Date of Award

5-2012

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Biomedical Sciences

Track

Cancer and Developmental Biology

Research Advisor

Linda M. Hendershot, Ph.D

Committee

Andrew M. Davidoff, M.D. Roderick Hori, Ph.D. Jill M. Lahti, Ph.D. Clinton F. Stewart, Pharm.D.

Keywords

ATF4, Hypoxia, Regulation, Tumor, Unfolded Protein Response (UPR), VEGF

Abstract

Tumor cells experience a limiting microenvironment due to inadequate vascularization that can affect the normal functioning of intracellular organelles. In the case of the endoplasmic reticulum, the limiting environment is further exacerbated by the high metabolic demands of the tumor cells, which together interfere with the proper maturation of nascent proteins synthesized there. The resultant accumulation of unfolded proteins activates a signal transduction pathway known as the Unfolded Protein Response, which serves primarily to protect the cell during stress and helps restore homeostasis to this organelle. As tumors expand resulting in regions that are a greater distance from functional blood vessels, they become increasingly hypoxic, which ultimately results in the activation of another stress pathway that is primarily regulated by the hypoxia inducible factor family of transcription factors (HIFs). This pathway protects cancer cells, in part, by up-regulating VEGFA, which stimulates blood flow to the tumor in a process known as angiogenesis. The interplay between these two stress pathways in tumor cell survival is relatively unexplored.

Microarray analysis of the unfolded protein response in a human medulloblastoma cell line revealed that, in addition to known targets, a large number of proangiogenic factors were upregulated. Real-Time PCR analyses confirmed that four of these factors, VEGFA, FGF2, angiogenin and IL8, were transcriptionally up-regulated in multiple cell lines by various ER stress inducers. Our studies on VEGFA regulation revealed that ATF4, a UPR-inducible transcription factor, bound to the mouse and human VEGFA promoters. Using a combination of mouse embryonic fibroblasts and human neuroblastoma cell lines that are deficient in this transcription factor, we demonstrated that ATF4 binds to the VEGFA promoter and contributes significantly to VEGFA expression in response to ER stress. We also found that VEGFA mRNA stability is increased in response to UPR activation, via activation of AMP kinase, demonstrating that increased mRNA levels occur at two regulatory points. In keeping with the increased mRNA levels, we found that VEGFA protein is secreted at levels as high as or higher than that achieved in response to hypoxia. Because the inadequate microenvironment experienced by solid tumors is expected to activate both UPR and HIF signaling pathways, we tested for possible interaction between them. Our studies show that VEGFA transcription rate and secreted protein levels are induced to a greater extent when both of these pathways are activated together as compared to each stress alone. Although we demonstrated a synergy between the two stress pathways experienced by tumor cells, surprisingly we found that this was not through the combined effects of the two different transcription factors, but instead that UPR activation can enhance HIF signaling, which has implications for other HIF targets that aid in tumor survival.

Our results indicate that the UPR plays a significant role in inducing positive regulators of angiogenesis. In the case of VEGFA expression this occurs at transcriptional, posttranscriptional and post-translational levels and is likely to have widespread implications for promoting angiogenesis in response to normal physiological cues as well as in pathological conditions like cancer.

DOI

10.21007/etd.cghs.2012.0243

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