Cancer Biology

STAMBPL1 activates the GRHL3/HIF1A/VEGFA axis through interaction with FOXO1 to promote angiogenesis in triple-negative breast cancer

Huan Fang
Huichun Liang
Chuanyu Yang
Dewei Jiang
Qianmei Luo
Wenming Cao author has email address
Huifeng Zhang author has email address
Ceshi Chen author has email address

Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, China
Kunming College of Life Sciences, University of Chinese Academy of Sciences, Kunming, China
The Department of Breast Medical Oncology, Zhejiang Cancer Hospital, Hangzhou, China
Department of Pharmacy, the First People’s Hospital of Yunnan Province/the Affiliated Hospital of Kunming University of Science and Technology, Kunming, China
Academy of Biomedical Engineering, Kunming Medical University, Kunming, China
The Third Affiliated Hospital, Kunming Medical University, Kunming, China

https://doi.org/10.7554/eLife.102433.2

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Figures and data

STAMBPL1 upregulates HIF1α in a non-enzymatic manner in TNBC cells.
(A) Western blot detected the protein levels of HIF1α when STAMBPL1 was knocked down under normoxia and hypoxia for 10 hours. (B-C) RNA samples were collected after 10 h of hypoxia treatment following the knockdown of STAMBPL1 in HCC1806 cells. RT‒qPCR experiments were performed to detect the mRNA levels of the HIF1α downstream targets VEGFA and GLUT1. (D-E) RNA samples of HCC1937 were collected as the above HCC1806 cells. RT‒ qPCR experiments were performed to detect the mRNA levels of the HIF1α downstream targets VEGFA and GLUT1. (F) Western blot detected the protein levels of HIF1α when STAMBPL1 /STAMBPL1-E292A was overexpressed under normoxia in HCC1806 and HCC1937 cells. *: P<0.05, **: P<0.01, ***: P<0.001, t test.

STAMBPL1 promotes HIF1A transcription and activates the HIF1α/VEGFA axis.
(A) Western blot analysis was performed to confirm the knockdown of STAMBPL1 in HCC1806 and HCC1937 cells. (B) RNA samples were collected after 10 h of hypoxia treatment following the knockdown of STAMBPL1 in HCC1806 cells. RT‒qPCR experiments were performed to detect the mRNA levels of HIF1α. (C) RNA samples of HCC1937 cells were collected as the above HCC1806 cells. RT‒qPCR experiments were performed to detect the mRNA levels of HIF1α. (D) Knocking down HIF1α using siRNAs in HCC1806 cells stably overexpressing STAMBPL1 under normoxia. Western blot was used to detect the protein levels of STAMBPL1 and HIF1α. (E) RT‒qPCR experiments were performed to detect the effect of HIF1α knockdown on the mRNA level of VEGFA mRNA in HCC1806 cells with STAMBPL1 overexpression under normoxia. (F) Knocking down HIF1α using siRNAs in HCC1937 cells stably overexpressing STAMBPL1 under normoxia. Western blot was used to detect the protein levels of STAMBPL1 and HIF1α. (G) RT‒qPCR experiments were performed to detect the effect of HIF1α knockdown on the mRNA level of VEGFA mRNA in HCC1937 cells with STAMBPL1 overexpression under normoxia. *: P<0.05, **: P<0.01, ***: P<0.001, t-test.

STAMBPL1 in TNBC cells enhances the activity of HUVECs and promotes TNBC angiogenesis.
(A) EDU assay was performed to detect the effect of HCC1806 conditioned medium (CM) with STAMBPL1 knockdown under normoxia or hypoxia on the proliferation of HUVECs. (B) Statistical analysis of the EdU assay results. (C) Wound healing assay was performed to detect the effect of HCC1806 CM with STAMBPL1 knockdown under normoxia or hypoxia on the migration of HUVECs. (D) Statistical analysis of the wound healing assay results.
(E) Tube formation assay was performed to detect the effect of HCC1806 CM with STAMBPL1 knockdown under normoxia or hypoxia on the tube formation of HUVECs. (F) Statistical analysis of the tube formation assay results. (G) The growth of TNBC xenograft tumors was evaluated by photographing the tumors in nude mice to assess the role of STAMBPL1. (H-I) The growth and weight of the transplanted tumors in the nude mice were statistically analyzed, and the STAMBPL1sh#1 and STAMBPL1sh#2 groups were compared with the shLuc group.
(J) An immunohistochemical assay was used to detect the expression of the angiogenesis marker CD31 in xenograft tumors. (K) The number of microvessels in the immunohistochemical experiments was statistically analyzed. *: P<0.05, **: P<0.01, ***: P<0.001, t-test.

