by CellTiter 96®AQueous One Solution Cell Proliferation Assay (Promega). Hypoxia state of T98G cells was determined via the evaluation of HIF-1α protein expression using HIF-1A ELISA Kit (Thermo Scientific). Tests were performed on cells treated with ZKK-3 and cultured in various oxygen conditions: 1/ normoxia (24 hours), 2/ hypoxia (24 hours), 3/ HBO (2 ATA, 1 hour followed by 23 hours of normoxia), 4/ double hypoxia, 5/ hypoxia/HBO. Results: Proliferation of T98G cells treated with ZKK-3 was significantly decreased after hyperbaric oxygenation in comparison to hypoxia conditions. Administration of HBO resulted also in statistically significant diminution of glioma cells’ viability. Expression of HIF-1α in T98G cells strongly depended on oxygen conditions: in hypoxia and double hypoxia the level of protein was significantly elevated compared with normoxia, while after hyperbaric oxygenation no changes or even decrease of HIF-1α level was observed. Comparison of double hypoxia and hypoxia/HBO groups showed that HBO addition after ZKK-3 supplementation caused significant reduction of HIF-1α expression. Conclusions: Treatment of malignant glioma cells with selected isothiourea derivative is significantly more efficient when combined with hyperbaric oxygen administration. Beneficial effect of HBO on decreasing HIF-1α expression allows to consider hyperbaric oxygenation as the way to overcome tumour hypoxia.ACKNOWLEDGEMENT: The research was supported by the KNOW-MMRC project and Foundation for the Development of Diagnostic and Therapy.

Ascorbate availability affects tumor implantation-take rate and increases tumor rejection in Gulo-/- mice.

In solid tumors, HIF1 upregulates the expression of hundreds of genes involved in cell survival, tumor growth, and adaptation to the hypoxic microenvironment. HIF1 stabilization and activity are suppressed by prolyl and asparagine hydroxylases, which require oxygen as a substrate and ascorbate as a cofactor. This has led us to hypothesize that intracellular ascorbate availability could modify the hypoxic HIF1 response and influence tumor growth. In this study, we investigated the effect of variable intracellular ascorbate levels on HIF1 induction in cancer cells in vitro, and on tumor-take rate and growth in the Gulo−/− mouse. These mice depend on dietary ascorbate, and were supplemented with 3,300 mg/L, 330 mg/L, or 33 mg/L ascorbate in their drinking water, resulting in saturating, medium, or low plasma and tissue ascorbate levels, respectively. In Lewis lung carcinoma cells (LL/2) in culture, optimal ascorbate supplementation reduced HIF1 accumulation under physiological but not pathological hypoxia. LL/2, B16-F10 melanoma, or CMT-93 colorectal cancer cells were implanted subcutaneously into Gulo−/− mice at a range of cell inocula. Establishment of B16-F10 tumors in mice supplemented with 3,300 mg/L ascorbate required an increased number of cancer cells to initiate tumor growth compared with the number of cells required in mice on suboptimal ascorbate intake. Elevated ascorbate intake was also associated with decreased tumor ascorbate levels and a reduction in HIF1α expression and transcriptional activity. Following initial growth, all CMT-93 tumors regressed spontaneously, but mice supplemented with 33 mg/L ascorbate had lower plasma ascorbate levels and grew larger tumors than optimally supplemented mice. The data from this study indicate that improved ascorbate intake is consistent with increased intracellular ascorbate levels, reduced HIF1 activity and reduced tumor initiation and growth, and this may be advantageous in the management of cancer.

Altered Expression of Angiotensinogen and Mediators of Angiogenesis in Ileal Crohn's Disease.

Angiotensin II (AII) is a powerful splanchnic vasoconstrictor with pro-inflammatory and pro-fibrotic properties. Angiotensin converting enzyme (ACE) inhibitors and AII Receptor Antagonists (ARBs) are therapeutic in animal models of colitis. The aim of this case-control study is to determine the expression of angiotensinogen and related genes in human ileal Crohn's disease. Using quantitative real-time polymerase chain reaction (RT-PCR), we measured mRNA expression levels of angiotensinogen (AGT), hypoxia inducible factor (HIF)1α and melanoma cell adhesion molecule (MCAM; CD146) in 101 human samples (69 biopsy, 12 resection) from affected ileum (inflamed CD cases, n=36) and unaffected ileum (non-inflamed CD cases, n=45 and controls, n=20). Immunohistochemistry for angiotensinogen was also performed. The study was of case-control design in a tertiary care setting. Ileal expression of AGT was lower in CD cases compared to controls (p<0.0001), in inflamed CD samples (p=0.017) and current smokers (p=0.02). HIF1α expression was lower in non-inflamed CD biopsy samples than controls (p=0.006). The presence of disease-associated NOD2 variants was associated with increased expression of markers of angiogenesis (HIF1α p=0.009; MCAM p=0.007) in inflamed CD samples. Angiotensinogen immunohistochemical staining supported the quantitative RT-PCR expression findings. Angiotensinogen expression is down regulated in human ileal CD, particularly in the presence of inflammation and current cigarette smoking, implicating the mesenteric vasculature and mucosal hypoxia as co-factors in ileal CD pathogenesis. A novel reduction in HIF1α expression in non-inflamed ileal mucosa in CD patients was also demonstrated.

