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

4 Chao Lu1#, Chune Ren1#, Tingting Yang1#, Yonghong Sun2#, Pengyun Qiao1, Dan Wang2, Shijun Lv2, Zhenhai Yu1*

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].

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.

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.

43 Implications: we demonstrate FBP1 as a novel regulator for Notch1 in breast cancer.

44 Key word

45 FBP1; Notch1; Interaction; Cell proliferation; Cancer;

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

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’-

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. GST

138 pull-down assay was performed as described previously (21).

139 Stable cell lines

140 The FBP1 shRNA (11) was generated with oligonucleotide 1#

141 5’-CCTTGATGGATCTTCCAACAT-3’, 2# 5’-CGACCTGGTTATGAACATGTT-3’. The Notch1

142 shRNA (24) was generated with oligonucleotide 5’-AAGTGTCTGAGGCCAGCAAGA-3’， The

143 control shNC was generated with oligonucleotide 5’-TTCTCCGAACGGTCACGT-3’, and the

144 plasmids were constructed by GenePharma company. The shRNA plasmids were co-transfected

145 with vectors expressing gag and vsvg genes into HEK293T cells. The viruses were harvested and

146 applied to breast cancer cells. The FBP1 knockdown cells were selected with puromycin, and

147 Notch1 knockdown cells were selected with G418 for more than 2 weeks.

148 Luciferase reporter assays

149 Cells were seeded onto 6-well plates, transfected with Flag-tagged FBP1 or shFBP1 together with

150 the reporter plasmid (HES1 promoter) and control pSV40-Renilla for 48hr, and the Cell extracts

151 were analyzed using the Dual Luciferase Assay System (Promega) according to the manufacturer’s

152 instructions (25).

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

154 Clone formation: the cells were seeded in 6-well plates (200-500 cells/plate) for 12-14 days. The

155 cells were then fixed with 4% formaldehyde, stained with crystal violet, and photographed. Wound

156 healing assays: cells were seeded in 6-well plates that were incubated in culture medium until a

157 monolayer was formed. The monolayer was then wounded by scratching with pipette tips and

158 washed with PBS. After 1 day, cells were photographed. Cell invasion and CCK-8 were performed

159 as described previously (23,26).

160 Immunohistochemistry (IHC)

161 Breast tissues were stained using human FBP1 and Notch1 antibodies. The staining was

162 performed as described previously(27). Additionally, IHC for Ki67 in mouse tumors from each

163 group was performed. The following proportion scores were assigned by the intensity (0–3) and

164 the percentage of cells with score of 0 (0-5%), 1 (6 to 25%), 2 (26 to 50%), 3 (51 to 75%) and 4

165 (76 to 100%). The staining grade was stratified as absent (score 0), weak (score 1 to 4), moderate

166 (score 5 to 8) or strong (score 9 to 12). The cutoff value for the low expression is set to﹤5. The

167 use of human breast tumor specimens and the database was approved by the Human Assurance

168 Committee of Affiliated Hospital of Weifang Medical University.

169 xenografts

170 2×106 stable indicated expressing MCF-7 cells were injected subcutaneously into the shoulder

171 sides of BALB/c nude mice (female 4-week-old). The injections were performed as described

172 previously (23,28). Tumor volumes were calculated using the formula: volume = (length×

173 width2)/2. The mice were sacrificed, and tumors were weighed prior to further histological

174 evaluation. The use of animals in this study was approved by the Animal Care and Use Committee

175 of Weifang Medical University.

176 TCGA database analyse

177 The gene expression data from the TCGA Breast Carcinoma project were assessed and visualized

178 by cBioPortal (https://www.cbioportal.org/). The Pearson correlations in mRNA expression levels

179 between NOTCH1 and FBP1 were computed.

180 Statistical analysis

181 The data analysis was performed by using statistical program SPSS software version 17.0

182 (Chicago, IL). Pearson correlation analysis was used to evaluate the relationship between two

183 variables. Pairwise comparisons were performed using a two-tailed Student t test. P values less

184 than 0.05 were considered significant.

