STAMBPL1 promotes triple-negative breast cancer angiogenesis by upregulating the transcription of GRHL3/HIF1α/VEGFA via FOXO1

doi:10.21203/rs.3.rs-4274439/v1

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STAMBPL1 promotes triple-negative breast cancer angiogenesis by upregulating the transcription of GRHL3/HIF1α/VEGFA via FOXO1

https://doi.org/10.21203/rs.3.rs-4274439/v1

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Anti-angiogenesis is a crucial therapeutic strategy for triple-negative breast cancer (TNBC), but the current targeted drugs are insufficient to meet clinical requirements. Our study has discovered that silencing the deubiquitinating enzyme STAMBPL1 can effectively inhibit the growth and angiogenesis of TNBC xenografts in nude mice. STAMBPL1 promotes the expression of HIF1α/VEGFA in TNBC through a non-enzymatic-dependent mechanism. STAMBPL1 interacts with the transcription factor FOXO1, which binds to the promoter of the GRHL3 gene, thereby positively regulating its transcription. Subsequently, GRHL3 binds to the HIF1α gene promoter to promote its transcription and angiogenesis. Moreover, we have demonstrated that the combination of FOXO1 inhibitor AS1842856 and VEGFR inhibitor Apatinib significantly inhibited the growth of transplanted tumors in nude mice. These findings indicate that the STAMBPL1/FOXO1/GRHL3/HIF1α/VEGFA axis provides potential therapeutic targets in TNBC.

Biological sciences/Cancer/Breast cancer

Biological sciences/Molecular biology/Transcription

STAMBPL1

FOXO1

GRHL3

HIF1α

VEGFA

Angiogenesis

Triple-negative breast cancer

Breast cancer is the most commonly diagnosed cancer and the leading cause of cancer-related deaths in women^[1]. Although the diagnosis and treatment of breast cancer has entered the era of molecular typing, there are still 35% of recurrence and metastasis due to treatment failure^[2]. The expression of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor (HER2) provides an effective target for endocrine and anti-HER2 treatment of breast cancer. In triple-negative breast cancer (TNBC), current studies have confirmed that anti-angiogenesis therapy, combined chemotherapy or immunotherapy have high clinical efficacy^[3].

Angiogenesis is a physiological process of forming new blood vessels on the basis of existing blood vessels^[4]. Angiogenesis plays an important role in growth, development, wound healing, and granulation tissue formation. In addition, angiogenesis also plays a crucial role in tumor growth, progression, and metastasis. The rapid growth of solid tumors requires blood to provide sufficient oxygen and nutrition, so the growth of solid tumors is usually accompanied by angiogenesis. Generally, when the diameter of the tumor is 1–2 mm, the tumor cells can obtain oxygen and nutrition from the surrounding matrix by passive diffusion. However, if there is no blood to continue to provide sufficient oxygen and nutrition for its division and growth, the growth of the tumor will be in a state of stagnation, and its diameter generally cannot exceed 2–3 mm, this is mainly because hypoxia leads to the death of cancer cells^[5]. In order to cope with hypoxic stress, cancer cells can promote the formation of new blood vessels in the normal tissues around the tumor by secreting angiogenesis activating factors such as VEGFA. Therefore, the study of angiogenesis in TNBC will help to prevent neovascularization from infiltrating into tumor tissues by blocking the sprouting of neovascularization in the early stage of tumor, so as to achieve the purpose of inhibiting tumor growth. In TNBC, inhibiting angiogenesis can also enhance the effectiveness of immunotherapy. It has been reported that angiogenesis is a key discriminative feature of patients with low CD8⁺ T cell infiltration, and its inhibitor could facilitate checkpoint blockade^[6]. Abnormal tumor vessels contribute significantly to the malignant characteristics of tumors, their ability to metastasize, evade the immune system, and resist therapy. Therefore, strategies aimed at “normalizing” these vessels in tumors are appealing, as they can improve vessel structure and function, delay tumor progression, and enhance the delivery and efficacy of cytotoxic therapies^[7].

The full name of STAMBPL1 is STAM binding protein-like1, also known as AMSH-LP or AMSH-FP. It is a member of the AMSH (Associated molecule with the SH3 domain of STAM) family and is widely expressed in various human tissues^[8]. STAMBPL1 protein contains nuclear localization signal (NLS), MPN (Mprl/Padl N-terminal) and JAMM domains, and belongs to the JAMM deubiquitinating enzyme family^[9]. Studies have shown that STAMBPL1 deubiquitinates the K63 chains of the early endosomal Clathrin, and STAMBPL1 lacking clathrin-binding ability does not localize to endosomes^[10]. Further studies showed that STAMBPL1 is highly expressed in gastric cancer and promotes the occurrence and development of gastric cancer^[11], and long non-coding RNA NEAT1 promotes the proliferation and migration of gastric cancer cells by up-regulating STAMBPL1^[12]. Targeting STAMBPL1 has been shown to promote the degradation of XIAP and trigger apoptosis in prostate cancer cells, suggesting that STAMBPL1 may be a potential therapeutic target for prostate cancer^[13]. Our previous studies have confirmed that STAMBPL1 promotes cisplatin resistance in TNBC cells by deubiquitinating and stabilizing phosphatase MKP-1 expression^[14]. However, the roles and mechanisms of STAMBPL1 in breast cancer angiogenesis have not been investigated.

FOXO1 belongs to the forkhead box transcription factor family. FOXO1 is an important homeostasis regulator in the process of organ response to stress^[15]. Its transcriptional activity is regulated by post-translational modification, including phosphorylation, acetylation, ubiquitination, methylation and O-glycosylation, which in turn affects its subcellular localization, stability and activity. In addition, multiple genes regulated by FOXO1 are associated with stem cell renewal, migration, invasion, differentiation, and oxidative stress, and are involved in life processes such as apoptosis, DNA damage/repair, tumorigenesis, angiogenesis, and glucose metabolism^[16]. Studies have shown that FOXO1 regulates the expression of VEGF in keratinocytes, and FOXO1 is necessary for angiogenesis in wound healing. FOXO1 also regulates CD36 transcription, endothelial cell differentiation and vascular maturation. Under physiological and pathological conditions, FOXO1 may play an important role in the formation of functional vascular networks by coupling the regulation of vascular endothelial cell CD36^[17]. At present, the role of FOXO1 in tumor progression remains controversial. On the one hand, most studies have shown that FOXO1 is a potential tumor suppressor involved in regulating the differentiation of a variety of cells and inhibiting tumor cell proliferation^[18]. On the other hand, FOXO1 contributes to maintain leukemia-initiating cells in acute and chronic myeloid leukemia, promotes the invasion and metastasis of colon cancer and breast cancer^[19]. Studies have shown that TRIB3 (Tribblepseudokinase 3) supports breast cancer stemness by suppressing FOXO1 degradation and enhancing SOX2 transcription^[20]. Our previous studies have also found that DNA damage chemotherapeutic drugs suppress basal-like breast cancer growth by down-regulating the transcription of the FOXO1-KLF5 axis^[21]. In addition, it has been reported that the FOXO1 inhibitor AS1842856 (a cell-permeable small molecule that directly binds to unphosphorylated FOXO1 protein to block its transcriptional activity) triggers apoptosis in basal-like breast cancer cells^[22], and the FOXO1 inhibitor AS1842856 alone or combined with Onapristone (PR antagonist), blunted phosphorylated PR, and tumorsphere formation in PR-A⁺ (T47D) and PR-B⁺ (MCF7 and BT474) cells^[23].