Oleanolic acid enhances the radiosensitivity of tumor cells under mimetic hypoxia through the reduction in intracellular GSH content and HIF-1α expression

We previously found that oleanolic acid (OA), a naturally pentacyclic triterpenoid, enhances the radiosensitizing effect on tumor cells. However, it is unclear whether or not OA enhances the radiosensitivity of hypoxic cells. Therefore, the aim of the present study was to further observe the influence of OA on hypoxic tumor cells, and the relative mechanism was also investigated. The radiosensitivity of rat glioma C6 cells and human lung cancer A549 cells with different treatments, under mimetic hypoxia, was evaluated by clonogenic assay. A micronucleus (MN) test, meanwhile, was utilized to observe the alteration in intracellular DNA damage. For determining the mechanism involved in the OA influence on the radiosensitivity of hypoxic cells, we determined the levels of intracellular reduced glutathione (GSH) using the glutathione reductase/5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) recycling assay. Simultaneously, the activities of γ-glutamylcysteine synthetase (γ-GCS) and GSH synthase (GSS), both enzymes for GSH synthesis, were tested using appropriate methods. Due to the involvement of hypoxia inducible factor-1α (HIF-1α) in the resistence of hypoxic cells to radiation damage, its levels were also observed by western blot method. The results from this study demonstrated that the clonogenic growth of irradiated cells was increased under mimetic hypoxia while the refractory effect of hypoxic cells to radiation was decreased following OA treatment. Moreover, the (MN) frequencies in the hypoxic cells treated with OA were augmented after irradiation compared with the cells without OA treatment. In the subsequent experiment, OA significantly reduced the biosynthesis of intracellular GSH via the attenuation of γ-GCS activity. Additionally, there was an obvious reduction in HIF-1α expression in irradiated cells treated with OA at different concentrations. In conclusion, OA significantly enhanced the radiosensitivity of tumor cells under mimetic hypoxia, through the reduction in intracellular GSH content and HIF-1α expression.

Hypoxia-Inducible Factor-1α and its Role in the Proliferation of Retinoblastoma Cells

In order to better understand the role of HIF-1α in the proliferation of the retinoblastoma cells, a siRNA knockdown of HIF-1α followed by a proliferation assay was performed. Further sequencing was then carried out in order to assess knockdown efficiency and expression of HIF-1α. Upregulation of HIF-1α gene expression in CoCl2-treated retinoblastoma cells was demonstrated via melting curve analysis from PCR tests and was further analyzed using western blot and densitometry analysis. Reduction of HIF-1α expression in retinoblastoma, post HIF-1α knockdown, was observed after siRNA transfection into Y-79 cells. Knockdown of HIF-1α resulted in a significant decrease in proliferation thereby demonstrating that HIF-1α is involved in promoting survival and proliferation in retinoblastoma cells. Stabilization of HIF-1α in retinoblastoma cells using CoCl2 was unsuccessful.

Abstract 1580: Molecular mechanisms of OSM-induced VEGF in breast cancer

Abstract Oncostatin M (OSM) is an interleukin-6 (IL-6) family cytokine that has been shown to induce expression of vascular endothelial growth factor (VEGF) in astroglioma and in hepatocellular and breast carcinoma. It is also known to induce VEGF in non-cancerous cells such as cardiac myocytes, hepatocytes, and smooth muscle cells. Under hypoxic conditions, VEGF expression is known to be dependent on the transcription factor hypoxia-inducible factor 1 alpha (HIF1α). Our current studies were designed to determine whether VEGF expression induced by OSM and other IL-6 family members, including IL-6 and leukemia inhibitory factor (LIF), is likewise dependent on HIF1α in human breast cancer cells. To analyze VEGF expression levels, MDA-MB-231 and T47D human breast cancer cells were cultured in the presence or absence of OSM, IL-6, and LIF, with or without a HIF1α siRNA, for 12 to 72 hrs. All three cytokines significantly induced HIF1α expression by Western blot analysis at each time point in both MDA-MD-231 and T47D cells. Conditioned media from these experiments was also analyzed using ELISA to determine HIF1α-induced VEGF expression. Treatment with OSM resulted in a two- to three-fold induction of secreted VEGF, while treatment with IL-6 and LIF showed no significant induction. In addition, OSM-induced VEGF expression was independent of HIF1α in MDA-MB-231 cells. This was indicated by unchanged secreted VEGF levels despite the reduction of HIF1α expression in siRNA treated groups. In T47D cells however, HIF1α siRNA-treatment for 72 hrs reduced VEGF levels by 40% suggesting that OSM-induced HIF1α may be partially responsible for VEGF expression in this cell line. To summarize, it is important to note that IL-6 and LIF did not induce VEGF expression in two commonly used human breast cancer cell lines, MDA-MB-231 and T47D. Furthermore, OSM-induced VEGF was not dependent on HIF1α in MDA-MB-231 cells, suggesting regulation of VEGF by an alternative molecular mechanism. Investigations are in progress to better understand mechanisms utilized by breast cancer cells for OSM-induced VEGF expression. Future results could lead to the potential design of OSM-targeted therapeutics. Funded by NIH R15CA137510, ACS RSG-09-276-01-CSM, Susan G. Komen for the Cure KG100513, and NIH/NCRR P20RR016454. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1580. doi:10.1158/1538-7445.AM2011-1580