185 Results

186 FBP1 is a novel binding partner of Notch1

187 As described previously, FBP1 as tumor suppressor can modulate the activity of Hif-1α via

188 physical interaction (11). To search for novel binding partners of FBP1, we overexpressed

189 HA-tagged FBP1 protein in MCF-7 cells and then performed mass spectrometry to identify the

190 associated proteins. Interestingly, we identified Notch1 as a novel interacting partner of FBP1

191 (Table S1). To confirm this binding, we performed co-immunoprecipitation (Co-IP) assay in

192 HEK293T and MCF-7 cells. The results detected the existence of interaction between endogenous and

193 exogenous FBP1 with ICN1 (Fig. 1A-D). Furthermore, GST-pull down assay demonstrated that

194 GST-ICN1 directly bound to His-FBP1 in vitro (Fig. 1E). Moreover, for the sake of validation which

195 region of the two proteins mediated the interaction, we constructed several truncation mutants of

196 Notch1 (Fig. 1F) and FBP1 (Fig. 1G) into GFP-tagged plasmids, which promoted protein fusion

197 expression, and were not easy to aggregate in the cells (11,29). The data demonstrated that the

198 residue 1761-2442aa of ICN1 and the residue 1-275aa (E1-E6) of FBP1 were required for their

199 interaction, but the residue 276-338aa (E7) of FBP1 was unnecessary for their interaction (Fig. 1H,

200 I). These results suggest that FBP1 could bind to Notch1 in breast cancer cells.

201 FBP1 promotes Notch1 degradation via the ubiquitin proteasome pathway

202 FBP1 could promote c-Myc degradation via the ubiquitin-proteasome pathway, which suggested

203 FBP1 could regulate some proteins stability. So we made a hypothesis that FBP1 could regulate

204 Notch1 protein stabilization. To test this hypothesis, we overexpressed Flag-tagged FBP1 with

205 HA-ICN1 in HEK293T cells. Interestingly, FBP1 could dramatically decrease Notch1 protein

206 level in a dose-dependent way (Fig. 2A). Further, to determine whether FBP1 affects Notch1

207 protein level independently of its enzyme activity, we overexpressed mutant Flag-tagged FBP1

208 (G260R) in HEK293T cells which had been proved its enzyme dead before (11). FBP1 (WT)

209 decreased Notch1 protein level compared to control, but FBP1 (G260R) action on Notch1 was not

210 different from FBP1 (WT), indicating that enzyme activity was not required for this regulation

211 (Fig. 2B). Moreover, FBP1 protein expression was more in the MCF-7 cells than in the MB231

212 cells, so we used MCF-7 cells for knockdown and MB231 cells for overexpression (Fig. S1A).

213 The effects of shRNA for FBP1 were detected in MCF-7 cells (Fig. S1B). In the later experiments,

214 we used shRNA-1# to knock down FBP1. As we expected, knockdown of FBP1 by shRNA

215 increased endogenous Notch1 protein level in MCF-7 cells (Fig. 2C). Conversely, overexpression

216 of Flag-tagged FBP1 decreased endogenous Notch1 protein level in MB231 cells (Fig. 2D).

217 Interestingly, FBP1 didn’t change mRNA levels of Notch1 (Fig. S1C). Further, we treated cells

218 with cycloheximide (CHX) to test the half-life of ICN1. The overexpression of Flag-tagged FBP1

219 evidently reduced the half-life of ICN1 compared with the overexpression of a control vector (Fig.

220 2E). To determine whether the proteasome pathway is required for regulation of Notch1 protein

221 stability, we transfected Flag-tagged FBP1 into MB231 cells. Forty-eight hours after transfection,

222 we treated the cells with CHX or MG132 for 8h, and tested the Notch1 protein level by western

223 blotting. MG132 inhibited the FBP1-mediated degradation of Notch1 protein (Fig. 2F). Because

224 the ubiquitin proteasome pathway contributes to Notch1 protein degradation (29), we examined

225 the effects of FBP1 knockdown or overexpression on Notch1 ubiquitination. FBP1 significantly

226 increased Notch1 ubiquitination, which was correlated with the decreased levels of Notch1

227 proteins (Fig. 2G, H). FBXW7 as a tumor suppressor is required for Notch1 degradation (30,31),

228 so we determine whether FBXW7 is required for FBP1 regulation of Notch1 degradation.

