Breast cancer is a leading cause of death in women worldwide, but the underlying mechanisms of breast tumorigenesis remain unclear. Fructose-1, 6-bisphosphatase 1 (FBP1), a rate-limiting enzyme in gluconeogenesis, was recently shown to be a tumor suppressor in breast cancer. However, the mechanisms of FBP1 as a tumor suppressor in breast cancer remain to be explored. Here we showed that FBP1 bound to Notch1 in breast cancer cells. Moreover, FBP1 enhanced ubiquitination of Notch1, further leading to proteasomal degradation via FBXW7 pathway. In addition, we found that FBP1 significantly repressed the transactivation of Notch1 in breast cancer cells. Functionally, Notch1 was involved in FBP1-mediated tumorigenesis of breast cancer cells in vivo and in vitro. Totally, these findings indicate that FBP1 inhibits breast tumorigenesis by regulating Notch1 pathway, highlighting FBP1 as a potential therapeutic target for breast cancer.

Implications:

We demonstrate FBP1 as a novel regulator for Notch1 in breast cancer.

Reprogramming metabolism is a hallmark of cancer, and maintains glucose homeostasis that is balanced by the catabolic glycolysis and anabolic gluconeogenesis pathways (1). Although main researches focus on glycolysis, Fructose-1, 6-biphosphatase (FBP), a rate-limiting enzyme in gluconeogenesis, is found to play an important role in tumorigenesis in many kinds of cancer (2). FBP catalyzes fructose-1, 6-bisphosphate (F-1, 6-P2) to fructose-6-phosphate (F6P) and this reaction is irreversible (3). FBP has two isoforms in mammalian cells, FBP1 and FBP2 (3). FBP1 consists of seven exons, and encodes a 362-amino acid protein and is mainly expressed in liver tissues; FBP2 is especially expressed in muscle (4). FBP1 functions as a tumor suppressor in many cancers through regulating aerobic glycolysis, such as hepatocellular carcinoma (5–8), pancreatic cancer (9, 10), kidney cancer (11), ovarian cancer (12), lung cancer (13), colorectal cancer (14), and breast cancer (15, 16). FBP1 is frequently downregulated in cancer progression. For example, FBP1 has a low expression in lung and breast cancers, which predicts a poor prognosis (13, 15). Furthermore, dysfunctions of NK cells by FBP1 induce inhibition of glycolysis during lung cancer progression (17). The key role of FBP1 in cancer is not only as an enzyme, but also as a coactivator for transcription factors to regulate target genes' expression. For example, FBP1 represses HIF-1α transcriptional activity via direct interaction (11). FBP1 functions as a novel regulator of Wnt/β-catenin pathway in breast cancer (15). In addition, FBP1 acts as a negative modulator of the IQGAP1–MAPK signaling axis in pancreatic ductal adenocarcinoma cells, and binding to the WW domain of IQGAP1 impedes IQGAP1-dependent ERK1/2 phosphorylation (pERK1/2) in a manner independent of FBP1 enzymatic activity (9), but the mechanisms of FBP1 as noncanonical functions of enzymes are still not fully understood in cancer.

Notch1 is highly expressed in many types of cancers, which was required for cancer cell proliferation and survival (18). The Delta-like 4 (DLL4) transmembrane ligands in adjoining cells can bind to and activate Notch1 receptor, and is cleaved by proteolytic enzymes within the membrane, thereby releasing its intracellular domain (ICN1, the activated form of Notch1; ref. 18). The ICN1 translocates into the nucleus to combine with coactivators and specifically promotes target genes' expression (19). The activated Notch receptor functions as an oncogene to regulate breast tumorigenesis and increased expression of Notch1 and its ligand Jagged-1 predicts poorer overall survival for women with breast cancer (20). Although the molecular events underlying Notch1 signal pathway have been well characterized, the mechanisms of regulation Notch1 protein stability have not been well understood.

In this study, we demonstrate that FBP1 is a crucial regulator of breast cancer tumorigenesis due to its interaction with Notch1. The underlying mechanisms of interaction include destabilization and inhibition of transcriptional activity of Notch1. Our in vivo and in vitro data suggest that Notch1 is required for FBP1-mediated breast tumorigenesis. Therefore, our study uncovers a rationale for the use of a FBP1/Notch1 pathway as the potential target for therapeutic intervention in breast cancer.

