Repression of rRNA gene transcription by endothelial SPEN deficiency normalizes tumor vasculature via nucleolar stress
Zi-Yan Yang, … , Tian Xiao, Hua Han
Zi-Yan Yang, … , Tian Xiao, Hua Han
Published August 22, 2023
Citation Information: J Clin Invest. 2023;133(20):e159860. https://doi.org/10.1172/JCI159860.
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Research Article Vascular biology

Repression of rRNA gene transcription by endothelial SPEN deficiency normalizes tumor vasculature via nucleolar stress

Abstract

Human cancers induce a chaotic, dysfunctional vasculature that promotes tumor growth and blunts most current therapies; however, the mechanisms underlying the induction of a dysfunctional vasculature have been unclear. Here, we show that split end (SPEN), a transcription repressor, coordinates rRNA synthesis in endothelial cells (ECs) and is required for physiological and tumor angiogenesis. SPEN deficiency attenuated EC proliferation and blunted retinal angiogenesis, which was attributed to p53 activation. Furthermore, SPEN knockdown activated p53 by upregulating noncoding promoter RNA (pRNA), which represses rRNA transcription and triggers p53-mediated nucleolar stress. In human cancer biopsies, a low endothelial SPEN level correlated with extended overall survival. In mice, endothelial SPEN deficiency compromised rRNA expression and repressed tumor growth and metastasis by normalizing tumor vessels, and this was abrogated by p53 haploinsufficiency. rRNA gene transcription is driven by RNA polymerase I (RNPI). We found that CX-5461, an RNPI inhibitor, recapitulated the effect of Spen ablation on tumor vessel normalization and combining CX-5461 with cisplatin substantially improved the efficacy of treating tumors in mice. Together, these results demonstrate that SPEN is required for angiogenesis by repressing pRNA to enable rRNA gene transcription and ribosomal biogenesis and that RNPI represents a target for tumor vessel normalization therapy of cancer.

Authors

Zi-Yan Yang, Xian-Chun Yan, Jia-Yu-Lin Zhang, Liang Liang, Chun-Chen Gao, Pei-Ran Zhang, Yuan Liu, Jia-Xing Sun, Bai Ruan, Juan-Li Duan, Ruo-Nan Wang, Xing-Xing Feng, Bo Che, Tian Xiao, Hua Han

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Figure 1

Endothelial SPEN deficiency represses EC proliferation and blunts angiogenesis.

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Endothelial SPEN deficiency represses EC proliferation and blunts angiog...
(A–C) HUVECs were transduced with NC or SPENi lentivirus expressing EGFP. Cell proliferation was determined by (A) EdU incorporation (n = 4) and (B) cell cycle analysis (n = 3). (C) ECs were recorded with a living-cell imaging workstation (Supplemental Figure 1F and Supplemental Videos, 1 and 2), and the relative track speeds of cells (n = 35 and 21 cell tracks for NC and SPENi, respectively) and cell perimeters (n = 6) were compared. Scale bar: 100 μm. (D) HUVECs were transduced with NC or SPENi lentivirus and subjected to RNA-Seq (n = 4). Cell cycle–related gene sets were analyzed by GSEA (color-coded gene sets are listed in Supplemental Figure 1H). (E and F) HUVECs were transduced with NC or SPENi lentivirus. The expression of angiogenesis-related genes was determined by (E) RT-qPCR (n = 4) and (F) immunoblotting (n = 5, except for n = 6 for ANGPT blots). β-Actin served as the loading control). (G) Sprouting was assessed by the microbead sprouting assay and quantitatively compared (n = 30 beads from 3 biological replicates). Scale bar: 100 μm. (H) The retinal vasculature of P6 pups was stained with anti-CD31 and photographed. The middle and bottom rows of images show the remodeling zone and angiogenic frontier of retinas, respectively. A, artery; V, vein; white arrows, vessel loops; yellow arrows, sprouts; yellow dashed lines, vascular radius. Scale bars: 100 μm. The EC area (n = 6), branch number (n = 6), and distal sprouts (n = 6) were quantified. (I) Immunostaining of mouse retinas after EdU labeling. EdU+ ECs were compared (n = 5). Scale bar: 100 μm. Data represent mean ± SEM. Unpaired 2-tailed Student’s t test was used.