Amino Acid Deprivation Promotes Tumor Angiogenesis through the GCN2/ATF4 Pathway

AbstractAs 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

Mild acidic stress induces Grp78 expression mainly and allows endothelial cell adaptation.

<p>HDMECs were maintained in acidic cultures (pH 7.0) for seven days; pH was adjusted by lactic acid (<b>A</b>) or HCl (<b>B</b>), HDMECs kept at regular pH 7.5 served as control. Cell lysates were collected for western blot analysis, β-actin expression was used as a loading control; Grp78 expression increased over time. <b>C</b>, <b>D</b>, RNA was collected using Qiagen RNAeasy kits. XBP1 mRNA splicing and CHOP mRNA levels remained unaltered after exposure to mild acidic stress for seven days. Values are means ± SEM. Columns, means of individual experiments; Bars, SEM. <b>E</b>, Proliferation rates were determined by SRB assay. Values are means ± SD. Columns, means of individual experiments; Bars, SD. <b>F</b>, HDMECs exposed to pH 7.0 for seven days and HDMECs kept at pH 7.5 for same period were stained with propidium iodide and cell cycle was analyzed by flow cytometry. No difference in cell cycle distribution was observed. Values are means ± SD. Columns, means of individual experiments; Bars, SD.</p

Acidic stress induces UPR in endothelial cells.

<p>Primary human endothelial cells, HDMECs, were maintained in acidic cultures for 48 hours; pH was adjusted by lactic acid (<b>A, B, C</b>) or HCl (<b>D, E, F</b>), HDMECs kept at regular pH 7.5 medium served as control. Cell lysates were collected for western blot analysis, β-actin expression was used as a loading control. <b>A, D</b>, Treatment with low pH increased protein expression of UPR markers: Grp78, ATF4, and <i>p</i>-elf2α at 48 hours (western blot analysis). <b>B, E</b>, Exposure to low pH increased XBP1 mRNA splicing levels at 48 hours. <b>C, F</b>. Acidic stress upregulated CHOP mRNA levels. RNA was collected using Qiagen RNAeasy kit, reverse transcribed and analyzed using RT-PCR. Values are means ± SEM. Columns, means of individual experiments; Bars, SEM.***p<0,001.</p

Tumor conditioned medium induces UPR in endothelial cells.

<p>Primary human endothelial cells, HDMECs, are maintained in conditioned medium obtained from oral squamous cell carcinoma UM-SCC-81B cell line (<b>A, B, C</b>) or U-87 glioblastoma cell line (<b>D, E, F</b>), HDMECs kept at regular medium served as control. Cell lysates were collected for western blot analysis, β-actin expression was used as a loading control. <b>A, D</b>, Treatment with CM increased protein expression of UPR markers: Grp78, ATF4, <i>p</i>-elf2α at 48 hours (western blot analysis). <b>B, E</b>, Exposure to CM increased XBP1 mRNA splicing levels at 48 hours. <b>C, F</b>, CM treatment upregulated CHOP mRNA levels. RNA was collected using Qiagen RNAeasy kit, reverse transcribed and analyzed using RT-PCR. Values are means ± SEM. Columns, means of individual experiments; Bars, SEM.**p<0.01, ***p<0.001.</p

Glucose-Regulated Protein 78 (Grp78) Confers Chemoresistance to Tumor Endothelial Cells under Acidic Stress - Figure 1

<p><b>In A</b>, LCM retrieval of endothelial cells from paraffin embedded tissue sections (a - before dissection, b - removal of blood cells, c- capture of endothelial cells). <b>B</b>, RNA purity control, using VEGFR2, a marker for endothelial cells, and E-cadherin, a marker for cells of ectodermal origin. <b>C, D, E</b>, real-time PCR to quantify Grp78, ATF4 and CHOP expression in TEC compared to NEC and primary endothelial cells. Specimens were obtained from human oral squamous cell carcinoma biopsies. Data presented from real-time PCR experiments reflect the expression levels of Grp78, ATF4 and CHOP normalized to β-tubulin. Values are means ± SD. Columns, means of individual experiments; Bars, SD. ***p<0.001.</p

Chronic exposure to mild acidic stress results in a chemoresistance phenotype through Grp78 expression.

<p><b>A</b>, HDMECs cultured under acidic stress for 7 days, with medium pH adjusted to 7.0 with lactic acid and HDMECs maintained at regular pH 7.5 for seven days (controls) were exposed to Sunitinib at different doses or the vehicle control (DMSO) for further 72 h and then examined for cell viability using the SRB assay. Vehicle treatment represents 100% viability. <b>B</b>, HDMECs were infected with control ShRNA (scshRNA) or ShRNA specifically targeted against human Grp78 (ShGrp78) or left uninfected; Four days after infection, cells were harvested for western blot analysis and probed for Grp78 and β-actin. <b>C</b>, Seven days after mild acidic stress (pH 7.0), scshRNA and ShGrp78 cells were treated with either vehicle or Sunitinib for further 72 h and then examined for cell viability using the SRB assay. Vehicle treatment represents 100% viability. <b>D</b>, HDMECs were maintained in acidic cultures (pH 7.0) for 7 days, HDMECs at regular pH 7.5 served as control. At day 7, cells were treated with either DMSO (Vehicle, 1%), or Sunitinib (0.25 µM) for an additional 48 hours after which cell lysates were collected for western blot analysis and probed for caspase 7 and β-actin. Previous exposure to acidic stress resulted in lower levels of cleaved caspase 7. <b>E</b>, ScshRNA and ShGrp78 cells were treated with Sunitinib (0.25 µM) for 48 hours after which cell lysates were collected for western blot analysis and probed for caspase 7 and α-tubulin. We observed increased caspase 7 activation in ShGrp78 cells treated with Sunitinib. <b>F</b>, Survival analysis showed increased cell death in ShGrp78 cells treated with Sunitinib. Values are means ± SEM. Columns, means of individual experiments; Bars, SEM. Significance is calculated by comparing drug-treated cells and vehicle control-treated cells. *p<0.05, **p<0.01, ***p<0.001.</p