Cell culture, plasmids, reagents, and antibodies

HEK293T, MCF-7, and MB231 cell lines were obtained from ATCC. All cell lines were cultured in DMEM (Hyclone) supplemented with 10% FBS (Hyclone), 100 U/mL penicillin, and 100 μg/mL streptomycin. All cell lines were maintained at 37°C in a humidified incubator containing 5% CO2. The identity of the cell lines was verified by short tandem repeat analysis. All cell lines were demonstrated to be free of contamination with Mycoplasma by PCR every 3 months. All cell lines were passaged no more than 10 times for use in experiments.

PCR-amplified human genes used in this study were cloned into pcDNA3.0/HA, pDNA3.1/Flag, pFlag-CMV4, pEGFP-C1, PET28a-His, or pGEX-4T-1. The mutants were generated by using overlap PCR. Mouse anti-Notch1, rabbit or mouse anti-FBP1, -HA, -GFP, -Flag, β-actin antibodies, or G418 were from Sigma. Rabbit anti-Notch1 antibody was from ProteinTech. Puromycin was from Gibco. Rabbit IgG and mouse IgG were from Santa Cruz Biotechnology.

Quantitative real-time PCR analysis

Total RNA isolation, reverse transcription (RT), and real-time PCR were conducted as described previously (21, 22). The following primer pairs were used for quantitative real-time PCR: MMP9, 5′-GCCTGCAACGTGAACATCT-3′(forward) and 5′-TCAAAGACCGAGTCCAGCTT-3′(reverse); JAG1, 5′-TGGTCAACGGCGAGTCCTTTAC-3′(forward) and 5′- GCAGTCATTGGTATTCTGAGCACAG-3′(reverse); HES1, 5′- ACGTGCGAGGGCGTTAATAC-3′(forward) and 5′-ATTGATCTGGGTCATGCAGTTG-3′(reverse); HEY1, 5′-CCGCTGATAGGTTAGGTCTCATTTG-3′(forward) and 5′-TCTTTGTGTTGCTGGGGCTG-3′(reverse); β-actin, 5′-ATGGCCACGGCTGCTTCCAGC-3′(forward) and 5′-CATGGTGGTGCCGCCAGACAG-3′(reverse).

Immunoprecipitation and Western blotting

Cells were lysed in NP-40 lysis buffer [150 mmol/L NaCl, 50 mmol/L Tris-HCl (pH 7.5), and 0.5% NP40] with multiple protease inhibitors (Sigma-Aldrich). Cell lysates were incubated with indicated antibodies and protein-A-agarose overnight at 4°C. Normal mouse or rabbit IgG was used as a negative control. The beads were washed five times with lysis buffer, eluted with loading buffer by boiling for 10 minutes at 100°C, and the samples were detected by Western blotting. The immunoprecipitation (IP) assay and Western blot analysis were performed as described previously (23).

GST pull-down assay

GST-tagged Notch1 and His-tagged FBP1 proteins were generated in BL21 (DE3). Purified His-tagged FBP1 protein was mixed with purified GST or GST-tagged Notch1 fusion protein in PBS binding buffer at 4°C for 2 hours, followed by the addition of glutathione-Sepharose 4B beads. After 1–3 hours of incubation, the beads were washed five times with PBS and eluted with loading buffer by boiling for 10 minutes at 100°C, and the samples were followed by Western blotting. GST pull-down assay was performed as described previously (21).

Stable cell lines

The FBP1 shRNA (11) was generated with oligonucleotide 1# 5′-CCTTGATGGATCTTCCAACAT-3′, 2# 5′-CGACCTGGTTATGAACATGTT-3′. The Notch1 shRNA (24) was generated with oligonucleotide 5′-AAGTGTCTGAGGCCAGCAAGA-3′. The control shNC was generated with oligonucleotide 5′-TTCTCCGAACGGTCACGT-3′ and the plasmids were constructed by GenePharma. The shRNA plasmids were cotransfected with vectors expressing gag and vsvg genes into HEK293T cells. The viruses were harvested and applied to breast cancer cells. The FBP1 knockdown cells were selected with puromycin and Notch1 knockdown cells were selected with G418 for more than 2 weeks.

Luciferase reporter assays

Cells were seeded onto 6-well plates, transfected with Flag-tagged FBP1 or shFBP1 together with the reporter plasmid (HES1 promoter) and control pSV40-Renilla for 48 hours, and the cell extracts were analyzed using the Dual Luciferase Assay System (Promega) according to the manufacturer's instructions (25).

Clone formation, wound-healing assays, cell invasion, and CCK-8

Clone formation

The cells were seeded in 6-well plates (200–500 cells/plate) for 12–14 days. The cells were then fixed with 4% formaldehyde, stained with crystal violet, and photographed.