229 Interestingly, FBP1 promoted FBXW7 binding to Notch1, which suggested that FBXW7 might be 8

230 involved in FBP1-mediated Notch1 protein stability (Fig. 2I). These findings suggest that FBP1

231 negatively regulates Notch1 protein level through the ubiquitin proteasome pathway.

232 FBP1 reduces Notch1 transcriptional activity

233 FBP1 could regulate Notch1 protein stability, so we determined whether FBP1 regulated

234 Notch1-mediated transcription. First, we analyzed the mRNA levels of Notch1 target genes after

235 Flag-tagged FBP1 overexpression. Overexpression FBP1 significantly reduced the mRNA levels

236 of previously defined Notch1 target genes, including MMP9, JAG1, HES1 and HEY1, in MB231

237 cells, and the change of gene expression was negatively correlated with this overexpression (Fig.

238 3A). Conversely, knockdown of FBP1 increased Notch1 target genes mRNA levels in MCF-7 cells

239 (Fig. 3B). Furthermore, by luciferase reporter assays, we found that FBP1 significantly reduced

240 the activity of human HSE1 promoter in breast cancer cells (Fig. 3C, D). Furthermore, knockdown

241 or overexpression of Notch1 could abolish FBP1-mediated mRNA levels in breast cancer cells

242 (Fig. 3E, F). Thus, our data reveal that FBP1 abrogates Notch1-mediated transcripts in breast

243 cancer cells.

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

245 To further validate the role of the Notch1/FBP1 signaling pathway in cell proliferation and

246 migration, FBP1-knockdown or overexpression breast cancer cells were transfected with shNotch1

247 or HA-tagged ICN1 vectors. The cell proliferation was significantly changed with knockdown or

248 overexpression of FBP1, but knockdown or overexpression of Notch1 could abrogate this cell

249 proliferation (Fig. 4A, B). Moreover, colony formation assay demonstrated that Notch1 was

250 required for FBP1-mediated cell proliferation (Fig. 4C). Further, cell migration was critical for

251 breast cancer development. Cell-scratch tests and transwell migration assays indicated that Notch1

252 was involved in FBP1-mediated inhibition of cell migration (Fig. 4D, E). These data indicate that

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

254 FBP1 inhibits breast tumor growth via Notch1 in vivo

255 To determine whether FBP1 inhibits breast tumor growth via Notch1 in vivo, athymic nude mice

256 subcutaneous engraftment assay was performed. Stable expression of shFBP1 MCF-7 cells

257 exhibited much larger size in tumor volume and tumor weight compared with the control shNC 9

258 group, but this promotion was abrogated by knockdown of Notch1 (Fig. 5A, B). Furthermore,

259 Notch1 knockdown on the FBP1-knockdown background reduced the rate of tumor growth (Fig.

260 5C). Moreover, we tested the proliferation biomarker Ki67 expression in these tumors. The

261 immunohistochemistry (IHC) staining results confirmed that knockdown Notch1 significantly

262 repressed the FBP1-mediated breast tumor growth in vivo (Fig. 5D). These data demonstrate that

263 FBP1 inhibits breast tumor growth via Notch1 in vivo.

264 FBP1 expression is negatively correlated with Notch1 in breast cancer

265 To determine the expressions of FBP1 and Notch1 in breast cancer tissues, we performed IHC

266 assays in 80 cases of breast cancer tissues, and 6 cases of normal breast tissues as control. Notably,

267 IHC staining indicated that FBP1 was more abundantly expressed in normal breast tissues (Fig.

268 6A, B), but Notch1 was expressed more in breast cancer tissues (Fig. 6C, D). Consistently, the

269 data showed that FBP1 protein level was negatively correlated with the tumor size and stage of

270 breast tumors, but Notch1 protein level was positively (Table 1). In addition, we demonstrated that

271 the correlation between FBP1 and Notch1 expression was negative in breast cancer (Fig. 6E). To