Abstract 2111: Expression pattern and mechanism of tumor suppression of Rab25

Abstract Introduction: Our previous work has shown that the loss of Rab25 occurs in estrogen receptor (ER) and progesterone receptor (PR) negative breast cancer cell lines and human tumor samples and seems to correlate with an active ras pathway. We have examined the expression levels of rab25 in breast tissue and have investigated the mechanism by which Rab25 exerts its anti-tumorigenic effect. Loss of Rab25 cooperates with mutant Ras to promote tumorigenesis. HIF-1α levels are reduced in Rab25 expressing cells. We have assessed Rab25 binding to the HIF-1α promoter and localized Rab25 to the nucleus, suggesting a novel mechanism for Rab25's antitumor activity and a novel function for Rab25. Materials and Methods: Cell culture – Normal mammary tissue and the RAO-3 cell line was grown in DFCI-1 medium. Cells were transduced with retroviral supernatant and selected. Expression of Rab25 in transduced cells was determined by RT-PCR and western blot. qPCR analysis- Expression of HIF-1α and Rab25 were quantified using real-time PCR with fluorescence detection. Luciferase report assay – We obtained the HIF-1α promoter fragment and inserted the promoter sequence in the pGL4 luciferase vector and the Luciferase Assay was then performed. Western blot- Protein extracts from both cytosolic and nuclear fraction were tested for Rab25 expression. Immunofluorescence- Frozen mammary tissue slides were stained with luminal and basal markers as well as antibody for Rab25 to confirm localization of Rab25 in both luminal and basal mammary epithelium. Results: 1. High level expression of Rab25 was found in luminal epithelium in normal human breast tissue. Basal epithelium also expressed Rab25. 2. Tumorigenic mammary epithelial cell lines, RAO-3 and RAO-4, have lost Rab 25 expression with nearly 1/10,000 the level of Rab25 of normal mammary basal epithelium. 3. Rab25 localizes to the nucleus and reduces luciferase activity from the HIF-1α promoter. 4. Expression of Rab25 substantially reduces HIF-1α levels. Conclusions: 1. Rab25 is a complex molecule that has been described as an intracellular transport protein. 2. It is also a tumor modulator. It can regulate tumor growth and angiogenesis. 3. Rab25 expression can be detected in both luminal epithelium and basal epithelium. 4. Our data indicate novel functions for Rab25 including reduction in HIF-1α expression and localization to the nucleus. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2111. doi:10.1158/1538-7445.AM2011-2111

ROS production and angiogenic regulation by macrophages in response to heat therapy

Purpose: It has been well established that inadequate blood supply combined with high metabolic rates of oxygen consumption results in areas of low oxygen tension (<1%) within malignant tumours and that elevating tumour temperatures above 39°C results in significant improvement in tumour oxygenation. Macrophages play a dual role in tumour initiation and progression having both pro-tumour and anti-tumour effects. However, the response of macrophages to heat within a hypoxic environment has not yet been clearly defined.Methods: Raw 264.7 murine macrophages were incubated under normoxia and chronic hypoxia at temperatures ranging from 37–43°C. Under normoxia at 41°C, macrophages start to release significant levels of superoxide. The combination of heat with hypoxia constitutes an additional stimulus leading to increased respiratory burst of macrophages.Results: The high levels of superoxide were found to be associated with changes in macrophage production of pro-angiogenic cytokines. While hypoxia alone (37°C) increased levels of hypoxia inducible factor-1α (HIF-1α) in macrophages, the combination of hypoxia and mild hyperthermia (39–41°C) induced a strong reduction in HIF-1α expression. The HIF-regulated vascular endothelial growth factor (VEGF) decreased simultaneously, revealing that heat inhibits both HIF-1α stabilization and transcriptional activity.Conclusion: The data suggest that temperatures which are readily achievable in the clinic (39–41°C) might be optimal for maximizing hyperthermic response. At higher temperatures, these effects are reversed, thereby limiting the therapeutic benefits of more severe hyperthermic exposure.