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A Non-Canonical Role of Fructose-1, 6-Bisphosphatase 1 Is Essential for Inhibition Of

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A Non-Canonical Role of Fructose-1, 6-Bisphosphatase 1 Is Essential for Inhibition Of Author Manuscript Published OnlineFirst on February 10, 2020; DOI: 10.1158/1541-7786.MCR-19-0842 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 1 A non-canonical role of fructose-1, 6-bisphosphatase 1 is essential for inhibition of 2 Notch1 in breast cancer 3 4 Chao Lu1#, Chune Ren1#, Tingting Yang1#, Yonghong Sun2#, Pengyun Qiao1, Dan Wang2, Shijun Lv2, Zhenhai Yu1* 5 6 1 Department of Reproductive Medicine, Affiliated Hospital of Weifang Medical University, Weifang, Shandong 7 Province, P.R. China. 8 2 Department of Pathology, Weifang Medical University, Weifang, Shandong Province, P.R. China. 9 #These authors contributed equally to this work. 10 *To whom correspondence should be addressed: Zhenhai Yu, Department of Reproductive Medicine, Affiliated 11 Hospital of Weifang Medical University, Weifang, Shandong Province, P.R. China. Fax: +86 5363083802, Tel: +86 12 5363081391, E-mail: [email protected]. 13 14 Running title: FBP1 inhibits Notch1 pathway in breast cancer. 15 16 17 Conflict of interest: The authors declare that they have no conflict of interest. 18 19 20 21 22 23 24 25 26 27 28 29 30 1 Downloaded from mcr.aacrjournals.org on September 26, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on February 10, 2020; DOI: 10.1158/1541-7786.MCR-19-0842 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 31 Abstract 32 Breast cancer is a leading cause of death in women worldwide, but the underlying mechanisms of 33 breast tumorigenesis remain unclear. Fructose-1, 6-bisphosphatase 1 (FBP1), a rate-limiting 34 enzyme in gluconeogenesis, was recently shown to be a tumor suppressor in breast cancer. 35 However, the mechanisms of FBP1 as a tumor suppressor in breast cancer remain to be explored. 36 Here we showed that FBP1 bound to Notch1 in breast cancer cells. Moreover, FBP1 enhanced 37 ubiquitination of Notch1, further leading to proteasomal degradation via FBXW7 pathway. In 38 addition, we found that FBP1 significantly repressed the transactivation of Notch1 in breast cancer 39 cells. Functionally, Notch1 was involved in FBP1-mediated tumorigenesis of breast cancer cells in 40 vivo and in vitro. Totally, these findings indicate that FBP1 inhibits breast tumorigenesis by 41 regulating Notch1 pathway, highlighting FBP1 as a potential therapeutic target for breast cancer. 42 43 Implications: we demonstrate FBP1 as a novel regulator for Notch1 in breast cancer. 44 Key word 45 FBP1; Notch1; Interaction; Cell proliferation; Cancer; 46 47 48 49 50 51 52 53 54 55 56 57 58 59 2 Downloaded from mcr.aacrjournals.org on September 26, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on February 10, 2020; DOI: 10.1158/1541-7786.MCR-19-0842 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 60 Introduction 61 Reprogramming metabolism is a hallmark of cancer, and keeps glucose homeostasis which is 62 balanced by the catabolic glycolysis and anabolic gluconeogenesis pathways (1). Although main 63 researches focus on glycolysis, Fructose-1, 6-biphosphatase (FBP), a rate-limiting enzyme in 64 gluconeogenesis, is found to play an important role in tumorigenesis in many kinds of cancer (2). 65 FBP catalyzes fructose-1, 6-bisphosphate (F-1, 6-P2) to fructose-6-phosphate (F6P), and this 66 reaction is irreversible (3). FBP has two isoforms in mammalian cells, FBP1 and FBP2 (3). FBP1 67 consists of seven exons, and encodes a 362-aminoacid protein, is mainly expressed in liver tissues, 68 and FBP2 is specially expressed in muscle (4). FBP1 functions as a tumor suppressor in many 69 cancers through regulating aerobic glycolysis, such as hepatocellular carcinoma (5-8), pancreatic 70 cancer (9,10), kidney cancer (11), ovarian cancer (12), lung cancer (13), colorectal cancer (14) and 71 breast cancer (15,16). FBP1 is frequently down-regulated in cancer progression. For example, 72 FBP1 has a low expression in lung and breast cancers, which predicts a poor prognosis (13,15). 73 Further, dysfunctions of Natural Killer Cells by FBP1 induce inhibition of glycolysis during lung 74 cancer progression (17). But the key role of FBP1 in cancer is not only as an enzyme but also as a 75 co-activitor for transcription factors to regulated target genes expression. For example, FBP1 76 represses HIF-1α transcriptional activity via direct interaction (11). FBP1 functions as a novel 77 regulator of Wnt/β-Catenin pathway in breast cancer (15). In addition, FBP1 acts as a negative 78 modulator of the IQGAP1–MAPK signaling axis in pancreatic ductal adenocarcinoma cells, and 79 binding to the WW domain of IQGAP1 impedes IQGAP1-dependent ERK1/2 phosphorylation 80 (pERK1/2) in a manner independent of FBP1 enzymatic activity (9). But the mechanisms of FBP1 81 as non-canonical functions of enzymes are still not fully understood in cancer. 