272 further validate our experimental results, we used the publicly available TCGA database in order

273 to get FBP1 gene expression data in 1904 breast invasive carcinoma samples. The FBP1 mRNA

274 levels were low expressed in nearly 7% breast invasive carcinoma samples (Fig. S2A). At the

275 same time, the KM plotter (http://kmplot.com/analysis/index.php?p=service&cancer=breast)

276 recurrence-free survival (RFS) indicated that a lower expression was closely correlated with poor

277 outcome in 3951 breast cancer patients (Fig. S2B). Further, we also compared the co-expression

278 between FBP1 and Notch1 in TCGA breast cancer samples. Interestingly, FBP1 and Notch1 were

279 negative correlated with each other in human breast cancer tissues (Fig. S2C). Thus, these data

280 show that the protein levels of FBP1 and Notch1 are negatively correlated in human breast cancer.

281 Discussion

282 FBP1 as a tumor suppressor played a crucial role in regulating gluconeogenesis and glycolysis,

283 which is highly expressed in multiple cancers (32). However, FBP1 is downregulated in many

284 kinds of cancers, the mechanisms of FBP1 as a tumor suppressor is not fully understood. Here, we

285 found FBP1 could interact with Notch1, and decreased its protein stability through the ubiquitin

286 proteasome pathway. Consistently, FBP1 decreased transcriptional activity of Notch1.

287 Furthermore, loss of FBP1 promoted breast cancer cell proliferation via enhancing Notch1

288 function in vitro and in vivo. Interestingly, FBP1 expression was negatively correlation with

289 Notch1 in breast cancer tissues. These findings reveal a new mechanism of FBP1 functions as a

290 tumor suppressor in breast cancer.

291 Although FBP1 is a catalytic enzyme, it regulates Notch1 functions independently of its enzyme

292 activity, which is consistent with previous study (11). FBP1 functions as a co-repressor for

293 transcription factors, including HIF-1α (11), c-MYC (33) and β-catenin (15), which represses

294 tumorigenesis in multiple cancers. Thus, there are still other transcription factors to be discovered.

295 Previous studies mostly focus on identifying downstream targets of Notch1, but less attention is

296 taken to understand the upstream mechanisms holding Notch1 activities, especially those involved

297 in Notch1 protein stabilization. From our finding, we demonstrate that FBP1 regulates Notch1

298 protein stability via promotion FBXW7 binding to Notch1. But the mechanism of this regulation

299 need to be conducted more in-depth research.

300 FBP1 is down-regulated in breast cancer, and inhibits breast tumor progression through multiple

301 pathways. For example, FBP1 modulates cell metabolism of breast cancer cells by inhibiting the

302 expression of HIF-1α (34). More, loss of FBP1 by snail-mediated repression provides metabolic

303 advantages in basal-like breast cancer (32). We uncovered the Notch1 is involved in FBP1

304 mediated-breast cancer tumorigenesis, which was consistent with previous studies (32,34).

305 However, a non-canonical role of Fructose-1, 6-bisphosphatase 1 is still unknown, which is worth

306 further investigation.

307 In conclusion, we demonstrate that Notch1 is a new binding partner for FBP1, and plays an

308 important role in FBP1-mediated tumorigenesis (Fig. 6F). Our data suggest that the FBP1/Notch1

309 protein complex may offer more opportunities to breast cancer prevention and therapies.

310 Conflict of interest: The authors declare that they have no conflicts of interest with the contents

311 of this article.

312 Acknowledgments: The study was supported by research grants from National Natural Science

313 Foundation of China (Grant no. 81972489), Shandong Province College Science and Technology

314 Plan Project (Grant no. J17KA254), Projects of medical and health technology development

315 program in Shandong province (Grant no. 2017WS398 and 2018WS057).

