Cancer cells express hypoxia-inducible factors (HIFs) in hypovascularized regions inside a tumor mass in response to hypoxia. HIFs activate more than 800 genes including those involved in angiogenesis, glycolysis, and growth factor signaling, which collectively facilitate cancer progression and metastasis. Indeed, increased expression of HIFs is an indicator for poor prognosis of cancer patients. In addition, embryonic stem cells (ESCs) maintain an undifferentiated state more efficiently in hypoxia than in normoxia. Therefore, it is important to understand the functions and regulation of HIFs for cancer therapy and regenerative medicine. The current project will identify and characterize long noncoding RNAs (lncRNAs) that are bound to HIF-1 in human breast cancer cells and embryonic stem cells (ESCs) cultured in hypoxia. LncRNAs are defined as RNAs that are longer than 200 bases and do not encode mRNA, rRNA, or tRNA. Chromatin immunoprecipitation and RNA sequencing were used to identify novel HIF-1-bound lncRNAs (collectively called R-HIFs) that have been mapped closely to genes encoding glycolysis enzymes. Cancer cells and ESCs use glycolysis as a primary metabolic pathway of glucose rather than oxidative phosphorylation in normoxia and this tendency is enhanced by HIFs in hypoxia. Enhanced glycolysis has an advantage of producing more metabolic intermediates necessary for rapid cell proliferation. Thus, the regulation of glycolytic enzymes by lncRNAs can serve as a novel target for cancer therapy. Currently virtually nothing is known about the involvement of lncRNAs in hypoxia or glycolysis. It was hypothesized that HIF-1 uses lncRNAs as novel coactivators to regulate its target genes, including glycolysis genes, in hypoxia and thereby promotes proliferation of breast cancer cells and ESCs. The following 3 aims have been proposed to test this hypothesis.
Aim 1 is designed to identify direct target genes of an R-HIF by screening of its DNA binding sites and by identification of genes whose expression level is altered by the knockdown of the R-HIF.
In Aim 2 the roles of other R-HIFs in hypoxic gene regulation will be determined by the combination of knockdown, analysis of their genome-wide binding sites, and identification of associated chromatin proteins.
In Aim 3 the roles of the R-HIFs in increased glycolysis and decreased oxidative phosphorylation in hypoxic cancer cells and ESCs will be determined through the analysis of energy and glucose metabolism. These studies are expected to establish lncRNAs as a novel entity essential for the regulation of glycolytic genes by HIF-1 in hypoxia.
This study will facilitate the understanding of the mechanistic link between hypoxia and cancer cell proliferation regulated by long noncoding RNA. It is expected to unravel novel therapeutic targets for various types of cancer.
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