STAMBPL1 promotes HIF1A transcription via the upregulation of GRHL3.
(A) Venn diagram showing the overlap of differentially expressed genes identified via RNA-seq analysis. (B) RT‒qPCR experiment was performed to detect the effect of STAMBPL1 knockdown on the mRNA levels of GRHL3 in HCC1806 cells. (C) In HCC1806 cells stably overexpressing STAMBPL1, GRHL3 was knocked down via siRNA, after which the protein was collected. Western blotting was performed to detect the effect of GRHL3 knockdown on the expression of HIF1α as STAMBPL1 overexpression under normoxia. (D-F) RT‒qPCR experiments were performed to detect the knockdown efficiency of GRHL3 and the effect of GRHL3 knockdown on the mRNA levels of HIF1α and its downstream target VEGFA when STAMBPL1 was overexpressing. (G) Statistical analysis of the EdU assay results. (H) Statistical analysis of the wound healing assay results. (I) Statistical analysis of the tube formation assay results. *: P<0.05, **: P<0.01, ***: P<0.001, t-test.

GRHL3 enhances HIF1A transcription by binding to its promoter.
(A) The JASPAR website was used to predict the potential binding sequence of the transcription factor GRHL3 to the HIF1α promoter. (B) Mutation pattern of the HIF1α promoter binding sequence.
(C) The protein level of HIF1α was detected in HCC1806 cells with GRHL3 overexpression by using Western blot. (D) ChIP‒PCR experiments conducted in HCC1806 cells stably overexpressing GRHL3 to detect the interaction between GRHL3 and the promoter of HIF1A gene. (E) A luciferase assay was performed in HEK293T cells to detect the effect of GRHL3 on the transcriptional activity of HIF1A promoter. (F) In HCC1806 cells, GRHL3 was knocked down by siRNA, and the cells were subjected to hypoxia for 4 hours. Western blotting was performed to detect the effect of GRHL3 knockdown on the protein level of HIF1α. (G-I) RT‒qPCR experiments were used to detect the effect of GRHL3 knockdown on the mRNA levels of HIF1α and its downstream target VEGFA. (J) In HCC1806 cells, GRHL3 was knocked down via siRNA, and the cells were then subjected to hypoxia for 24 hours. The conditioned medium was collected and used to treat HUVECs. EdU assays were performed to detect the effect of GRHL3 knockdown CM on the proliferation of HUVECs. Statistical analysis of the EdU assay results was performed. (K) Wound healing assays were performed to detect the effect of GRHL3 knockdown CM on the migration of HUVECs. Statistical analysis of the wound healing assay results was performed. (L) The tube formation assay was performed to detect the effect of GRHL3 knockdown CM on the tube formation of HUVECs. Statistical analysis of the tube formation assay results was performed. *: P<0.05, **: P<0.01, ***: P<0.001, t-test.