316 Author Contributions：Z.Y. designed research; C.L., T.Y., C.R., Q.P. X.H, and L.W.

317 performed research; D.W., C.L., Y.S. and S.L. contributed new reagents/analytic tools; Z.Y.,

318 C.L. and T.Y. analyzed data; Z.Y. wrote and revised the paper.

319 Reference 320 1. Petersen MC, Vatner DF, Shulman GI. Regulation of hepatic glucose metabolism in health and 321 disease. Nat Rev Endocrinol 2017;13:572-87 322 2. Hunter RW, Hughey CC, Lantier L, Sundelin EI, Peggie M, Zeqiraj E, et al. Metformin 323 reduces liver glucose production by inhibition of fructose-1-6-bisphosphatase. Nat Med 324 2018;24:1395-406 325 3. Erion MD, van Poelje PD, Dang Q, Kasibhatla SR, Potter SC, Reddy MR, et al. MB06322 326 (CS-917): A potent and selective inhibitor of fructose 1,6-bisphosphatase for controlling 327 gluconeogenesis in type 2 diabetes. Proc Natl Acad Sci U S A 2005;102:7970-5 328 4. Kaur R, Dahiya L, Kumar M. Fructose-1,6-bisphosphatase inhibitors: A new valid approach 329 for management of type 2 diabetes mellitus. Eur J Med Chem 2017;141:473-505 330 5. Chen R, Li J, Zhou X, Liu J, Huang G. Fructose-1,6-Bisphosphatase 1 Reduces (18)F FDG 331 Uptake in Hepatocellular Carcinoma. Radiology 2017;284:844-53 332 6. Liu GM, Li Q, Zhang PF, Shen SL, Xie WX, Chen B, et al. Restoration of FBP1 suppressed 333 Snail-induced epithelial to mesenchymal transition in hepatocellular carcinoma. Cell Death 334 Dis 2018;9:1132 335 7. Hirata H, Sugimachi K, Komatsu H, Ueda M, Masuda T, Uchi R, et al. Decreased Expression 336 of Fructose-1,6-bisphosphatase Associates with Glucose Metabolism and Tumor Progression 337 in Hepatocellular Carcinoma. Cancer Res 2016;76:3265-76 338 8. Yang LN, Ning ZY, Wang L, Yan X, Meng ZQ. HSF2 regulates aerobic glycolysis by 339 suppression of FBP1 in hepatocellular carcinoma. Am J Cancer Res 2019;9:1607-21 340 9. Jin X, Pan Y, Wang L, Ma T, Zhang L, Tang AH, et al. Fructose-1,6-bisphosphatase Inhibits 341 ERK Activation and Bypasses Gemcitabine Resistance in Pancreatic Cancer by Blocking 342 IQGAP1-MAPK Interaction. Cancer Res 2017;77:4328-41 343 10. Yang C, Zhu S, Yang H, Deng S, Fan P, Li M, et al. USP44 suppresses pancreatic cancer 344 progression and overcomes gemcitabine resistance by deubiquitinating FBP1. Am J Cancer 345 Res 2019;9:1722-33 346 11. Li B, Qiu B, Lee DS, Walton ZE, Ochocki JD, Mathew LK, et al. Fructose-1,6-bisphosphatase 347 opposes renal carcinoma progression. Nature 2014;513:251-5 348 12. Xiong X, Zhang J, Hua X, Cao W, Qin S, Dai L, et al. FBP1 promotes ovarian cancer 349 development through the acceleration of cell cycle transition and metastasis. Oncol Lett 350 2018;16:1682-8 351 13. Sheng H, Ying L, Zheng L, Zhang D, Zhu C, Wu J, et al. Down Expression of FBP1 Is a 352 Negative Prognostic Factor for Non-Small-Cell Lung Cancer. Cancer Invest 2015;33:197-204 353 14. Li Q, Wei P, Wu J, Zhang M, Li G, Li Y, et al. The FOXC1/FBP1 signaling axis promotes 354 colorectal cancer proliferation by enhancing the Warburg effect. Oncogene 2019;38:483-96 355 15. Li K, Ying M, Feng D, Du J, Chen S, Dan B, et al. Fructose-1,6-bisphosphatase is a novel 12

