As tumors continue to grow and exceed their blood supply, nutrients become limited leading to deficiencies in amino acids (AAD), glucose (GD), and oxygen (hypoxia). These alterations result in significant changes in gene expression. While tumors have been shown to overcome the stress associated with GD or hypoxia by stimulating vascular endothelial growth factor (VEGF)-mediated angiogenesis, the role of AAD in tumor angiogenesis remains to be elucidated. We found that in human tumors, the expression of the general control non-derepressible 2 (GCN2, an AAD sensor) kinase is elevated at both protein and mRNA levels. In vitro studies revealed that VEGF expression is universally induced by AAD treatment in all five cell lines tested (five of five). This is in contrast to two other angiogenesis mediators interleukin-6 (two of five) and fibroblast growth factor 2 (two of five) that have a more restricted expression. Suppressing GCN2 expression significantly decreased AAD-induced VEGF expression. Silencing activating transcription factor 4 (ATF4), a downstream transcription factor of the GCN2 signaling pathway, is also associated with strong inhibition of AAD-induced VEGF expression. PKR-like kinase, the key player in GD-induced unfolded protein response is not involved in this process. In vivo xenograft tumor studies in nonobese diabetic/severe combined immunodeficient mice confirmed that knockdown of GCN2 in tumor cells retards tumor growth and decreases tumor blood vessel density. Our results reveal that the GCN2/ATF4 pathway promotes tumor growth and angiogenesis through AAD-mediated VEGF expression and, thus, is a potential target in cancer therapy.
Abbreviations
AAD
amino acid deprivation
ATF4
activating transcription factor 4
GCN2
general control non-derepressible 2
GD
glucose deprivation
HNSCC
human head and neck squamous cell carcinoma
IL-6
interleukin-6
LCM
laser capture microdissection
IHC
immunohistochemistry
MEF
mouse embryonic fibroblast
SCID
severe combined immunodeficient
PERK
PKR-like kinase
UPR
unfolded protein response
VEGF
vascular endothelial growth factor
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This work was supported by The Sharon and Lauren Daniels Cancer Research Fund, the Office of the Provost, University of Michigan, grant P50-CA97248 from the National Institutes of Health/National Cancer Institute (NIH/NCI), R01-DE21139 from the NIH/National Institute of Dental and Craniofacial Research (NIDCR), and the University of Michigan Cancer Center Support grant (P30 CA046592).