HIF-1α In Tumors Research Articles

Steppogenin suppresses tumor growth and sprouting angiogenesis through inhibition of HIF-1α in tumors and DLL4 activity in the endothelium

BackgroundHypoxia is a characteristic feature of many solid tumors. As an adaptive response to hypoxia, tumor cells activate hypoxia-inducible factor-1α (HIF-1α). Under hypoxic conditions, angiogenesis mediated by HIF-1α is involved in the growth and metastasis of tumor cells. During the angiogenic process, differentiated tip endothelial cells (ECs) characterized by high expression of DLL4 promote angiogenic germination through filopodia. Inhibitors of HIF-1α or DLL4 have been widely studied PurposeWe tried to find inhibitors targeting both HIF-1α and DLL4 in tumor which have not yet been developed. Study designIn this study, we examined a natural compound that inhibits sprouting angiogenesis and tumor growth by targeting both HIF-1α and DLL4 under hypoxic conditions. MethodsAfter examining cell viability of 70 selected natural compounds, we assessed the effects of compounds on HIF-1α and DLL4 transcriptional activity using a dual-luciferase reporter assay. Western blot analysis, immunofluoresecnt assay and real-time qPCR were performed to identify expression of proteins, such as HIF-1α and DLL4, as well as HIF-1α target genes under hypoxic conditions. In vitro angiogenesis assay and in vivo allograft tumor experiment were performed to investigate inhibition of tumor growth through anti-angiogenic activity. ResultsAmong these compounds, steppogenin, which is extracted from the root bark of Morus alba l, respectively inhibited the transcriptional activity of HIF-1α under hypoxic conditions in HEK293T cells and vascular endothelial growth factor (VEGF)-induced DLL4 expression in vascular ECs in a dose-dependent manner. In tumor cells and retinal pigment epithelial cells, steppogenin significantly suppressed HIF-1α protein levels under hypoxic conditions as well as VEGF-induced DLL4 expression in ECs. Furthermore, steppogenin suppressed hypoxia-induced vascular EC proliferation and migration as well as VEGF-induced sprouting of EC spheroids. ConclusionThese results suggest that the natural compound steppogenin could potentially be used to treat angiogenic diseases, such as those involving solid tumors, because of its dual inhibition of HIF-1α and DLL4.

EXTH-41. THE microRNA miR-138 MAY BE RESPONSIBLE FOR SOME OF THE EFFECTS OF METABOLIC KETOSIS ON MALIGNANT GLIOMA

Abstract We and others have demonstrated that altering metabolism through the use of a ketogenic diet (KD) has numerous effects in mouse models of malignant glioma including slowed growth, reduced hypoxia and angiogenesis, enhanced survival and potentiation of the effects of radiation and chemotherapy. These effects are recapitulated in vitro when the ketone β-hydroxybutyrate (BHB) is added to mouse and human malignant glioma cells grown under high glucose conditions. BHB has been demonstrated to be an epigenetic modifier, providing a potential mechanism for the pluripotent effects of ketones even in the presence of high glucose. Therefore, we have analyzed alterations in microRNA (miRNA) expression in tumors and cells treated with a KD or BHB, respectively. MicroRNAs are short, single-stranded, noncoding RNAs that posttranscriptionally control gene expression. Each miRNA can affect the expression of many genes and they are implicated in all hallmarks of cancer. We found a number of miRNAs deregulated in tumor tissue from mice fed a KD, particularly mmu-miR-138 which was increased over 30-fold relative to tumors from mice fed a standard diet. MiR-138 is involved in a variety of anti-tumor effects that correlate with our data from KD treated gliomas. We have shown that ketones radiosensitize glioma cells, potentially through a reduction in DNA damage repair. MiR-138 has been shown to reduce the expression of H2AX which plays a key role in sensing and repairing DNA damage. Hypoxia inducible factor-1α (HIF-1α) is also a target of miR-138, and we have demonstrated a reduction in hypoxia, angiogenesis and the expression of HIF-1α in tumors from mice fed a KD. Furthermore, when we genetically engineered tumor cells to express miR-138 and injected them into mice, only necrotic tumor foci were found. Thus, we suggest that miR-138 is an important regulatory mechanism for the anti-tumor actions of metabolic ketosis in glioma.

HIF-1α acts as a molecular target for simvastatin cytotoxicity in B16.F10 melanoma cells cultured under chemically induced hypoxia.

Our previous studies reported that one of the main mechanisms of the antitumor activity of simvastatin (SIM) in B16.F10 murine melanoma cells was associated with strong suppression of the constitutive cell production of the α subunit of the heterodimeric transcription factor hypoxia-inducible factor (HIF)-1. Thus, the present study aimed to broaden this finding under hypoxic conditions induced by incubation of B16.F10 cells with cobalt chloride, when the constitutive production of HIF-1α in these melanoma cells is amplified by inducible expression of this factor. The data demonstrated that the SIM antiproliferative effects on melanoma cells were mediated mainly via strong suppressive actions on the B16.F10 cell capacity to support tumor angiogenesis and inflammation, as a result of a high inhibition of the inducible expression of HIF-1α. However, the constitutive expression of HIF-1α was not affected by SIM, probably due to the lack of effect of this statin on nuclear factor-κB production in B16.F10 cancer cells at the concentration tested. Additionally, the present study noted slight reducing effects of SIM on tumor oxidative stress, which may contribute to the main inhibitory action of this statin on HIF-1α production in hypoxic tumor cells. Collectively, these data are valuable for future anticancer strategies based on SIM administration in combination with cytotoxic drugs that are able to counteract the constitutive expression of HIF-1α in tumors.