356 regulator of Wnt/beta-Catenin pathway in breast cancer. Biomed Pharmacother 357 2016;84:1144-9 358 16. Fu D, Li J, Wei J, Zhang Z, Luo Y, Tan H, et al. HMGB2 is associated with malignancy and 359 regulates Warburg effect by targeting LDHB and FBP1 in breast cancer. Cell Commun Signal 360 2018;16:8 361 17. Cong J, Wang X, Zheng X, Wang D, Fu B, Sun R, et al. Dysfunction of Natural Killer Cells 362 by FBP1-Induced Inhibition of Glycolysis during Lung Cancer Progression. Cell Metab 363 2018;28:243-55 e5 364 18. Lobry C, Oh P, Aifantis I. Oncogenic and tumor suppressor functions of Notch in cancer: it's 365 NOTCH what you think. J Exp Med 2011;208:1931-5 366 19. Previs RA, Coleman RL, Harris AL, Sood AK. Molecular pathways: translational and 367 therapeutic implications of the Notch signaling pathway in cancer. Clin Cancer Res 368 2015;21:955-61 369 20. Reedijk M, Odorcic S, Chang L, Zhang H, Miller N, McCready DR, et al. High-level 370 coexpression of JAG1 and NOTCH1 is observed in human breast cancer and is associated 371 with poor overall survival. Cancer Res 2005;65:8530-7 372 21. Yu Z, Zhao X, Huang L, Zhang T, Yang F, Xie L, et al. Proviral insertion in murine 373 lymphomas 2 (PIM2) oncogene phosphorylates pyruvate kinase M2 (PKM2) and promotes 374 glycolysis in cancer cells. J Biol Chem 2013;288:35406-16 375 22. Ren C, Yang T, Qiao P, Wang L, Han X, Lv S, et al. PIM2 interacts with tristetraprolin and 376 promotes breast cancer tumorigenesis. Mol Oncol 2018;12:690-704 377 23. Yang T, Ren C, Qiao P, Han X, Wang L, Lv S, et al. PIM2-mediated phosphorylation of 378 hexokinase 2 is critical for tumor growth and paclitaxel resistance in breast cancer. Oncogene 379 2018;37:5997-6009 380 24. Rizzo P, Miao H, D'Souza G, Osipo C, Song LL, Yun J, et al. Cross-talk between notch and the 381 estrogen receptor in breast cancer suggests novel therapeutic approaches. Cancer Res 382 2008;68:5226-35 383 25. Yu Z, Ge Y, Xie L, Zhang T, Huang L, Zhao X, et al. Using a yeast two-hybrid system to 384 identify FTCD as a new regulator for HIF-1alpha in HepG2 cells. Cell Signal 2014;26:1560-6 385 26. Yang T, Ren C, Lu C, Qiao P, Han X, Wang L, et al. Phosphorylation of HSF1 by PIM2 386 Induces PD-L1 Expression and Promotes Tumor Growth in Breast Cancer. Cancer Res 387 2019;79:5233-44 388 27. Han X, Ren C, Yang T, Qiao P, Wang L, Jiang A, et al. Negative regulation of AMPKalpha1 389 by PIM2 promotes aerobic glycolysis and tumorigenesis in endometrial cancer. Oncogene 390 2019;38:6537-49 391 28. Yu Z, Huang L, Qiao P, Jiang A, Wang L, Yang T, et al. PKM2 Thr454 phosphorylation 392 increases its nuclear translocation and promotes xenograft tumor growth in A549 human lung 393 cancer cells. Biochem Biophys Res Commun 2016;473:953-8 394 29. Wang Z, Hu Y, Xiao D, Wang J, Liu C, Xu Y, et al. Stabilization of Notch1 by the Hsp90 395 Chaperone Is Crucial for T-Cell Leukemogenesis. Clin Cancer Res 2017;23:3834-46 396 30. Tsunematsu R, Nakayama K, Oike Y, Nishiyama M, Ishida N, Hatakeyama S, et al. Mouse 397 Fbw7/Sel-10/Cdc4 is required for notch degradation during vascular development. J Biol 398 Chem 2004;279:9417-23 399 31. Thompson BJ, Buonamici S, Sulis ML, Palomero T, Vilimas T, Basso G, et al. The SCFFBW7