The Grainyhead gene encodes a highly conserved transcription factor family that was originally discovered by a mutant of Drosophila, which failed to form a complete neural tube closure process. Abnormal head granulosa cells lead to a granular head phenotype, which is also the reason for the naming of the family^[24]. GRHL1, GRHL2 and GRHL3 were found to be homologous to Drosophila GRH^{[25, 26]}. GRHL transcription factor is a part of the LSF/GRH transcription factor family, which contains two branches, the GRH subfamily composed of GRHL1, GRHL2 and GRHL3 and the LSF/CP2 subfamily composed of TFCP2, LBP1a and LBP-9 transcription factors^[27]. GRHL1-3 contains an N-terminal transcriptional activation domain, a central CP2 DNA binding domain with structural similarity to p53, and a C-terminal dimer domain with protein-like folding^{[28, 29]}. GRHL3 not only plays an important role in normal growth and development, but also has certain physiological significance in the development of cancer. Studies have shown that GRHL3 is expressed in human endothelial cells and is necessary for endothelial cell migration^[30]. In mammary epithelial cells, BRD4 maintains the epithelial phenotype of the basement by promoting the expression of p63 and GRHL3 ^[31]. Additionally, studies have shown that GRHL3 plays a tumor-promoting role in breast cancer^[32]. However, several studies have found that GRHL3 is highly expressed in the early stage of breast cancer, while the expression level in the late stage is significantly reduced, and GRHL3-positive breast cancer patients have a better prognosis^[33]. Therefore, the function and mechanism of GRHL3 in breast cancer are still unclear, and further research is required.

In this study, we demonstrated that STAMBPL1 interacts with FOXO1 to promote TNBC angiogenesis by upregulating the transcription of GRHL3/HIF1α/VEGFA. Our findings suggest that STAMBPL1 and FOXO1 might serve as promising therapeutic targets for TNBC.

STAMBPL1 promotes TNBC angiogenesis

To test whether STAMBPL1 promotes angiogenesis and tumor growth in vivo, we performed animal experiments in nude mice. HCC1806 cells with stable STAMBPL1 knockdown were orthotopically inoculated into the fat pad of 6-week-old female mice (n = 4). Western blotting was performed to detect the knockdown effect of STAMBPL1 protein in animal experiments (Figure S1A). We observed that depletion of STAMBPL1 significantly suppressed breast cancer cell growth in vivo, which is consistent with our previous report^[14]. STAMBPL1 significantly inhibited the tumor growth based on the tumor volume and the tumor weight (Fig. 1A-C). We also demonstrated that STAMBPL1 significantly decreased the microvessel numbers in the xenograft tumors, as assessed by CD31 immunohistochemistry (Fig. 1D-E).

To investigate whether STAMBPL1 promotes angiogenesis in vitro, we used siRNA to knock down STAMBPL1 in HCC1806 and HCC1937 cells. Following hypoxia treatment, we collected the conditioned medium and used it to treat HUVECs (Human umbilical vein endothelial cells). We then conducted DNA synthesis, wound healing, and tube formation assays in vitro. As a result, conditioned medium derived from either HCC1806 (Fig. 1F-K) or HCC1937 (Figure S1B-G) cells under hypoxia significantly promoted the proliferation, migration, and tube formation of HUVECs compared with conditioned medium under normoxia. Importantly, knockdown of STAMBPL1 significantly inhibited the proliferation, migration and tube formation of HUVECs. These results demonstrate that STAMBPL1 promotes TNBC angiogenesis in vitro and in vivo.

STAMBPL1 promotes TNBC angiogenesis by promoting HIF1α/VEGFA transcription

HIF1α is well known to play a key role in angiogenesis. We constructed stable STAMBPL1 and STAMBPL1-E292A (a mutant without deubiquitination enzyme activity) overexpression HCC1806 and HCC1937 cells, and found that overexpression of STAMBPL1 could promote the protein expression of HIF1α, in an enzyme independent manner (Fig. 2A). Additionally, knockdown of STAMBPL1 significantly decreased HIF1α protein expression levels under hypoxia treatment in both cell lines (Fig. 2B). Meanwhile, we also found that knockdown of STAMBPL1 significantly decreased HIF1α and VEGFA/GLUT1 (two well-known HIF1α downstream target genes) mRNA levels under hypoxia treatment in both cell lines (Figs. 2C-E and S2A-C). To investigate whether STAMBPL1 promotes TNBC angiogenesis by promoting the transcription of HIF1α, we performed rescue experiments in HCC1806 and HCC1937 cells. We observed that overexpression of STAMBPL1 promoted the proliferation, migration and tube formation of HUVECs, which could be blocked by knockdown of HIF1α (Figs. 2F-K and S2D-I). At the same time, we also examined the expression of HIF1α and VEGFA and found that overexpression of STAMBPL1 promoted the expression of HIF1α and VEGFA, which could be rescued by knocking down HIF1α (Figs. 2L-M and S2J-K). These results demonstrate that STAMBPL1 promotes TNBC angiogenesis by promoting HIF1α transcription.

STAMBPL1 promotes TNBC angiogenesis by promoting HIF1α transcription mediated by GRHL3

In the previous results, we observed that STAMBPL1 positively regulates the transcriptional level of HIF1α. To determine the transcription factor responsible for mediating HIF1α transcription by STAMBPL1, we performed RNA-seq analysis after knocking down STAMBPL1 in HCC1806 cells following 10 h of hypoxia treatment (Fig. 3A). It turns out that the mRNA expression of transcription factor GRHL3 was significantly decreased in HCC1806 (Fig. 3B) and in HCC1937 (Figure S3A). To further investigate the regulatory role of GRHL3 on HIF1α transcription, we predicted the GRHL3 binding sequence at the HIF1α gene promoter using the JASPAR website (Figure S3B). Subsequently, we cloned the wild-type (WT) and mutant promoter sequences into the pGL3-BASIC luciferase reporter plasmid (Fig. 3C). Luciferase assays showed that GRHL3 can indeed bind to the putative binding site and activate HIF1α gene promoter (Fig. 3D). We also verified that GRHL3 bound to the putative binding site of the HIF1α gene promoter by ChIP-PCR experiments (Fig. 3E).