Wound-healing assays

Cells were seeded in 6-well plates that were incubated in culture medium until a monolayer was formed. The monolayer was then wounded by scratching with pipette tips and washed with PBS. After 1 day, cells were photographed. Cell invasion and CCK-8 were performed as described previously (23, 26).

IHC

Breast tissues were stained using human FBP1 and Notch1 antibodies. The staining was performed as described previously (27). In addition, IHC for Ki67 in mouse tumors from each group was performed. The following proportion scores were assigned by the intensity (0–3) and the percentage of cells with score of 0 (0%–5%), 1 (6%–25%), 2 (26%–50%), 3 (51%–75%), and 4 (76%–100%). The staining grade was stratified as absent (score 0), weak (score 1–4), moderate (score 5–8), or strong (score 9–12). The cut-off value for the low expression is set to <5. The use of human breast tumor specimens and the database was approved by the Human Assurance Committee of Affiliated Hospital of Weifang Medical University (Weifang, Shandong Province, P.R. China).

Xenografts

A total of 2 × 106 stable indicated expressing MCF-7 cells were injected subcutaneously into the shoulder sides of BALB/c nude mice (female, 4-week-old). The injections were performed as described previously (23, 28). Tumor volumes were calculated using the formula: volume = (length × width2)/2. The mice were sacrificed and tumors were weighed prior to further histologic evaluation. The use of animals in this study was approved by the Animal Care and Use Committee of Weifang Medical University (Weifang, Shandong Province, P.R. China).

The Cancer Genome Atlas database analyses

The gene expression data from The Cancer Genome Atlas (TCGA) Breast Carcinoma project were assessed and visualized by cBioPortal (https://www.cbioportal.org/). The Pearson correlations in mRNA expression levels between NOTCH1 and FBP1 were computed.

Statistical analysis

The data analysis was performed by using statistical program SPSS software version 17.0. Pearson correlation analysis was used to evaluate the relationship between two variables. Pairwise comparisons were performed using a two-tailed Student t test. P values less than 0.05 were considered significant.

FBP1 is a novel binding partner of Notch1

As described previously, FBP1 as tumor suppressor can modulate the activity of Hif-1α via physical interaction (11). To search for novel binding partners of FBP1, we overexpressed HA-tagged FBP1 protein in MCF-7 cells and then performed mass spectrometry to identify the associated proteins. Interestingly, we identified Notch1 as a novel interacting partner of FBP1 (Supplementary Table S1). To confirm this binding, we performed coimmunoprecipitation (co-IP) assay in HEK293T and MCF-7 cells. The results detected the existence of interaction between endogenous and exogenous FBP1 with ICN1 (Fig. 1AD). Furthermore, GST-pull down assay demonstrated that GST-ICN1 directly bound to His-FBP1 in vitro (Fig. 1E). Moreover, for the sake of validation which region of the two proteins mediated the interaction, we constructed several truncation mutants of Notch1 (Fig. 1F) and FBP1 (Fig. 1G) into GFP-tagged plasmids, which promoted protein fusion expression, and were not easy to aggregate in the cells (11, 29). The data demonstrated that the residue 1,761–2,442 aa of ICN1 and the residue 1–275 aa (E1–E6) of FBP1 were required for their interaction, but the residue 276–338 aa (E7) of FBP1 was unnecessary for their interaction (Fig. 1H and I). These results suggest that FBP1 could bind to Notch1 in breast cancer cells.

