STAMBPL1 upregulates HIF1α in a non-enzymatic manner in TNBC cells.

(A) After STAMBPL1 was knocked down in HCC1806 and HCC1937 cells and then subjected to hypoxia for 10 h, STAMBPL1 knockdown downregulated the protein level of HIF1α. (B-C) RNA samples were collected after 10 h of hypoxia treatment following the knockdown of STAMBPL1 in HCC1806 cells. RT‒qPCR experiments revealed that the knockdown of STAMBPL1 downregulated the mRNA levels of the HIF1α downstream targets VEGFA and GLUT1. (D-E) RNA samples were collected after 10 hours of hypoxia treatment following the knockdown of STAMBPL1 in HCC1937 cells. RT‒qPCR experiments revealed that the knockdown of STAMBPL1 downregulated the mRNA levels of the HIF1α downstream targets VEGFA and GLUT1. (F) Overexpression of STAMBPL1/STAMBPL1-E292A upregulated HIF1α protein expression in HCC1806 and HCC1937 cells stably overexpressing STAMBPL1/STAMBPL1-E292A under normoxia. *: 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 revealed that the knockdown of STAMBPL1 could downregulate the mRNA levels of HIF1α. (C) RNA samples were collected after 10 hours of hypoxia treatment following the knockdown of STAMBPL1 in HCC1937 cells. RT‒qPCR experiments revealed that the knockdown of STAMBPL1 downregulated the mRNA levels of HIF1α. (D) HCC1806 cells stably overexpressing STAMBPL1 were used to knock down HIF1α via siRNA. The protein was then collected and subjected to Western blotting experiments. The results showed that the overexpression of STAMBPL1 promoted the expression of HIF1α, which could be rescued by the knockdown of HIF1α. (E) HIF1α was knocked down in HCC1806 cells stably overexpressing STAMBPL1 via siRNA. The RNA was then collected and subjected to RT‒ qPCR experiments. The results showed that the overexpression of STAMBPL1 promoted the expression of the VEGFA mRNA, which could be rescued by the knockdown of HIF1α. (F) In HCC1937 cells stably overexpressing STAMBPL1, HIF1α was knocked down via siRNA, and protein was collected. Western blotting experiments revealed that the overexpression of STAMBPL1 promoted HIF1α expression, which could be rescued by the knockdown of HIF1α. (G) In HCC1937 cells stably overexpressing STAMBPL1, HIF1α was knocked down via siRNA, and RNA was collected. RT‒qPCR experiments revealed that the overexpression of STAMBPL1 promoted VEGFA mRNA expression, which could be rescued by the knockdown of HIF1α. *: 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) In HCC1806 cells, STAMBPL1 was knocked down via siRNA, and the cells were then subjected to hypoxia for 24 hours. The conditioned medium (CM) collected from these cells was used to treat HUVECs. EdU assays revealed that the conditioned medium after STAMBPL1 knockdown inhibited the proliferation of HUVECs. (B) Statistical analysis of the EdU assay results. (C) In HCC1806 cells, STAMBPL1 was knocked down via siRNA, and the cells were then subjected to hypoxia for 24 hours. The CM collected from these cells was used to treat HUVECs. Wound healing assays demonstrated that the conditioned medium after STAMBPL1 knockdown inhibited the migration of HUVECs. (D) Statistical analysis of the wound healing assay results. (E) In HCC1806 cells, STAMBPL1 was knocked down via siRNA, and the cells were then subjected to hypoxia for 24 hours. The CM collected from these cells was used to treat HUVECs. A tube formation assay revealed that the conditioned medium after STAMBPL1 knockdown inhibited 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. Knockdown of STAMBPL1 significantly inhibited tumor growth. (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 experiments demonstrated that the knockdown of STAMBPL1 resulted in decreased 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 experiments revealed that the overexpression of STAMBPL1 promoted the expression of HIF1α, which could be rescued by the knockdown of GRHL3. (D-F) In HCC1806 cells stably overexpressing STAMBPL1, GRHL3 was knocked down via siRNA, and RNA was subsequently collected. RT‒qPCR experiments revealed that the overexpression of STAMBPL1 upregulated the mRNA expression of GRHL3, HIF1α and its downstream target VEGFA, which could be rescued by the knockdown of GRHL3. (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) Overexpression of GRHL3 in HCC1806 cells stably overexpressing GRHL3 resulted in increased protein expression of HIF1α. (D) ChIP‒PCR experiments conducted in HCC1806 cells stably overexpressing GRHL3 revealed that GRHL3 could bind to the