To investigate whether GRHL3 promotes the expression of HIF1α and VEGFA, we established stable GRHL3 overexpression HCC1806 and HCC1937 cells. We observed that overexpression of GRHL3 up-regulated the protein level of HIF1α (Figs. 3F and S3C). Additionally, knockdown of GRHL3 significantly decreased HIF1α protein expression under hypoxia treatment in both cell lines (Figs. 3G and S3D). Furthermore, knockdown of GRHL3 significantly decreased HIF1α/VEGFA mRNA levels under hypoxia treatment in both cell lines (Figs. 3H-J and S3E-G).

To further test whether GRHL3 promotes angiogenesis and tumor growth in vivo, we conducted animal experiments in nude mice. HCC1806 cells with stable GRHL3 knockdown were orthotopically inoculated into the fat pad of 6-week-old female mice (n = 4). RT-qPCR was performed to detect the knockdown effect of GRHL3 in animal experiments (Figure S3H). Our results showed that knockdown of GRHL3 significantly inhibited the tumor growth based on the tumor volume and the tumor weight (Figs. 3K-L and S3I). Additionally, immunohistochemistry analysis with CD31 staining indicated a significant decrease in microvessel numbers in the xenograft tumors with GRHL3 knockdown (Figs. 3M-N).

To investigate whether GRHL3 further promotes angiogenesis in vitro, we knocked down GRHL3 in HCC1806 and HCC1937 cells and treated them with hypoxia. Then the conditioned medium was collected and used to treat HUVECs. As a result, conditioned medium derived from either HCC1806 or HCC1937 cells under hypoxia significantly promoted the proliferation, migration, and tube formation of HUVECs compared with conditioned medium under normoxia (Figs. 3O-Q and S3J-R). Importantly, knockdown of GRHL3 significantly inhibited the proliferation, migration and tube formation of HUVECs. These results suggest that GRHL3 promotes TNBC angiogenesis.

GRHL3 promotes TNBC angiogenesis by promoting HIF1α transcription

Since GRHL3 promotes the transcription of HIF1α/VEGFA and promote TNBC angiogenesis. Then, we wondered whether GRHL3 promotes TNBC angiogenesis by HIF1α. We performed rescue experiments in HCC1806 and HCC1937 cells. Overexpression of GRHL3 promoted the proliferation, migration and tube formation of HUVECs, which could be blocked by knockdown of HIF1α (Figs. 4A-F and S4A-F). Meanwhile, we also detected the expression of VEGFA, and found that overexpression of GRHL3 promoted the expression of HIF1α and VEGFA, while knockdown of HIF1α could block the VEGF induction (Figs. 4G-H and S4G-H). These results indicate that GRHL3 promotes TNBC angiogenesis by promoting HIF1α transcription.

STAMBPL1 promotes TNBC angiogenesis by promoting GRHL3 transcription

Next, we asked whether STAMBPL1 promotes TNBC angiogenesis by promoting the transcription of GRHL3. We also performed rescue experiments in HCC1806 and HCC1937 cells. We found that overexpression of STAMBPL1promoted the proliferation, migration and tube formation of HUVECs, which could be blocked by knockdown of GRHL3 (Figs. 5A-F and S5A-F). Meanwhile, we also found that overexpression of STAMBPL1 promoted the expression of HIF1α and VEGFA, and knockdown of GRHL3 blocked this phenomenon (Figs. 5G-J and S5G-J). These results suggest that STAMBPL1 promotes TNBC angiogenesis by promoting GRHL3 transcription.

STAMBPL1 promotes TNBC angiogenesis by promoting GRHL3/HIF1α/VEGFA transcription through FOXO1

The next question is how STAMBPL1 promotes the transcription of GRHL3. Previous studies have shown that STAMBPL1 can be localized in the nucleus ^[9], leading us to speculate that STAMBPL1 may interact with a transcription factor to regulate the transcription of GRHL3. It has been reported that FOXO1 is an upstream transcription factor for GRHL3 ^[31]. Therefore, we hypothesized that STAMBPL1 interacts with FOXO1 to promote the transcription of GRHL3. To investigate this, we collected HCC1937 cell lysates and performed immunoprecipitation using an anti-FOXO1 antibody and found that endogenous FOXO1 protein interacted with endogenous STAMBPL1 protein (Fig. 6A). We further mapped the regions of STAMBPL1 and FOXO1 proteins responsible for the interaction (Figures S6A-B) by generating a series of Flag-FOXO1/GST-fused STAMBPL1 deletion mutants. We transfected them into HEK293T cells and performed immunoprecipitation assays and GST-pull down assays by using Flag-M2 beads and glutathione beads, respectively. To map the domains of STAMBPL1 that interact with FOXO1, we transfected no-tagged full-length FOXO1 into HEK293T cells with full-length or truncated STAMBPL1. We found that the N-terminus (1–140 aa) of the STAMBPL1 protein interacted with FOXO1 (Figure S6C). As shown in Figure S6D, the FOXO1 protein (250–596aa) interacted with STAMBPL1. Finally, we confirmed that STAMBPL1 and FOXO1 were colocalized using immunofluorescence staining (Fig. 6B). Taken together, these results suggest that STAMBPL1 interacts with FOXO1.

To investigate whether FOXO1 promotes the expression of GRHL3, HIF1α and VEGFA in TNBC cells, we knocked down FOXO1 in HCC1806 and HCC1937 cells and then treated the cells with hypoxia. As expected, knockdown of FOXO1 inhibited hypoxia induced HIF1α protein expression and GRHL3, HIF1α and VEGFA mRNA levels (Figs. 6C-F and S6E-H). Next, we knocked down FOXO1 in HCC1806 and HCC1937 cells that stably overexpressed STAMBPL1. Consistently, overexpression of STAMBPL1 promoted the protein expression of HIF1α, which could be rescued by knocking down FOXO1 (Figs. 6G and S6I). Additionally, knockdown of FOXO1also blocked the mRNA expression of GRHL3, HIF1α and VEGFA, induced by STAMBPL1 (Figs. 6H-J).

Furthermore, we constructed stable HCC1806 and HCC1937 cells overexpressing FOXO1 and then knocked down GRHL3. The results showed that overexpression of FOXO1 promoted the protein expression of HIF1α, which was attenuated by GRHL3 knockdown (Figs. 6K and S6J). Similarly, FOXO1 overexpression increased the mRNA expression of GRHL3, HIF1α, and VEGFA, and knockdown of GRHL3 blocked the induction of HIF1α and VEGFA by FOXO1 (Figs. 6L-N).