400 ubiquitin ligase complex as a tumor suppressor in T cell leukemia. J Exp Med 401 2007;204:1825-35 402 32. Dong C, Yuan T, Wu Y, Wang Y, Fan TW, Miriyala S, et al. Loss of FBP1 by Snail-mediated 403 repression provides metabolic advantages in basal-like breast cancer. Cancer Cell 404 2013;23:316-31 405 33. Wang B, Fan P, Zhao J, Wu H, Jin X. FBP1 loss contributes to BET inhibitors resistance by 406 undermining c-Myc expression in pancreatic ductal adenocarcinoma. J Exp Clin Cancer Res 407 2018;37:224 408 34. Shi L, He C, Li Z, Wang Z, Zhang Q. FBP1 modulates cell metabolism of breast cancer cells 409 by inhibiting the expression of HIF-1alpha. Neoplasma 2017;64:535-42 410 411 Figure Legends

412 Fig. 1. FBP1 is a novel binding partner of Notch1.

413 (A, B) Co-IP assay showed the interaction between exogenous FBP1 and ICN1 in HEK293T cells.

414 (C, D) Co-IP assay showed the interaction between endogenous FBP1 and ICN1 in MCF-7 cells.

415 (E) GST-pulldown assays showed that purified GST-ICN1 had physical interaction with

416 His-FBP1.

417 (F) Schematic representation of ICN1 truncation mutants.

418 (G) Schematic representation of FBP1 truncation mutants.

419 (H) Co-IP assay showed the interaction between HA-tagged FBP1 and GFP-tagged ICN1

420 truncation mutants in HEK293T cells.

421 (I) Co-IP assay showed the interaction between HA-tagged ICN1 and GFP-tagged FBP1

422 truncation mutants in HEK293T cells.

423 All experiments were repeated at least 3 times.

424 Fig. 2. FBP1 promotes Notch1 degradation via the ubiquitin proteasome pathway.

425 (A, B) HEK293T cells were transfected with indicated plasmids. Western blot tested whole cell

426 lysate after 72hr transfection.

427 (C) MCF-7 cells were stably expressed shRNA-FBP1 followed by western blot.

428 (D) MB231 cells were transfected with indicated plasmids. Western blot tested whole cell lysate

429 after 72hr transfection.

430 (E) MB231 cells were transfected with Flag-tagged FBP1. After 48hr transfection, cells were

431 treated with 100μg/mL cycloheximide (CHX) for the indicated amount of time followed by

432 western blot. The data represent mean ± SD of three independent experiments, *p<0.05.

433 (F) MB231 cells were transfected with Flag-tagged FBP1. After 48hr transfection, cells were

434 treated with 20μg/mL of MG132 for 8hr followed by IP and western blot.

435 (G) MCF-7 cells were stably expressed shRNA-FBP1. Cells were treated with 20μg/mL of

436 MG132 for 8hr followed by IP and western blot.

437 (H) MB231 cells were transfected with Flag-tagged FBP1. After 48hr transfection, cells were

438 treated with CHX or MG132 for another 8hr incubation followed by western blot.

439 (I) MB231 cells were transfected with Flag-tagged FBP1. After 48hr transfection, Co-IP assay

440 showed the interaction between FBXW7 and ICN1.

441 All experiments were repeated at least 3 times.

442 Fig. 3. FBP1 reduces Notch1 transcriptional activity.

443 (A) MB231 cells were transfected with Flag-tagged FBP1. After 72hr transfection, qRT-PCR was

444 performed to analyze Notch1-targeted mRNA levels.

445 (B) MCF-7 cells were stably expressed shRNA-FBP1. qRT-PCR was performed to analyze

446 Notch1-targeted mRNA levels.

447 (C) Notch1 reporter activity in MB231 cells with or without Flag-tagged FBP1.

448 (D) Notch1 reporter activity in MCF-7 cells with or without stably expressed shRNA-FBP1.

449 (E) MCF-7 cells were stably expressed shRNA-NC or shRNA-FBP1 with shRNA-Notch1.

450 qRT-PCR was performed to analyze Notch1-targeted mRNA levels.