promoter region of the target gene HIF1A. (E) A luciferase assay performed in HEK293T cells indicated that GRHL3 could bind to the promoter sequence of HIF1A and increase its transcriptional activity. (F) In HCC1806 cells, GRHL3 was knocked down by siRNA, and the cells were subjected to hypoxia for 4 hours. Western blotting experiments revealed that the knockdown of GRHL3 downregulated the protein level of HIF1α. (G-I) In HCC1806 cells, GRHL3 was knocked down by siRNA, followed by hypoxia treatment for 4 hours, after which RNA samples were collected. RT‒qPCR experiments revealed that GRHL3 knockdown was effective and that the knockdown of GRHL3 could downregulate 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 revealed that the conditioned medium after GRHL3 knockdown inhibited the proliferation of HUVECs. Statistical analysis of the EdU assay results was performed. (K) 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. Wound healing assays revealed that the conditioned medium after GRHL3 knockdown inhibited the migration of HUVECs. Statistical analysis of the wound healing assay results was performed. (L) 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. The results of the tube formation assay revealed that conditioned medium from GRHL3-knockdown HUVECs inhibited tube formation. 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 experiments revealed that the overexpression of GRHL3 promoted the expression of HIF1α, which could be rescued by the knockdown of HIF1α. (B) In HCC1806 cells stably overexpressing GRHL3, HIF1α was knocked down by siRNA, and then the RNA was collected. RT‒qPCR experiments revealed that the overexpression of GRHL3 promoted the expression of the VEGFA mRNA, which could be rescued by the knockdown of HIF1α. (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 assays revealed that the overexpression of GRHL3 promoted the proliferation of HUVECs, which could be rescued by the knockdown of HIF1α. (D) 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. A wound healing assay revealed that the overexpression of GRHL3 promoted the migration of HUVECs, which could be rescued by the knockdown of HIF1α. (E) 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. The tube formation assay revealed that the overexpression of GRHL3 promoted the tube formation of HUVECs, which could be rescued by the knockdown of HIF1α. (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. The results showed that the knockdown of GRHL3 significantly inhibited tumor growth. (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 experiments revealed that the knockdown of FOXO1 in HCC1806 cells followed by hypoxia treatment for 4 hours inhibited HIF1α protein expression. (B-D) RT‒ qPCR experiments revealed that the knockdown of FOXO1 in HCC1806 cells followed by hypoxia treatment for 4 hours decreased the mRNA levels of GRHL3/HIF1α/VEGFA. (E) In HCC1806 cells stably overexpressing STAMBPL1, protein samples were collected after FOXO1 was knocked down via siRNA. Western blotting experiments revealed that the overexpression of STAMBPL1 promoted the expression of HIF1α, which could be rescued by the knockdown of FOXO1. (F-H) In HCC1806 cells stably overexpressing STAMBPL1, RNA samples were collected after the knockdown of FOXO1 via siRNA. RT‒qPCR experiments revealed that the overexpression of STAMBPL1 promoted the mRNA expression of GRHL3, HIF1α and VEGFA, which could be rescued by the knockdown of FOXO1. (I) An endogenous ChIP‒PCR assay was performed using an anti-FOXO1 antibody in HCC1806 cells. (J) A luciferase assay performed in HEK293T cells revealed that FOXO1 was able to bind to the promoter sequence of GRHL3 and increase its transcriptional activity. (K) A luciferase assay conducted in HEK293T cells revealed that STAMBPL1 enhanced 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 cotransfected 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) TCGA database analysis revealed high expression levels of STAMBPL1, FOXO1, and GRHL3 in TNBC. (D) The effect of STAMBPL1 overexpression in HCC1806 cells was detected by Western blotting. (E) The effect of drug treatment on transplanted tumors in nude mice was evaluated by photographing the tumors. The overexpression of STAMBPL1 promoted tumor growth, whereas the combination of the FOXO1 inhibitor AS1842856 (10 mg/kg, every two days) and the VEGFR inhibitor apatinib (50 mg/kg, every two days) significantly inhibited tumor growth. (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.