Next, we utilized the JASPAR website to identify the binding sequence of the transcription factor FOXO1 at the GRHL3 gene promoter (Figure S6K). We demonstrated that FOXO1 can bind to the GRHL3 gene promoter and promote its transcriptional activity (Figs. 6O-P). Moreover, STAMBPL1 enhanced the activation of the GRHL3 gene promoter by FOXO1, as demonstrated by luciferase assays (Fig. 6Q). Finally, we knocked down FOXO1 in HCC1806 cells stably overexpressing STAMBPL1 and performed ChIP-PCR experiments to validate that STAMBPL1 was recruited to the GRHL3 gene promoter through FOXO1 to activate the transcription (Fig. 6R). Taken together, STAMBPL1 promotes TNBC angiogenesis by promoting GRHL3/HIF1α/VEGFA transcription by FOXO1.

The combination of FOXO1 inhibitor AS1842856 and VEGFR inhibitor Apatinib significantly inhibits the growth of tumors in nude mice

Through analysis of the TCGA database, we found that STAMBPL1, FOXO1 and GRHL3 were highly expressed in TNBC compared to non-TNBC tumors (Figs. 7A-C). To evaluate the potential therapeutic effect of combining the FOXO1 inhibitor AS1842856 and the VEGFR inhibitor Apatinib in vivo, we performed animal experiments in nude mice. HCC1806 cells with stable STAMBPL1 overexpression (Fig. 7D) were orthotopically inoculated into the fat pad of 6-week-old female mice (n = 16/group). Once the tumor mass reached approximately 50 mm³, each group was divided into four subgroups to receive VEGFR inhibitor Apatinib (50 mg/kg, once two days), FOXO1 inhibitor AS1842856 (10 mg/kg, once two days), a combination of both drugs, and vehicle control for 20 days. We observed that overexpression of STAMBPL1 promoted breast cancer cell growth in vivo, and the inhibitory effect of FOXO1 inhibitor and VEGFR inhibitor alone on transplanted tumors in mice was not significant, but the growth of transplanted tumors in nude mice could be significantly inhibited by both drugs in combination (Figs. 7E-G). Meanwhile, these drugs treatment had no effect on the body weight of mice (Fig. 7H). These results suggest that the combination of FOXO1 inhibitor AS1842856 and VEGFR inhibitor Apatinib can effectively inhibit the growth of transplanted tumors in nude mice. The Fig. 7I is the work model of this study.

In this study, we have discovered that STAMBPL1 promotes TNBC angiogenesis by promoting the transcription of HIF1α/VEGFA independently of its enzyme activity. Through RNA-seq analysis, we have identified that STAMBPL1 positively regulates the transcription of GRHL3. We have further demonstrated that GRHL3 binds to the promoter of the HIF1α gene, thereby promoting its transcription. Additionally, we have discovered that STAMBPL1 interacts with FOXO1 to facilitate the transcription of GRHL3/HIF1α/VEGFA. Importantly, we have confirmed that the combination of the FOXO1 inhibitor AS1842856 and the VEGFR inhibitor Apatinib effectively inhibits tumor growth in nude mice. These findings suggest that both STAMBPL1 and FOXO1 may serve as potential therapeutic targets for inhibiting angiogenesis in TNBC. This study is the first to uncover the role of STAMBPL1 in promoting angiogenesis through the FOXO1/GRHL3/HIF1α axis. Furthermore, it has identified a novel transcription factor, GRHL3, which regulates the transcription of HIF1α. These findings provide valuable insights for developing a new therapeutic strategy to target TNBC.

The role of STAMBPL1 in tumors has not been fully recognized. Recent studies have reported its involvement in the epithelial-mesenchymal transition of various cancers, and its absence has been shown to affect the mesenchymal phenotype of lung and breast cancer^[34]. Our previous research has demonstrated that STAMBPL1 can stabilize MKP1 through deubiquitination, and the deletion of STAMBPL1 and MKP1 increases the sensitivity of breast cancer cells to cisplatin^[14], suggesting that STAMBPL1 may be a potential therapeutic target for breast cancer. However, the mechanism by which STAMBPL1 activates the transcriptional activity of FOXO1 has not been addressed, as STAMBPL1 does not stabilize the FOXO1 protein through its deubiquitinating enzyme activity. We speculate that STAMBPL1 may recruit other co-activators to the FOXO1 transcription complexes. It will be important to develop a Stambpl1 knockout mouse model to investigate the exact role of STAMBPL1 in TNBC. Additionally, we also need to find HIF1α and FOXO1 antibodies suitable for immunohistochemistry to detect their expression in TNBC clinical samples.

Several studies reported that FOXO1 inhibits tumor angiogenesis^[35–39]. Studies have found that M2 macrophage-derived exosomal miR-942 promotes the migration and invasion of lung adenocarcinoma cells and facilitates angiogenesis by binding to FOXO1 to alleviate the inhibition of β-catenin, in which up-regulation of FOXO1 induced a decrease in cell invasion and angiogenesis in vitro^[35]. FOXO1 inhibits gastric cancer growth and angiogenesis under hypoxic conditions via inactivation of the HIF1α-VEGF pathway, possibly in association with SIRT1^[36]. Cancer-associated fibroblasts (CAFs)-derived extracellular vesicles (EVs) delivered miR-135b-5p into colorectal adenocarcinoma cells to downregulate FOXO1 and promote HUVECs proliferation, migration, and angiogenesis^[37]. Colorectal cancer cell-derived exosomes overexpressing miR-183-5p promote proliferation, migration and tube formation of HMEC-1 (human microvascular endothelial cell) cells through inhibiting FOXO1^[38]. Bladder cancer cells-derived exosomal miR-1247-3p facilitates angiogenesis through inhibiting FOXO1 expression^[39]. However, the role of FOXO1 in breast cancer angiogenesis has not been studied, and our study found that FOXO1 can promote the expression of HIF1α and VEGFA, suggesting that it may play a role in promoting angiogenesis in breast cancer.

Studies have demonstrated that the AKT-FOXO1 signaling pathway regulates the expression of GRHL3. To investigate this, the researchers utilized a previously published ChIP-seq dataset for FOXO1 from human endometrial stromal cells. They observed that FOXO1 occupied BRD4-bound enhancers near the GRHL3 gene. Subsequently, they confirmed that the removal of EGF and Insulin from the growth medium significantly increased the expression of GRHL3, but administration of the FOXO1 inhibitor AS1842856 partially blocked the induced expression of GRHL3^[31]. These results suggest that FOXO1 plays a regulatory role as the upstream of GRHL3, and our study also confirmed that FOXO1 promotes the transcriptional of GRHL3 by binding to its promoter.