451 (F) MB231 cells were over-expressed Flag-tagged FBP1 or Flag-tagged FBP1 with HA-ICN1.

452 qRT-PCR was performed to analyze Notch1-targeted mRNA levels.

453 All data represent mean ± SD of three independent experiments, *p<0.05.

454 Fig. 4. Notch1 is involved in FBP1-mediated cell proliferation and migration in vitro.

455 (A) The growth curves of stable shRNA-FBP1 MCF-7 cells with or without stably expression of

456 shRNA-Notch1 were measured with CCK8 assay.

457 (B) The growth curves of overexpression Flag-tagged FBP1 MB231 cells with or without

458 expression of HA-tagged ICN1 were measured with CCK8 assay.

459 (C) Crystal violet staining assay evaluated the ability of colony formation in breast cancer cells

460 with expressing indicated proteins. 461 (D) Wound-healing assay evaluated the ability of migration in breast cancer cells with expressing 462 indicated proteins.

463 (E) Transwell assay evaluated the ability of invasion in breast cancer cells with expressing

464 indicated proteins.

465 All data represent mean ± SD of three independent experiments, *p<0.05.

466 Fig. 5. FBP1 inhibits breast tumor growth via Notch1 in vivo.

467 (A) The images of tumors were shown to compare the tumor size of each groups.

468 (B, C) Stably transfected MCF-7 cells with indicated proteins showed a significant reduction of

469 tumor weights (B) and tumor volume (C) compared with control group.

470 (D) Representative images of H/E staining and Ki67 staining of tumor samples (Scale bar, 20μm).

471 All data represent mean ± SD of six independent experiments, *p<0.05.

472 Fig. 6. FBP1 expression is negatively correlated with Notch1 in breast cancer.

473 (A, B) Histopathologic sections of breast tissues were stained with anti-FBP1 (A) or Notch1 (B)

474 antibodies. Representative images of indicated breast tissues are shown (scale bar, 20μm).

475 (C, D) Semi-quantitative immunohistochemical analysis of 6 normal and 80 breast tissues for

476 FBP1 or Notch1. *p<0.05.

477 (E) FBP1 and Notch1 expression are negatively correlated in breast patient samples.

478 (F) Schematic diagram of the proposed FBP1-Notch1 signaling pathway.

479 Table 1 Analysis of correlation between FBP1 or Notch1 protein levels and clinicopathological

480 parameters of breast cancer patients.

Table 1. Analysis of correlation between FBP1 and Notch1 protein levels and clinicopathological parameters of breast cancer patients FBP1 expression Notch1 expression

Variable n Low High P-value n Low High P-value

0.318 0.122 Age

36 28 8 36 12 24 ≤50y

44 38 6 44 8 36 >50y

Tumor size 0.017 0.004 34 24 10 34 14 20 ≤2 cm

46 42 4 46 6 40 >2 cm

0.003 0.007 TNM stage

52 38 14 52 18 34 Ⅰ-Ⅱ

28 28 0 28 2 26 Ⅲ-Ⅳ

ER status 0.174 0.791 50 39 11 50 12 38 ＋

30 27 3 30 8 22 －

PR status 0.318 0.606 44 38 6 44 12 32 ＋

36 28 8 36 8 28 －

HER2 0.704 0.441 42 34 8 42 12 30 ＋

－ 38 32 6 38 8 30

A non-canonical role of fructose-1, 6-bisphosphatase 1 is essential for inhibition of Notch1 in breast cancer

Chao Lu, Chune Ren, Tingting Yang, et al.

Mol Cancer Res Published OnlineFirst February 10, 2020.

Updated version Access the most recent version of this article at: doi:10.1158/1541-7786.MCR-19-0842

Supplementary Access the most recent supplemental material at: Material http://mcr.aacrjournals.org/content/suppl/2020/02/08/1541-7786.MCR-19-0842.DC1

Author Author manuscripts have been peer reviewed and accepted for publication but have not yet Manuscript been edited.

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://mcr.aacrjournals.org/content/early/2020/02/08/1541-7786.MCR-19-0842. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.