GRHL3 enhances HIF1A transcription by binding to its promoter.
(A) In HCC1806 cells stably overexpressing GRHL3, HIF1α was knocked down by siRNA, and the protein was subsequently collected. Western blotting was performed to detect the protein level of GRHL3-Flag and HIF1α. (B) RT‒qPCR experiments were performed to detect the effect of HIF1α knockdown on the mRNA level of VEGFA as GRHL3 overexpression. (C) HCC1806 cells stably overexpressing GRHL3 were used for the knockdown of HIF1α via siRNA. The conditioned medium was then collected and used to treat HUVECs. EdU assay was performed to detect the effect of CM with HIF1α knockdown on the proliferation of HUVECs as GRHL3 overexpression. (D) Wound healing assay was performed to detect the effect of CM with HIF1α knockdown on the migration of HUVECs as GRHL3 overexpression. (E) The tube formation assay was performed to detect the effect of CM with HIF1α knockdown on the tube formation of HUVECs as GRHL3 overexpression. (F) RT‒qPCR was used to detect the knockdown of GRHL3 in HCC1806 cells. (G) Photographs of xenograft tumors from nude mice were taken to evaluate the role of GRHL3 in the growth of TNBC xenograft tumors. (H) The weights of the transplanted tumors from the nude mice were statistically analyzed, and the shGRHL3 1# and shGRHL3 2# groups were compared with the shLuc group. (I) The growth of transplanted tumors in nude mice was statistically analyzed, and the shGRHL3 1# and shGRHL3 2# groups were compared with the shLuc group. (J) An immunohistochemical assay was performed to detect the expression of the angiogenesis marker CD31 in xenograft tumors. (K) Statistical analysis of the number of microvessels in the immunohistochemical experiments. *: P<0.05, **: P<0.01, ***: P<0.001, t-test.

STAMBPL1 mediates GRHL3 transcription by interacting with FOXO1.
(A) Western blotting was used to detect the HIF1α protein expression when FOXO1 was knocking down in HCC1806 cells followed by hypoxia treatment for 4 hours. (B-D) RT‒qPCR experiments were performed to detect the effect of FOXO1 knockdown on the mRNA levels of GRHL3/HIF1α/VEGFA in HCC1806 cells under hypoxia for 4 hours. (E) In HCC1806 cells stably overexpressing STAMBPL1, protein samples were collected after FOXO1 was knocked down via siRNA. Western blotting was performed to detect the effect of FOXO1 knockdown on the expression of HIF1α as STAMBPL1 overexpression. (F-H) RT‒qPCR experiments were performed to detect the effect of FOXO1 knockdown on the mRNA expression of GRHL3, HIF1α and VEGFA as STAMBPL1 overexpression. (I) An endogenous ChIP‒PCR assay was performed using an anti-FOXO1 antibody in HCC1806 cells. (J) A luciferase assay was performed in HEK293T cells to detect the effect of FOXO1 on the transcriptional activity of GRHL3 promoter. (K) A luciferase assay was conducted in HEK293T cells to detected the effect of STAMBPL1 on the activation of the GRHL3 promoter by FOXO1. (L) ChIP‒PCR experiments were performed in HCC1806 cells stably overexpressing STAMBPL1 after knocking down FOXO1 via siRNA. (M) PCDH-STAMBPL1-3×Flag and PCDH-FOXO1-3×Flag plasmids were co-transfected into HEK293T cells, and then, immunofluorescence experiments were performed. Red represents STAMBPL1 staining, green represents FOXO1 staining, and blue represents DAPI staining. (N) Endogenous STAMBPL1 protein was immunoprecipitated from HCC1937 cells via an anti-FOXO1 antibody. Immunoglobulin G (IgG) served as the negative control. Endogenous STAMBPL1 was detected via western blotting. *: P<0.05, **: P<0.01, ***: P<0.001, t-test.

The combination of VEGFR and FOXO1 inhibitors synergistically suppresses TNBC xenograft growth. (A-C)
Metabric database analysis in BCIP revealed high expression levels of STAMBPL1, FOXO1, and GRHL3 in TNBC (n = 319) compared with non-TNBC (n = 1661). (D) The effect of STAMBPL1 overexpression in HCC1806 cells was detected by Western blotting. (E) Nude mice with tumors received the FOXO1 inhibitor AS1842856 (10 mg/kg, every treatments. The effect of the drug treatments on the transplanted tumors was assessed by imaging the tumors. (F-G) The weight and growth of the transplanted tumors in the nude mice were statistically analyzed. The vector control group and the STAMBPL overexpression group were compared. The nondrug group and the combined drug group in the vector control group were compared. The nondrug group and the combined drug group in the STAMBPL overexpression group were compared. (H) The final weights of the nude mice were statistically analyzed. *: P<0.05, **: P<0.01, ***: P<0.001, t-test.

The working model of this study.
STAMBPL1 interacts with FOXO1 to promote TNBC angiogenesis by activating the GRHL3/HIF1α/VEGFA axis.

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