GRHL3 is a highly conserved epidermal-specific developmental transcription factor, has recently gained attention in the field of cancer research^[40]. Until now, only a few genes regulated by GRHL3 had been identified in the cancer research. Studies have shown that GRHL3 levels are significantly reduced in human skin and head and neck squamous cell carcinomas and suggest that GRHL3 is a key tumor suppressor pathway in squamous cell carcinomas^[41]. GRHL3 was also found to be induced by TNF-α in the mammarycarcinoma cell line MCF-7 and identified as a TNFα-induced endothelial cell migration factor with a promigratory activity as high as VEGF^[42]. However, the specific role of GRHL3 in breast cancer is unclear. Studies have confirmed that GRHL3 strongly stimulated endothelial cell migration, which is consistent with an angiogenic, pro-tumorigenic function^[42]. In our study, we demonstrated that GRHL3 promotes TNBC angiogenesis. HIF1α, a known regulator of angiogenesis, is regulated by growth factors^{[43, 44]}, cytokines^[45]and mitogens^{[46, 47]}. The EGFR/Akt pathway is a known positive regulator of HIF1α, potentially through mTOR^[43] or independent of it^{[48, 49]}. While the mechanisms regulating HIFα protein expression have been extensively studied, those modulating HIF1α transcriptional activity remain unclear. Our study reveals for the first time that GRHL3 promotes transcriptional activity by binding to the promoter of the HIF1α gene.

In summary, STAMBPL1 promoted TNBC angiogenesis by activating the GRHL3/HIF1A pathway via FOXO1 in a non-enzymatic manner. These findings highlight the significant role of STAMBPL1 in TNBC angiogenesis and suggest that targeting the STAMBL1/FOXO1/GRHL3/HIF1α/VEGFA axis could be a potential therapeutic strategy to inhibit angiogenesis in TNBC.

Cell lines and reagents

All cell lines used in this study, including HCC1806, HCC1937, and HEK293T cells, were purchased from ATCC (American Type Culture Collection, Manassas, VA, USA) and validated by STR (short tandem repeat) analysis and these cell lines tested negative for mycoplasma contamination. HCC1806 and HCC1937 cells were cultured in RPMI 1640 medium supplemented with 5% FBS. HEK293T cells were cultured in DMEM (Thermo Fisher, Grand Island, USA) with 5% FBS at 37°C with 5% CO₂. Primary HUVECs were maintained in EGM-2 BulletKit (CC-3162, Lonza, USA). AS1842856 (Cat#HY-100596), Apatinib (Cat#HY-13342S) were purchased from MCE (New Jersey, USA).

Plasmid construction and stable STAMBPL1, GRHL3 and FOXO1 overexpression

We constructed the full-length STAMBPL1/GRHL3/FOXO1genesand then subcloned them into the pCDH lentiviral vector. The packaging plasmids (including pMDLg/pRRE, pRSV-Rev, and pCMV-VSV-G) and pCDH-STAMBPL1/GRHL3/FOXO1 expression plasmid were cotransfected into HEK293T cells (2 ×10⁶ in 10 cm plate) to produce lentivirus. Following transfection for 48 hours, the lentivirus was collected and used to infect HCC1806 and HCC1937 cells. Forty-eight hours later, puromycin (2 μg/ml) was used to screen the cell populations.

Stable knockdown of STAMBPL1 and GRHL3

The pSIH1-H1-puro shRNA vector was used to expressSTAMBPL1, GRHL3 andluciferase(LUC)shRNAs.STAMBPL1shRNA#1,5’-GGAGCATCAGAGATTGATA-3’;STAMBPL1shRNA#2,5’-GCTGCTATGCCTGACCATA-3’;GRHL3shRNA#1,5’-CCTTGAGCTTCCTCTATGA-3’;GRHL3shRNA#2,5’-AGAGGAAGATGCGCGATGA-3’;LuciferaseshRNA,5’-CUUACGCUGAGUACUUCGA-3’;HCC1806 and HCC1937 cells were infected with lentivirus. Stable populations were selected using 1 to 2 mg/mL puromycin. The knockdown effect was evaluated by Western blotting.

RNA interference

The siRNA target sequences used in this study are as follows:STAMBPL1siRNA#1,5’-GGAGCATCAGAGATTGATA-3’;STAMBPL1siRNA#2,5’-GCTGCTATGCCTGACCATA-3’;GRHL3siRNA#1,5’-CCTTGAGCTTCCTCTATGA-3’;GRHL3siRNA#2,5’-AGAGGAAGATGCGCGATGA-3’;HIF1αsiRNA#1,5’-AAGAGGTGGATATGTCTGG-3’;HIF1αsiRNA#2,5’-CGTCGAAAAGAAAAGTCTCTT-3’;FOXO1siRNA#1,5’-CCCAGAUGCCUAUACAAAC-3’;FOXO1siRNA#2,5’-CTCAAATGCTAGTACTATTAG-3’. All siRNAs were synthesized by RiboBio (RiboBio, China) and transfected at a final concentration of 50 nM.

Western blotting (WB) and antibodies

The WB procedure has been described in our previous study^[⁵⁰^]. The anti-STAMBPL1 (sc-376526) and anti-GAPDH (sc-25778) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The anti-FOXO1 (#2880S) andanti-HIF1α (#36169S) antibodies were purchased from CST. Anti-β-actin (A5441) and anti-GST (G7781) antibodies were purchased from Sigma‒Aldrich (St Louis, MO, USA). The anti-Flag (ab205606) antibody was purchased from Abcam.

The real-time polymerase chain reaction (RT-qPCR)

Total RNA was extracted using TRIzol (Invitrogen, 15596026). 1 μg of total RNA was reversely transcribed to cDNA according to manufacturer's instructions of the HiScript II QRT SuperMix for qPCRkit (Vazyme, R223-01). For the quantitative PCR (RT-qPCR), the SYBR Green Select Master Mix system (Applied Biosystems, 4472908, USA) was used on the ABI-7900HT system (Applied Biosystems, 4351405). The primers of PCR are as follow: the 18S forward is 5′-CTCAACACGGGAAACCTCAC-3′, the 18S reverse is 5′-CGCTCCACCAACTAAGAACG-3′; the HIF1α forward is 5′-AAGTCTGCAACATGGAAGGTAT-3′, the HIF1α reverse is 5′-TGAGGAATGGGTTCACAAATC-3′; the VEGFA forward is 5′-TGAGGAATGGGTTCACAAATC-3′, the VEGFA reverse is 5′-ATCTGCATGGTGATGTTGGA-3′; the GLUT1 forward is 5′-TCGTCGGCATCCTCATCGCC-3′, the GLUT1 reverse is 5′-CCGGTTCTCCTCGTTGCGGT-3′; the GRHL3 forward is 5′-GGGGCTGAGGAATGCGATCT-3′, the GRHL3 reverse is 5′-AATTTTGCCGTCCAGCTCCC-3′.

