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. 2013 Jan 1;132(1):29-41.
doi: 10.1002/ijc.27666. Epub 2012 Jun 26.

Overcoming evasive resistance from vascular endothelial growth factor a inhibition in sarcomas by genetic or pharmacologic targeting of hypoxia-inducible factor 1α

Yeo-Jung Kim  1 , Hae-June Lee, Tae-Min Kim, T S Karin Eisinger-Mathason, Alexia Y Zhang, Benjamin Schmidt, Daniel L Karl, Michael S Nakazawa, Peter J Park, M Celeste Simon, Sam S Yoon
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Overcoming evasive resistance from vascular endothelial growth factor a inhibition in sarcomas by genetic or pharmacologic targeting of hypoxia-inducible factor 1α (V体育2025版)

V体育平台登录 - Yeo-Jung Kim et al. Int J Cancer. .
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Abstract

Increased levels of hypoxia and hypoxia-inducible factor 1α (HIF-1α) in human sarcomas correlate with tumor progression and radiation resistance. Prolonged antiangiogenic therapy of tumors not only delays tumor growth but may also increase hypoxia and HIF-1α activity. In our recent clinical trial, treatment with the vascular endothelial growth factor A (VEGF-A) antibody, bevacizumab, followed by a combination of bevacizumab and radiation led to near complete necrosis in nearly half of sarcomas. Gene Set Enrichment Analysis of microarrays from pretreatment biopsies found that the Gene Ontology category "Response to hypoxia" was upregulated in poor responders and that the hierarchical clustering based on 140 hypoxia-responsive genes reliably separated poor responders from good responders VSports手机版. The most commonly used chemotherapeutic drug for sarcomas, doxorubicin (Dox), was recently found to block HIF-1α binding to DNA at low metronomic doses. In four sarcoma cell lines, HIF-1α shRNA or Dox at low concentrations blocked HIF-1α induction of VEGF-A by 84-97% and carbonic anhydrase 9 by 83-93%. HT1080 sarcoma xenografts had increased hypoxia and/or HIF-1α activity with increasing tumor size and with anti-VEGF receptor antibody (DC101) treatment. Combining DC101 with HIF-1α shRNA or metronomic Dox had a synergistic effect in suppressing growth of HT1080 xenografts, at least in part via induction of tumor endothelial cell apoptosis. In conclusion, sarcomas respond to increased hypoxia by expressing HIF-1α target genes that may promote resistance to antiangiogenic and other therapies. HIF-1α inhibition blocks this evasive resistance and augments destruction of the tumor vasculature. .

