ATF5 and HIF1α cooperatively activate HIF1 signaling pathway in esophageal cancer

Abstract

Background: Esophageal cancer (ESCA) is one of the most common cancers worldwide and has a very poor prognosis. Hypoxia-inducible factor 1 (HIF1) signaling pathway plays a critical role in tumorigenesis and is therefore considered a potential therapeutic target in the treatment of many cancers. Activating transcription factor 5 (ATF5) facilitates the expression of various genes and has been extensively studied for its potential role in cancer treatment.

Methods: The expression level of ATF5 in clinic sample was detected by quantitative real time PCR and immunohistochemistry. ATF5 biological function was investigated by western blot, cell cycle analysis, cell viability assay, luciferase reporter assays, colony formation assay, transwell assay, wound healing assay, tube formation assay, and ELISA assay. CHIP and Re-CHIP assay, GST-pulldown, and RNA-sequencing were used to study the cross-talks between ATF5 and HIF1 complex. Mouse xenograft study was utilized to study the correlation of ATF5 and tumor growth in vivo. Student's t-test or Chi-square test was used for statistical analysis.

Results: Here, we first found ATF5 was dramatically upregulated in ESCA cancer and related with poor survival time. Next, we found that the expression level of ATF5 had a positive relationship with the proliferation, migration, and invasion ability of ESCA cells. Besides, we innovatively found that ATF5 functions as a novel coactivator in HIF1 transcription complex by binding to HIF1α. Further, we demonstrated that silencing ATF5 phenocopies HIF1α knockdown in tumorigenic properties in vitro and inhibited ESCA tumor angiogenesis and proliferation in vivo.

Conclusion: Herein, we found ATF5 as a novel component of the HIF1 transcription complex. The findings of the present study may provide new insights into the development of a novel and more efficient therapeutic strategy against ESCA. Video abstract.

Keywords: ATF5; Esophageal cancer; HIF1.

Fig. 1

ATF5 is upregulated in ESCA. a ATF5 expression level in normal and ESCA tissues via the Oncomine database. b–c Kaplan–Meier plot of the overall survival via the GEPIA database (b) and TCGA database (c) Patients with ESCA were classified by ATF5 expression level. d mRNA level of ATF5 in 16 paired ESCA tissues. e–g Analysis of ATF5 expression level in paired ESCA tissues via IHC; Scale bar 100 μm; *p < 0.01 (f) and *p < 0.01 (g)

Fig. 2

ATF5 overexpression promotes the proliferation, migration, and invasion ability of ESCA cells. a Analysis of ATF5 expression level in vector versus ATF5 cells by western blot. b Analysis of cell cycle in vector versus ATF5 cells by PI assay. c Analysis of cell proliferation ability in vector versus ATF5 cells by CCK8 assay; *p < 0.01. d Analysis of tumorigenicity in vector versus ATF5 cells by colony formation assay; *p < 0.01. e The migration ability of identified cells stably expressing the empty vector or ATF5 was analyzed through a wound healing assay; Scale bar 100 μm; p < 0.01. f Analysis of invasion and migration ability in vector versus ATF5 cells by Transwell assay; Scale bar 100 μm (left) or 50 μm (right)

Fig. 3

Silencing of ATF5 inhibits the proliferation, migration, and invasion ability of ESCA cells. a Analysis of ATF5 expression level in shnc versus shATF5 cells by western blot. b Analysis of cell cycle in shnc versus shATF5 cells by PI assay. c Analysis of cell proliferation ability in shnc versus shATF5 cell by CCK8 assay; *p < 0.01. d Analysis of tumorigenicity in shnc versus shATF5 cells by colony formation assay; *p < 0.01. e Analysis of invasion and migration ability of shnc versus shATF5 cells by Transwell assay; Scale bar 100 μm(up) or 50 μm(down). f The migration ability of identified cells stably expressing shnc and shATF5 was analyzed through a wound healing assay; Scale bar 100 μm; p < 0.01

Fig. 4

The novel role of ATF5 in the HIF1 signaling pathway. a Detection of the expression level of identified proteins in shATF5 versus shnc cells by western blot in hypoxia. b Detection of the expression level of identified mRNAs in shATF5 versus shnc cells by RT-PCR; p < 0.01 in hypoxia. c Evaluating the secretion of VEGFA in the identified cells by ELISA; p < 0.01 in hypoxia. d Dual-luciferase assays were performed to detect the luciferase activation of identified genes in ESCA cells transfected with shnc and shKDM4C; p < 0.01 in hypoxia. e Investigating the tube formation ability of HUVECs induced by supernatant from medium of identified cells in hypoxia; Scale bar 100 μm; p < 0.01

Fig. 5

ATF5 acts as a novel coactivator in the HIF1 transcription complex by binding to HIF1α. a, b Examining the interaction between ATF5 and HIF1 target gene promoters by CHIP in hypoxia. c, d Investigating the interaction between HIF1 transcriptional complex and VEGFA promoter in shnc and shATF5 cells by CHIP in hypoxia; p < 0.01. e Investigating the interaction between ATF5 and HIF1 transcriptional complex on endogenous VEGFA promoters by ChIP/Re-ChIP in hypoxia. f Whole-cell extracts of KYSE30 cells were collected for IP analysis using the indicated antibodies, followed by IB analysis in hypoxia. g Investigating which protein of HIF1 transcriptional complex directly binding to ATF5 by GST pull down. h Whole-cell extracts of shATF5 KYSE30 cells were collected for IP analysis using the indicated antibodies, followed by IB analysis in hypoxia

Fig. 6

Inhibition ATF5 phenocopies HIF1α knockdown in tumorigenic properties in vitro. a The gene sequence of a dominant-negative ATF5 (D/N-ATF5). b Western blot showing the expression levels of the identified proteins in hypoxia. c Dual-luciferase assay showing the activation ability of identified gene promoters in identified cells in hypoxia; p < 0.01. d CCK8 assay showing the proliferation ability of identified cells in hypoxia; p < 0.01. e Transwell assay showing the invasion and migration ability of the identified cells in hypoxia; Scale bar 100 μm. f Tube formation ability of HUVECs induced by supernatant from medium of identified the cells in hypoxia; Scale bar 100 μm; p < 0.01

Fig. 7

Silencing of ATF5 inhibited tumor angiogenesis and tumor proliferation in vivo. a The tumor growth curve among the four groups; p < 0.01. b, c Representative tumor images among the four groups (b), the right graph is showing the weight of the tumor (c); p < 0.01. d, e. Representative IHC staining of the identified protein among the four groups; the right graph showing the micro-vessel density of the tumors in the groups; Scale bar 100 μm. f Western blot assay showing the expression levels of the identified proteins in xenograft tumors. g Proposed model: ATF5/HIF1α interaction enhancing the binding of the HIF1 transcriptional complex on the promoters of the HIF1 target genes