Cell proliferation and migration assays

To detect the proliferation of HUVECs, we used the Click-iT®EdUAlexaFluor 647 Imaging Kits (Invitrogen) according to the manufacturer’s protocol. Briefly, HUVECs were seeded on coverslips (BD Biosciences) at 0.5×10⁵ cells per well. On the next day, the supernatants were discarded and the cells were cultured with the CM. Six hours later, the cells were incubated with EdU in CM for 4 hours, followed by fixing and staining. For each sample, three random fields were observed by using fluorescence microscopy, and the total numbers of cells and EdU positive cells were counted. To detect the migration of HUVECs, we performed the wound-healing assay. Twenty-four hours after seeding, the supernatants of HUVECs were discarded and the cells were scratched and cultured with the CM for 24 hours. Wound closure was imaged under microscopy. For each image, the gap width was analyzed by Image J.

Tube formation assays

HUVECs (1 × 10⁴) in CM were seeded onto Matrigel (BD Biosciences)-coated μ-Slide angiogenesis (ibidiGmbH, Munich, Germany). At 6 hours after seeding, images were taken under microscopy, and then were analyzed with Image J. The total branch length were measured.

Chromatin immunoprecipitation assays

After the sample preparation is completed, the subsequent experimental steps refer to the instructions in the SimpleChIP (R) Plus Kit (CST, # 9005). The PCR primers for amplifying the interest region on the HIF1αgene promoter were as follows:5′-GACTGACAGGCTTGAAGTTTATGC-3′and5′-TGTTGCTGTAAACTTCAAGGGAAA-3′, and the PCR primers for amplifying the interest region on the GRHL3 gene promoter were as follows:5′-TTCTATCCCTTCTGTGCTGACCA-3′and5′-TGTGCCAGACCCTACTCTGGG-3′.

Dual-luciferase report assays

The DNA fragments HIF1α and GRHL3 were amplified using MCF10A cell genomic DNA as PCR template and then cloned into pGL3-Basic vector, respectively. HEK293T cells were seeded in 24-plates and transfected with pCDH-GRHL3-3×Flag or pCDH-FOXO1-3×Flag and pGL3 luciferase reporter plasmids (both 600 ng/well) together with pCMV-Renilla control (5ng/well). After transfection for 48 hours, cell lysates were collected and the luciferase activities were detected by using the dual-luciferase reporter assay system (Promega, USA). For the WT HIF1α promoter (with the GRHL3 binding motif), the primers of PCR are as follow: the forward is 5′-gctagcccgggctcgagatctCCACTGCGCTCCAGCCTG-3′; the reverse is 5′-cagtaccggaatgccaagcttCCTCAGACGAGGCAGCACTG-3′. For the mutant HIF1αpromoter (without the GRHL3 binding motif), the primers of PCR are as follow: the forward is 5′-TCTTTCCCTGAGGCCTTCCTATATGCTTAT-3′; the reverse is 5′-ATAAGCATATAGGAAGGCCTCAGGGAAAGA-3′. For the WTGRHL3 promoter (with the FOXO1 binding motif), the primers of PCR are as follow: the forward is 5′-gctagcccgggctcgagatctATTAACAAGGGTGACTGAAGAGGG-3′; the reverse is 5′-cagtaccggaatgccaagcttTGGAGGTATACCTCAACAGGTGC-3′. For the mutant GRHL3 promoter (without the FOXO1 binding motif), the primers of PCR are as follow: the forward is 5′-CTCCCCCACCAAACAAAGAAGGAGAACACCCC-3′; the reverse is 5′-GGGGTGTTCTCCTTCTTTGTTTGGTGGGGGAG-3′.

Immunofluorescence staining

HEK293T cells plated on cell culture slides were transfected with STAMBPL1 and FOXO1 expression plasmids. Two days after transfection, cells were fixed in 3.7% polyformaldehyde at 4 °C overnight. After blocking with 5% BSA at room temperature for 1 hours, cells were stained with anti-STAMBPL1 (mouse) and anti-FOXO1 (rabbit) antibodies at 4 °C overnight. Subsequently, cells were stained with both Alexa Fluor647-labeled anti-mouse secondary antibody and FITC-labeled anti-rabbit antibody (ZSGB-Bio, Beijing, China) at room temperature for 1 hours. Nuclei were stained using DAPI (Biosharp, BL739A) for 15 min. Images were captured using a confocal microscope.

Immunoprecipitation and GST pull-down

For endogenous protein interaction, cell lysates were first incubated with anti-FOXO1 antibodies or rabbit IgG (2729S;Cell Signaling Technology) and then incubated with Protein A/G Magnetic Beads (HY-K0202; MCE).For the GST pull down assay, cell lysates were directly incubated with GlutathioneSepharose 4B (10312185; Cytiva) overnight at 4°C. For the IP-Flag assay, cell lysates were directly incubated with anti-Flag Magnetic Beads (HY-K0207; MCE) overnight at 4°C. The precipitates were washed four times with 1 ml of lysis buffer, boiled for 10 minutes with 1×SDS sample buffer, and subjected to WB analysis.

Xenograft experiments

We purchased 5- to 6-week-old female BALB/cnude mice from SLACCAS (Changsha, China). HCC1806-shLuc, HCC1806-shSTAMBPL1, or HCC1806-shGRHL3 cells, and HCC1806-PCDH-Vector, HCC1806-PCDH-STAMBPL1(1 × 10⁶ in Matrigel (BD Biosciences, NY, USA)) were implanted into the mammary fat pads of the mice. When the tumor volume reached approximately 50 mm³, the nude mice were randomly assigned to the control and treatment groups (n = 4/group). The control group was given vehicle alone, and the treatment group received FOXO1 inhibitor AS1842856 (10 mg/kg), VEGFR inhibitor Apatinib (50 mg/kg), and FOXO1 inhibitor AS1842856 was combined with VEGFR inhibitor Apatinib via intragastric administration every two days for 20 days. The tumor volume was calculated as follows: tumor volume was calculated by the formula: (π×length × width²)/6.