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Figures

Figure 1
Figure 1
(a) A Gene Set Enrichment Analysis plot of the “Response to hypoxia” category from Gene Ontology is shown (top). Genes (x-axis) are ordered by their t-statistics comparing poor and good responders. The upregulated and downregulated genes in poor responders are placed on the left and right, respectively. The enrichment score (y-axis) is a cumulative sum reflecting the degree of over-representation for the genes in this category compared to the rest of the genes; a high enrichment score indicates the presence of hypoxia-related genes among the genes that are significantly different in the good/poor responder phenotype. The locations of the 28 hypoxia-related genes are shown in the middle bar, with the gene symbols for the 11 genes that contribute to the maximum value of the enrichment score shown in gray. The relative expression levels of the 11 genes are also shown in a heat map (bottom). Red and blue represent the relative upregulation and downregulation, respectively, compared to its average expression. (b) Supervised hierarchical clustering analysis of 140 hypoxia-responsive genes. Poor and good responders are indicated by red and green, respectively. This analysis demonstrates that the vast majority of good and poor responders can be differentiated based on the expression of hypoxia-related genes. Dendogram is shown at top. (c) Relative mRNA levels of VEGF-A in sarcomas before and after bevacizumab (BV) treatment. Relative value is in relation to the lowest level of expression, which was assigned a value of 1. Poor and good responders are indicated by red and green boxes, respectively. Bars represent standard deviation. *p < 0.05 compared to pretreatment level.
Figure 2
Figure 2
(a) Western blot analysis of HIF-1α in human and mouse sarcoma cell lines in 21% oxygen (normoxia) and 0.5% oxygen (hypoxia). GAPDH blot serves as loading control. (b) Relative mRNA levels of VEGF-A, CA9 and FOXM1 in human and mouse sarcoma cell lines under normoxic and hypoxic conditions. (c) VEGF-A protein secreted into media of human and sarcoma cell lines under normoxic and hypoxic conditions. For relative value, expression level under normoxia for each cell line assigned a value of 1. Bars represent standard deviation. *p < 0.05 compared to normoxia level.
Figure 3
Figure 3
(a) Photos of HT1080 sarcoma xenografts treated with control IgG or DC101 and stained for hypoxia using Hypoxyprobe™-1. Length of treatment (Tx) and tumor sizes at time of harvesting are listed. (b) Percent hypoxia of H1080 xenografts at various sized tumors after treatment with control IgG or DC101. (c) Percentage of nuclei in various sized tumors treated with control IgG or DC101 staining for HIF-1α or HIF-2α. Bars represent standard deviation. *p < 0.05 compared to control IgG group; ns, not significant (p > 0.05). (d) Photos of HT1080 sarcoma xenografts treated with control IgG or DC101 and stained for hypoxia HIF-1a. Tumor sizes at time of harvesting are listed. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Figure 4
Figure 4
(a) Western blot analysis of HIF-1α in HT1080 cells in 21% oxygen (normoxia) and 0.5% oxygen (hypoxia) following treatment with HIF-1α shRNA or scrambled (Scr) control shRNA. GAPDH blot serves as loading control. Proliferation (b) and migration (c) of HT1080 cells after transduction with HIF-1α shRNA or scrambled (Scr) shRNA. (d) Growth of HT1080 cell transduced with HIF-1α shRNA or scrambled (Scr) shRNA following subcutaneous injection in athymic nude mice. Some groups treated with DC101 and IgG were used as antibody treatment control. Metronomic doxorubicin (Dox) was given to one group. (e) Relative mRNA levels of CA9 in HT1080 tumor groups. Bars represent standard deviation. *p < 0.05 compared to control group; ns, not significant (p > 0.05); **p < 0.05 compared to Scr shRNA + IgG control group, HIF-1α shRNA + IgG group and Scr shRNA + DC101 group.
Figure 5
Figure 5
(a) Relative mRNA levels of CA9 or FOXM1 in human and mouse sarcoma cell lines under normoxia, hypoxia or hypoxia with doxorubicin (10 μM). (b) VEGF-A protein secreted into media of human and sarcoma cell lines under normoxia, hypoxia or hypoxia with doxorubicin (10 μM). (c) Growth of HT1080 fibrosarcoma xenografts in vivo. HT1080 cells were injected subcutaneously, and growth was monitored for 17 days. (d) Relative mRNA levels of CA9 in HT1080 tumors. (e) Percent necrosis in HT1080 tumors as determined following H&E staining. For relative values, expression level under normoxia for each cell line or control group assigned a value of 1. Bars represent standard deviation. *p ≤ 0.05 compared to control group; **p < 0.05 compared to control group and to other treatment groups.
Figure 6
Figure 6
(a) Photos of CD31 immunohistochemical staining of HT1080 tumors along with graph showing microvessel density. Scale bar = 100 mm. (b) Overall apoptosis in HT1080 tumor groups. (c)Photos of coimmunofluorescence for CD31 and TUNEL along with the graph of endothelial cell-specific apoptosis. Scale bar = 7 mm. Arrows point to TUNEL-positive and CD31-positive cells. Bars represent standard deviation. *p ≤ 0.05 compared to control IgG group; **p < 0.05 compared to control IgG and DC101 groups.
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