FBP1 promotes Notch1 degradation via the ubiquitin proteasome pathway

FBP1 could promote c-Myc degradation via the ubiquitin–proteasome pathway, which suggested FBP1 could regulate some proteins stability. So we made a hypothesis that FBP1 could regulate Notch1 protein stabilization. To test this hypothesis, we overexpressed Flag-tagged FBP1 with HA-ICN1 in HEK293T cells. Interestingly, FBP1 could dramatically decrease Notch1 protein level in a dose-dependent way (Fig. 2A). Furthermore, to determine whether FBP1 affects Notch1 protein level independently of its enzyme activity, we overexpressed mutant Flag-tagged FBP1 (G260R) in HEK293T cells that had been proven enzyme dead before (11). FBP1 (WT) decreased Notch1 protein level compared with control, but FBP1 (G260R) action on Notch1 was not different from FBP1 (WT), indicating that enzyme activity was not required for this regulation (Fig. 2B). Moreover, FBP1 protein expression was more in the MCF-7 cells than in the MB231 cells, so we used MCF-7 cells for knockdown and MB231 cells for overexpression (Supplementary Fig. S1A). The effects of shRNA for FBP1 were detected in MCF-7 cells (Supplementary Fig. S1B). In the later experiments, we used shRNA-1# to knock down FBP1. As we expected, knockdown of FBP1 by shRNA increased endogenous Notch1 protein level in MCF-7 cells (Fig. 2C). Conversely, overexpression of Flag-tagged FBP1 decreased endogenous Notch1 protein level in MB231 cells (Fig. 2D). Interestingly, FBP1 did not change mRNA levels of Notch1 (Supplementary Fig. S1C). Furthermore, we treated cells with cycloheximide (CHX) to test the half-life of ICN1. The overexpression of Flag-tagged FBP1 evidently reduced the half-life of ICN1 compared with the overexpression of a control vector (Fig. 2E). To determine whether the proteasome pathway is required for regulation of Notch1 protein stability, we transfected Flag-tagged FBP1 into MB231 cells. Forty-eight hours after transfection, we treated the cells with CHX or MG132 for 8 hours, and tested the Notch1 protein level by Western blotting. MG132 inhibited the FBP1-mediated degradation of Notch1 protein (Fig. 2F). Because the ubiquitin proteasome pathway contributes to Notch1 protein degradation (29), we examined the effects of FBP1 knockdown or overexpression on Notch1 ubiquitination. FBP1 significantly increased Notch1 ubiquitination, which was correlated with the decreased levels of Notch1 proteins (Fig. 2G and H). FBXW7 as a tumor suppressor is required for Notch1 degradation (30, 31), so we determine whether FBXW7 is required for FBP1 regulation of Notch1 degradation. Interestingly, FBP1 promoted FBXW7 binding to Notch1, which suggested that FBXW7 might be involved in FBP1-mediated Notch1 protein stability (Fig. 2I). These findings suggest that FBP1 negatively regulates Notch1 protein level through the ubiquitin proteasome pathway.

FBP1 reduces Notch1 transcriptional activity

FBP1 could regulate Notch1 protein stability, so we determined whether FBP1 regulated Notch1-mediated transcription. First, we analyzed the mRNA levels of Notch1 target genes after Flag-tagged FBP1 overexpression. Overexpressing FBP1 significantly reduced the mRNA levels of previously defined Notch1 target genes, including MMP9, JAG1, HES1, and HEY1, in MB231 cells, and the change of gene expression was negatively correlated with this overexpression (Fig. 3A). Conversely, knockdown of FBP1 increased Notch1 target genes' mRNA levels in MCF-7 cells (Fig. 3B). Furthermore, by luciferase reporter assays, we found that FBP1 significantly reduced the activity of human HSE1 promoter in breast cancer cells (Fig. 3C and D). Furthermore, knockdown or overexpression of Notch1 could abolish FBP1-mediated mRNA levels in breast cancer cells (Fig. 3E and F). Thus, our data reveal that FBP1 abrogates Notch1-mediated transcripts in breast cancer cells.

Notch1 is involved in FBP1-mediated cell proliferation and migration in vitro

To further validate the role of the Notch1/FBP1 signaling pathway in cell proliferation and migration, FBP1-knockdown or -overexpressing breast cancer cells were transfected with shNotch1 or HA-tagged ICN1 vectors. The cell proliferation was significantly changed with knockdown or overexpression of FBP1, but knockdown or overexpression of Notch1 could abrogate this cell proliferation (Fig. 4A and B). Moreover, colony-forming assay demonstrated that Notch1 was required for FBP1-mediated cell proliferation (Fig. 4C). Furthermore, cell migration was critical for breast cancer development. Cell scratch tests and transwell migration assays indicated that Notch1 was involved in FBP1-mediated inhibition of cell migration (Fig. 4D and E). These data indicate that Notch1 is involved in FBP1-mediated cell proliferation and migration in vitro.

FBP1 inhibits breast tumor growth via Notch1 in vivo

To determine whether FBP1 inhibits breast tumor growth via Notch1 in vivo, athymic nude mice subcutaneous engraftment assay was performed. Stable expression of shFBP1 MCF-7 cells exhibited much larger size in tumor volume and tumor weight compared with the control shNC group, but this promotion was abrogated by knockdown of Notch1 (Fig. 5A and B). Furthermore, Notch1 knockdown on the FBP1-knockdown background reduced the rate of tumor growth (Fig. 5C). Moreover, we tested the proliferation biomarker Ki67 expression in these tumors. The IHC staining results confirmed that knockdown Notch1 significantly repressed the FBP1-mediated breast tumor growth in vivo (Fig. 5D). These data demonstrate that FBP1 inhibits breast tumor growth via Notch1 in vivo.