Immunohistochemical staining

The xenograft tumor tissues were fixed in 3.7% formalin solution. The immunohistochemistry assay was performed on 4-μm-thick paraffin sections after the pressure-cooking for antigen retrieval. The anti-CD31 primary antibody (1:400, Abcam, ab28364) was used. After 12 hours, the slides were washed three times with PBS and incubated with secondary antibodies (hypersensitive enzyme-labeled goat anti-mouse/rabbit IgG polymer (OriGene, China) at room temperature for 20 min, DAB concentrate chromogenic solution (1:200 dilution of concentrated DAB chromogenic solution), counterstained with 0.5% hematoxylin, dehydrated with graded concentrations of ethanol for 3 min each (70%–80%–90%–100%),and finally stained with dimethyl benzene immunostained slides were evaluated by light microscopy, and the number of microvessel with positive CD31 expression was counted.

Statistical analysis

All graphs were created using GraphPad Prism software version 8.0. Comparisons between two independent groups were assessed by two-tailed Student’s t-test. One-way analysis of variance with least significant differences was used for multiple group comparisons. P-values of <0.05, 0.01 or 0.001 were considered to indicate a statistically significant result, comparisons significant at the 0.05 level are indicated by*, at the 0.01 level are indicated by **, or at the 0.001 level are indicated by ***.

Ethics

Animal feeding and experiments were approved by the animal ethics committee of Kunming Institute of Zoology, Chinese Academy of Sciences (IACUC-RE-2023-04-002).

Declaration of interests

The authors declare no conflicts of interest.

Acknowledgments

This work was supported by the National Key R&D Program of China (2020YFA0112300, 2023YFA1800500), National Natural Science Foundation of China (U2102203 to CC, 82203413 to HL), Biomedical Projects of Yunnan Key Science and Technology Program (202302AA310046 to CC), Yunnan Fundamental Research Projects (202101AS070050 to CC, 202201AT070290 to HL), and Yunnan Revitalization Talent Support Program (Yunling Shcolar Project to CC), Yunnan (Kunming) Academician Expert Workstation (grant No. YSZJGZZ-2020025 to CC), Kunming University of Science and Technology-the First People’s Hospital of Yunnan Province Joint Major Project (No.KUST-KH2022005Z), Center of Clinical Pharmacy of the First People’s Hospital of Yunnan Province Open Project (No. 2023YJZX-YX04).

Data availability statement

The authors confirm that the data supporting the findings of this study are available within the article and its supplementary materials.