82 Notch1 is highly expressed in many types of cancers, which was required for cancer cell 83 proliferation and survival (18). The Delta-like 4 (DLL4) transmembrane ligands in adjoining cells 84 can bind to and activate Notch1 receptor, and is cleaved by proteolytic enzymes within the 85 membrane, thereby releasing its intracellular domain (ICN1, the activated form of Notch1) (18). 86 The ICN1 translocates into the nucleus to combine with co-activators and specifically promotes 87 target genes expression (19). The activated Notch receptor functions as an oncogene to regulate 88 breast tumorigenesis, and increased expression of Notch1 and its ligand Jagged-1 predicts poorer 89 overall survival for women with breast cancer (20). Although the molecular events underlying 3 Downloaded from mcr.aacrjournals.org on September 26, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on February 10, 2020; DOI: 10.1158/1541-7786.MCR-19-0842 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 90 Notch1 signal pathway have been well characterized, the mechanisms of regulation Notch1 91 protein stability have not been well understood. 92 In this study, we demonstrate that FBP1 is a crucial regulator of breast cancer tumorigenesis due to 93 its interaction with Notch1. The underlying mechanisms of interaction include destabilization and 94 inhibition transcriptional activity of Notch1. Our in vivo and in vitro data suggest that Notch1 is 95 required for FBP1-mediated breast tumorigenesis. Therefore, our study uncovers a rationale for 96 the use of a FBP1/Notch1 pathway as the potential target for therapeutic intervention in breast 97 cancer. 98 Materials and Methods 99 Cell culture, plasmids, reagents and antibodies 100 HEK293T, MCF-7 and MB231 cell lines were obtained from ATCC (Manassas, VA, USA). All 101 cell lines were cultured in DMEM medium (HyClone) supplemented with 10% FBS (HyClone), 102 100U/ml penicillin and 100μg/ml Streptomycin. All cell lines were maintained at 37°C in a 103 humidified incubator containing 5% CO2. The identity of the cell lines was verified by short 104 tandem repeat analysis. All cell lines were demonstrated to be free of contamination with 105 mycoplasma by PCR every three months. All cell lines were passaged no more than 10 times for 106 use in experiments. 107 PCR-amplified human genes used in this study were cloned into pcDNA3.0/HA, pDNA3.1/Flag, 108 pFlag-CMV4, pEGFP-C1, PET28a-His, or pGEX-4T-1. The mutants were generated by using 109 overlap PCR. Mouse anti-Notch1, rabbit or mouse anti-FBP1, -HA, -GFP, -Flag, β-actin 110 antibodies or G418 were from Sigma. Rabbit anti-Notch1 antibody was from Proteintech. 111 Puromycin was from GBICO. Rabbit IgG and mouse IgG were from Santa Cruz Biotechnology. 112 Quantitative Real-Time PCR analysis 113 Total RNA isolation, reverse transcription (RT), and real-time PCR were conducted as described 114 previously (21,22). The following primer pairs were used for quantitative real-time PCR: MMP9, 115 5’-GCCTGCAACGTGAACATCT-3’(forward) and 116 5’-TCAAAGACCGAGTCCAGCTT-3’(reverse); JAG1, 117 5’-TGGTCAACGGCGAGTCCTTTAC-3’(forward) and 5’- 4 Downloaded from mcr.aacrjournals.org on September 26, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on February 10, 2020; DOI: 10.1158/1541-7786.MCR-19-0842 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 118 GCAGTCATTGGTATTCTGAGCACAG-3’(reverse); HES1, 5’- 119 ACGTGCGAGGGCGTTAATAC-3’(forward) and 120 5’-ATTGATCTGGGTCATGCAGTTG-3’(reverse); HEY1, 121 5’-CCGCTGATAGGTTAGGTCTCATTTG-3’(forward) and 122 5’-TCTTTGTGTTGCTGGGGCTG-3’(reverse); β-actin, 123 5’-ATGGCCACGGCTGCTTCCAGC-3’(forward) and 124 5’-CATGGTGGTGCCGCCAGACAG-3’(reverse). 125 Immunoprecipitation and western blotting 126 Cells were lysed in NP-40 lysis buffer (150 mM NaCl, 50 mM Tris-HCl [pH 7.5], and 0.5% NP40) 127 with multiple protease inhibitors (Sigma-Aldrich). Cell lysises was incubated with indicated 128 antibodies and protein-A-agarose overnight at 4℃. Normal mouse or rabbit IgG was used as 129 negative control. The beads were washed five times with lysed buffer, and eluted with loading 130 buffer by boiling for 10 min at 100℃, and the samples were detected by Western blotting. The IP 131 assay and western blot were performed as described previously (23). 132 GST pull-down assay 133 GST-tagged Notch1 and His-tagged FBP1 proteins were generated in BL21 (DE3). Purified 134 His-tagged FBP1 protein was mixed with purified GST or GST-tagged Notch1 fusion protein in 135 PBS binding buffer at 4°C for 2 h, followed by the addition of glutathione-Sepharose 4B beads. 136 After 1-3 h of incubation, the beads were washed five times with PBS and eluted with loading 137 buffer by boiling for 10 min at 100℃, and the samples were followed by Western blotting.
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