FBP1 expression is negatively correlated with Notch1 in breast cancer

To determine the expressions of FBP1 and Notch1 in breast cancer tissues, we performed IHC assays in 80 cases of breast cancer tissues, and 6 cases of normal breast tissues as control. Notably, IHC staining indicated that FBP1 was more abundantly expressed in normal breast tissues (Fig. 6A and B), but Notch1 was expressed more in breast cancer tissues (Fig. 6C and D). Consistently, the data showed that FBP1 protein level was negatively correlated with the tumor size and stage of breast tumors, but Notch1 protein level was positively (Table 1). In addition, we demonstrated that the correlation between FBP1 and Notch1 expression was negative in breast cancer (Fig. 6E). To further validate our experimental results, we used the publicly available TCGA database to get FBP1 gene expression data in 1,904 breast invasive carcinoma samples. The FBP1 mRNA levels were low, expressed in nearly 7% breast invasive carcinoma samples (Supplementary Fig. S2A). At the same time, the Kaplan–Meier plotter (http://kmplot.com/analysis/index.php?p=service&cancer=breast) recurrence-free survival (RFS) indicated that a lower expression was closely correlated with poor outcome in 3,951 patients with breast cancer (Supplementary Fig. S2B). Furthermore, we also compared the coexpression between FBP1 and Notch1 in TCGA breast cancer samples. Interestingly, FBP1 and Notch1 were negative correlated with each other in human breast cancer tissues (Supplementary Fig. S2C). Thus, these data show that the protein levels of FBP1 and Notch1 are negatively correlated in human breast cancer.

FBP1 as a tumor suppressor played a crucial role in regulating gluconeogenesis and glycolysis, which is highly expressed in multiple cancers (32). However, FBP1 is downregulated in many kinds of cancers, the mechanisms of FBP1 as a tumor suppressor is not fully understood. Here, we found FBP1 could interact with Notch1, and decreased its protein stability through the ubiquitin proteasome pathway. Consistently, FBP1 decreased transcriptional activity of Notch1. Furthermore, loss of FBP1 promoted breast cancer cell proliferation via enhancing Notch1 function in vitro and in vivo. Interestingly, FBP1 expression was negatively correlation with Notch1 in breast cancer tissues. These findings reveal a new mechanism of FBP1 functions as a tumor suppressor in breast cancer.

Although FBP1 is a catalytic enzyme, it regulates Notch1 functions independently of its enzyme activity, which is consistent with previous study (11). FBP1 functions as a corepressor for transcription factors, including HIF-1α (11), c-MYC (33), and β-catenin (15), which represses tumorigenesis in multiple cancers. Thus, there are still other transcription factors to be discovered. Previous studies mostly focus on identifying downstream targets of Notch1, but less attention is taken to understand the upstream mechanisms holding Notch1 activities, especially those involved in Notch1 protein stabilization. From our findings, we demonstrate that FBP1 regulates Notch1 protein stability via promoting FBXW7 binding to Notch1, but the mechanism of this regulation needs more in-depth research.

FBP1 is downregulated in breast cancer and inhibits breast tumor progression through multiple pathways. For example, FBP1 modulates cell metabolism of breast cancer cells by inhibiting the expression of HIF-1α (34). Moreover, loss of FBP1 by snail-mediated repression provides metabolic advantages in basal-like breast cancer (32). We uncovered that Notch1 is involved in FBP1-mediated breast cancer tumorigenesis, which was consistent with previous studies (32, 34). However, a noncanonical role of Fructose-1, 6-bisphosphatase 1 is still unknown, which is worth further investigation.

In conclusion, we demonstrate that Notch1 is a new binding partner for FBP1 and plays an important role in FBP1-mediated tumorigenesis (Fig. 6F). Our data suggest that the FBP1/Notch1 protein complex may offer more opportunities to breast cancer prevention and therapies.

No potential conflicts of interest were disclosed.

Conception and design: Z. Yu

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): C. Lu, C. Ren, T. Yang, Y. Sun, D. Wang, S. Lv, Z. Yu

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): C. Lu, C. Ren, T. Yang, Y. Sun, P. Qiao, Z. Yu

Writing, review, and/or revision of the manuscript: C. Lu, Z. Yu

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): C. Lu, C. Ren, T. Yang, Y. Sun, Z. Yu

The study was supported by research grants from National Natural Science Foundation of China (grant no. 81972489), Shandong Province College Science and Technology Plan Project (grant no. J17KA254), Projects of Medical and Health Technology Development Program in Shandong Province (grant no. 2017WS398 and 2018WS057).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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