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Figuresupplement1.tif
Figure S1. STAMBPL1 promotes TNBC angiogenesis (A)Western blot analysis was performed to confirm the knockdown of STAMBPL1 in HCC1806 cells. (B) HCC1937 cells were transfected with STAMBPL1 siRNA and subjected to hypoxia for 24 hours. The conditioned medium collected from these cells was then used to treat HUVECs. EdU assay revealed that the conditioned medium after STAMBPL1 knockdown inhibited the proliferation of HUVECs. (C) Statistical analysis of the EdU assay results. (D) HCC1937 cells were transfected with STAMBPL1 siRNA and subjected to hypoxia for 24 hours. The conditioned medium collected from these cells was used to treat HUVECs. Wound healing assay demonstrated that the conditioned medium after STAMBPL1 knockdown inhibited the migration of HUVECs. (E) Statistical analysis of the wound healing assay results. (F) HCC1937 cells were transfected with STAMBPL1 siRNA and subjected to hypoxia for 24 hours. The conditioned medium collected from these cells was used to treat HUVECs. Tube formation assay showed that the conditioned medium after STAMBPL1 knockdown inhibited tube formation of HUVECs. (G) Statistical analysis of the tube formation assay results. *: P<0.05, **: P<0.01, ***: P<0.001, t-test.
Figuresupplement21.tif
Figure S2. STAMBPL1 promotes TNBC angiogenesis by promoting HIF1α/VEGFA transcription (A-C) RNA samples were collected after 10 hours of hypoxia treatment following knockdown of STAMBPL1 in HCC1937 cells. RT-qPCR experiments revealed that knockdown of STAMBPL1 downregulated the mRNA levels of HIF1α and its downstream targets VEGFA and GLUT1. (D-E) HCC1806 and HCC1937 cells with stable overexpression of STAMBPL1, HIF1α was knocked down using siRNA, and then the conditioned medium was collected and used to treat HUVECs. The EdU assay results showed that overexpression of STAMBPL1 promoted the proliferation of HUVECs, which could be rescued by knockdown of HIF1α. (F-G) HCC1806 and HCC1937 cells with stable overexpression of STAMBPL1, HIF1α was knocked down using siRNA, and then the conditioned medium was collected and used to treat HUVECs. The wound healing assay results showed that overexpression of STAMBPL1 promoted the migration of HUVECs, which could be rescued by knockdown of HIF1α. (H-I) HCC1806 and HCC1937 cells with stable overexpression of STAMBPL1, HIF1α was knocked down using siRNA, and then the conditioned medium was collected and used to treat HUVECs. The tube formation assay results showed that overexpression of STAMBPL1 promoted the tube formation of HUVECs, which could be rescued by knockdown of HIF1α. (J) In HCC1937 cells with stable overexpression of STAMBPL1, HIF1α was knocked down using siRNA, and protein was collected. Western blotting experiments showed that overexpression of STAMBPL1 promoted HIF1α expression, which could be rescued by knockdown of HIF1α. (K)In HCC1937 cells with stable overexpression of STAMBPL1, HIF1α was knocked down using siRNA, and RNA was collected. RT-qPCR experiments showed that overexpression of STAMBPL1 promoted VEGFA mRNA expression, which could be rescued by knockdown of HIF1α. *: P<0.05, **: P<0.01, ***: P<0.001, t-test.
Figuresupplement22.tif
Figuresupplement31.tif
Figure S3. STAMBPL1 promotes TNBC angiogenesis by promoting HIF1α transcription mediated by GRHL3 (A)RT-qPCR experiments showed that knockdown of STAMBPL1 inhibited GRHL3 mRNA levels in HCC1937 cells. (B) The JASPAR website predicted the potential binding sequence of the transcription factor GRHL3 to the HIF1α promoter. (C)Overexpression of GRHL3 upregulated the protein expression of HIF1α in HCC1937 cells with stable GRHL3 overexpression. (D) In HCC1937 cells, knockdown of GRHL3 using siRNA and treated with hypoxia for 4 hours. Western blotting experiments showed that knockdown of GRHL3 could down-regulate the protein level of HIF1α. (E-G) In HCC1937 cells, GRHL3 was knocked down by siRNA, followed by hypoxia treatment for 4 hours, and then RNA samples were collected. RT-qPCR experiments showed that GRHL3 knockdown was effective, and knockdown of GRHL3 could down-regulate the mRNA levels of HIF1α and its downstream target VEGFA. (H) RT-qPCR was used to detect the knockdown of GRHL3 in HCC1806 cells. (I) The growth of transplanted tumor in nude mice were statistically analyzed, and the shGRHL3 1# and shGRHL3 2# groups were compared with the shLuc group. (J-L) In HCC1806 and HCC1937 cells, GRHL3 was knocked down by siRNA and then treated with hypoxia for 24 hours, and then the conditioned medium was collected to treat HUVECs, EdU assay showed that the conditioned medium after knocking down GRHL3 inhibited the proliferation of HUVECs. Statistical analysis of the EdU assay results was performed. (M-O)In HCC1806 and HCC1937 cells, GRHL3 was knocked down by siRNA and then treated with hypoxia for 24 hours, and then the conditioned medium was collected to treat HUVECs，wound healing assay showed that the conditioned medium after knocking down GRHL3 inhibited the migration of HUVECs. Statistical analysis of the wound healing assay results was performed. (P-R) In HCC1806 and HCC1937 cells, GRHL3 was knocked down by siRNA and then treated with hypoxia for 24 hours, and then the conditioned medium was collected to treat HUVECs, the tube formation assay showed that the conditioned medium after knocking down GRHL3 inhibited 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.
Figuresupplement32.tif
Figuresupplement4.tif
Figure S4. GRHL3 promotes TNBC angiogenesis by promoting HIF1α transcription (A) In HCC1937 cells stably overexpressing GRHL3, HIF1α was knocked down using siRNA. The conditioned medium collected from these cells was then used to treat HUVECs. EdU assay results revealed that the overexpression of GRHL3 promoted the proliferation of HUVECs, which could be rescued by HIF1α knockdown. (B)In HCC1937 cells stably overexpressing GRHL3, HIF1α was knocked down using siRNA, and the conditioned medium was collected to treat HUVECs. Wound healing assay results showed that GRHL3 overexpression enhanced the migration of HUVECs, which could be rescued by HIF1α knockdown. (C) In HCC1937 cells stably overexpressing GRHL3, HIF1α was knocked down using siRNA, and the conditioned medium was collected to treat HUVECs. Tube formation assay showed that overexpression of GRHL3 promoted the tube formation of HUVECs, which could be rescued by HIF1α knockdown. (D) Statistical analysis of EdU assay results. (E) Statistical analysis of wound healing assay results. (F)Statistical analysis of tube formation assay results. (G) In HCC1937 cells stably overexpressing GRHL3, HIF1α was knocked down using siRNA, and then the protein was collected. Western blotting experiments showed that overexpression of GRHL3 promoted the expression of HIF1α, which could be rescued by knockdown of HIF1α. (H) In HCC1937 cells stably overexpressing GRHL3, HIF1α was knocked down using siRNA and then the RNA was collected. RT-qPCR experiments showed that overexpression of GRHL3 promoted the mRNA expression of VEGFA, which could be rescued by knockdown of HIF1α. *: P<0.05, **: P<0.01, ***: P<0.001, t-test.
Figuresupplement5.tif
Figure S5. STAMBPL1 promotes TNBC angiogenesis by promoting GRHL3transcription (A) In HCC1937 cells stably overexpressing STAMBPL1, GRHL3 was knocked down using siRNA, and then the conditioned medium was collected to treat HUVECs. EdU assay showed that overexpression of STAMBPL1 promoted the proliferation of HUVECs, which could be rescued by knockdown of GRHL3. (B) In HCC1937 cells stably overexpressing STAMBPL1, HIF1α was knocked down using siRNA, and then the conditioned medium was collected to treat HUVECs. Wound healing assay showed that overexpression of STAMBPL1 promoted the migration of HUVECs, which could be rescued by knockdown of GRHL3. (C) In HCC1937 cells stably overexpressing STAMBPL1, GRHL3 was knocked down using siRNA, and then the conditioned medium was collected to treat HUVECs. Tube formation assay showed that overexpression of STAMBPL1 promoted the tube formation of HUVECs, which could be rescued by knockdown of GRHL3. (D) Statistical analysis of EdU assay. (E) Statistical analysis of wound healing assay. (F) Statistical analysis of tube formation assay. (G) In HCC1937 cells stably overexpressing STAMBPL1, GRHL3 was knocked down using siRNA, and then the protein was collected. Western blotting experiments showed that overexpression of STAMBPL1 promoted the expression of HIF1α, which could be rescued by knockdown of GRHL3. (H-J) In HCC1937 cells stably overexpressing STAMBPL1, GRHL3 was knocked down using siRNA and then the RNA was collected. RT-qPCR experiments showed that overexpression of STAMBPL1 promoted the mRNA expression of GRHL3, HIF1α and its downstream target VEGFA, which could be rescued by knockdown of GRHL3. *: P<0.05, **: P<0.01, ***: P<0.001, t-test.
Figuresupplement6.tif
Figure S6. STAMBPL1 promotes TNBC angiogenesis by promoting GRHL3/HIF1α/VEGFAtranscription through FOXO1 (A) The truncated structure diagram of STAMBPL1. (B) The truncated structure diagram of FOXO1. (C) The full-length and truncated plasmids of pEBG-STAMBPL1-GST and pCDH-FOXO1-no tag were transfected into HEK293T cells, and the proteins were collected for GST-pull down assay at 48 hours after transfection. (D)The full-length and truncated plasmids of pCDH-FOXO1-3×Flag and pEBG-STAMBPL1-GST were transfected into HEK293T cells, and the proteins were collected for IP-Flag assay at 48 hours after transfection. (E) Western blotting experiments showed that knockdown of FOXO1 in HCC1937 cells followed by hypoxia treatment for 4 hours inhibited HIF1α protein expression. (F-H)RT-qPCR experiments showed that knockdown of FOXO1 in HCC1937 cells followed by hypoxia treatment for 4 hours inhibited the mRNA levels of GRHL3/HIF1α/VEGFA. (I)In HCC1937 cells stably overexpressing STAMBPL1, protein samples were collected after knockdown of FOXO1 with siRNA. Western blotting experiments showed that overexpression of STAMBPL1 promoted the expression of HIF1α, which could be rescued by knockdown of FOXO1. (J) In HCC1937 cells stably overexpressing FOXO1, protein samples were collected after knockdown of GRHL3 with siRNA. Western blotting experiments showed that overexpression of FOXO1 promoted the expression of HIF1α, which could be rescued by knockdown of GRHL3. (K) The JASPAR website predicts the possible binding sequence of transcription factor FOXO1 to the GRHL3 promoter and its mutation pattern map. *: P<0.05, **: P<0.01, ***: P<0.001, t-test.

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STAMBPL1 promotes triple-negative breast cancer angiogenesis by upregulating the transcription of GRHL3/HIF1α/VEGFA via FOXO1

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STAMBPL1 promotes TNBC angiogenesis

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