Abstract 3745: Validation of the co-expression of breast cancer stem cell markers with HIF-1α in tumors.

Abstract Stem-like breast cancer cells (SBCCs) are drug resistant, invasive, and likely to lead to tumor recurrence and repopulation. High expression of the adhesion molecule CD44, the drug transporter ABCG2, and of the enzyme ALDH1A1 are well-established markers associated with SBCC-enriched tumor populations [1]. Hypoxic tumor microenvironments are frequently associated with increased aggressiveness and resistance to chemo and radiation therapy. Hypoxia results in the stabilization of the hypoxia inducible factor -1 (HIF-1), a transcription factor that activates a battery of genes, including those associated with SBCCs, that help cancer cells to survive, repopulate and finally metastasize to distant location. Recently, we reported the role of hypoxia and HIF-1α in regulating the expression of CD44 and its variant isoforms in triple negative breast cancer [2]. Here we have validated the association between hypoxia and CD44 expression in these tumors. We used tumors derived from MDA-MB-231 cells genetically engineered to express red fluorescent protein (tdtomato) under the control of hypoxia response element (231-HRE-RFP). Optical imaging (Nikon fluorescence microscope) was performed to detect hypoxia in fresh tissue slices, followed by immunohistochemistry (IHC) staining for HIF-1α, CD44 and ABCG2 expression in 5μm thickness adjacent sections from paraffin embedded 231-HRE-RFP tumors. Slides were scanned on an Image Scope digital scanner. Analysis for HIF-1 α nuclear staining was performed by drawing regions of interest (ROI) on scanned images using manufacturer supplied macro (Aperio Technologies Inc. CA, USA). For co-registration and quantification studies, ROI drawn images of HIF-1α and CD44 were co-registered to the bright field and fluorescent optical images using an in-house program developed in MATLAB (Mathworks Inc.). Statistical analysis (t-test) was performed using Microsoft Excel 2010 (Microsoft Inc. Seattle, USA). Following co-registration, intensely fluorescing regions of 231-HRE-RFP tumors were found to be associated with elevated nuclear HIF-1α expression and higher CD44 membrane expression. A trend of increased optical intensity (p≤0.09) and significantly increased CD44 pixel intensity (p≤0.05) was observed in the high HIF-1α ROI compared to the low HIF-1α ROI. Work is under way to co-register other breast cancer stem cell markers such as ABCG2 and ALDH1A1 in these tumors. These data further highlight the role of hypoxia in engendering a stem-like phenotype, and the potential importance of targeting hypoxia to minimize the burden of cells with stem-like characteristics in tumors. All animal protocols were approved by the JHU animal care and use committee. This work was supported by NIH R01CA136576 and P50 CA103175. 1. Al-Hajj, M et al., Proc Natl Acad Sci U S A, 2003. 2. Krishnamachary.B. et al., PLoS One 2012;7(8)e44078- Citation Format: Balaji Krishnamachary, Samata Kakkad, Marie-France Penet, Keve Zoltani, Venu Raman, Mayur Gadiya, Yelena Mironchik, Flonne Wildes, Zaver M. Bhujwalla. Validation of the co-expression of breast cancer stem cell markers with HIF-1α in tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3745. doi:10.1158/1538-7445.AM2013-3745

Farnesyltransferase Inhibitor Manumycin Targets IL1β-Ras-HIF-1α Axis in Tumor Cells of Diverse Origin

We have recently reported that Ras acts as an intermediate coactivator in IL-1β-mediated hypoxia-inducible factor-1α (HIF-1α) activation in glioblastoma multiforme (GBM). Since HIF-1α plays a crucial role in linking inflammatory and oncogenic pathways, we investigated whether this IL1β-Ras-HIF-1α signaling axis observed in GBM also exists in other tumors of diverse origin under normoxia. Treatment with IL-1β induced Ras in non-GBM cell lines A549 (lung), HeLa (cervical), and HepG2 (liver), and inhibition of Ras activity attenuated HIF-1α activity. Our findings suggest that Ras links IL-1β and HIF-1α in tumors of diverse origin. As we have previously reported that the farnesyltransferase inhibitor manumycin decreases Ras activity in glioma cells, we investigated whether manumycin could regulate IL-1β-mediated HIF-1α activation. Manumycin abrogated IL-1β-induced HIF-1α activation in both glioma and non-glioma tumor cells. In addition, manumycin also decreased IL-1β induced pro-inflammatory responses in tumor cells.