D-mannose suppresses the angiogenesis and progression of colorectal cancer

doi:10.21203/rs.3.rs-3913237/v1

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D-mannose suppresses the angiogenesis and progression of colorectal cancer

https://doi.org/10.21203/rs.3.rs-3913237/v1

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Angiogenesis is an important factor influencing the development of solid tumors, and vascular endothelial growth factor receptor-2 (VEGFR2) is a central regulator of angiogenesis. Antibodies and inhibitors against VEGFR2 have been widely used in various malignancies. However, the regulatory mechanism of VEGFR2 has not been fully clarified. Here, we show that D-mannose can significantly inhibit angiogenesis and tumor growth by degrading VEGFR2. Specifically, D-mannose inactivates GSK3β by promoting the phosphorylation of GSK3β at Ser9, enhances nuclear translocation of TFE3, and promotes lysosomal biogenesis, thereby increasing lysosomal-mediated degradation of VEGFR2. Thus, D-mannose significantly suppressed proliferation, migration and capillary formation of human umbilical vein endothelial cells (HUVEC) in vitro. Oral administration of D-mannose dramatically inhibited angiogenesis and tumor growth in mice. Our findings reveal a previously unrecognized anti-tumor mechanism of D-mannose by destabilizing VEGFR2 and provide a new strategy for clinical treatment of colorectal cancer (CRC).

D-mannose

VEGFR2

GSK3β

TFE3

CRC

lysosome

Colorectal cancer (CRC) is a common malignant tumor of the gastrointestinal tract. Recent statistics showed that the incidence of CRC has risen to the third highest overall incidence and the second highest mortality rate¹. Despite advances in screening procedures and adjuvant therapy, the incidence, prevalence and mortality of CRC remain high¹. Therefore, developing new treatments and drugs for CRC is urgently needed.

Angiogenesis plays a crucial role in the growth, recurrence and metastasis of CRC². The formation of new blood vessels provides nutrients and oxygen for tumors, thus promoting the rapid proliferation of cancer cells³. Various growth factors, cytokines, and mechanical cues are implicated in the induction of angiogenesis within tumors⁴. The main pro-angiogenic drivers are vascular endothelial growth factor (VEGF) and its receptors VEGFR1–3⁵, among which, VEGFR2 has been identified as a critical component of VEGF-mediated angiogenesis⁶.

VEGFR2 is a large 151 kDa membrane protein consisting of 7 extracellular immunoglobulin (Ig)-like domains, a single transmembrane helix, and a split intracellular kinase domain⁷. VEGFR2 has been implicated in angiogenesis in many solid tumors, including breast cancer, colon cancer, hepatoma, and gastric cancer^{8 2, 9, 10}. VEGF induces receptor dimerization and subsequent phosphorylation by binding to VEGFR2, thereby maintaining downstream signaling. Phosphorylation of VEGFR2 promotes downstream activation of ERK1/2, AKT, and eNOS, which are critical for HUVEC proliferation, migration, and angiogenesis¹¹. Therefore, VEGFR2 blocking therapies that reduce its activity or stability and downstream signaling are of great clinical importance.

D-mannose is a six-carbon sugar, a C-2 epimer of glucose, which enters mammalian cells via the same transporters as glucose¹². When D-mannose enters the cell, it can be phosphorylated by hexokinase (HK) and produces mannose-6-phosphate (M6P), which can either enter the glycolytic pathway directly or provide a substrate for glycosylation¹². A number of studies have shown that D-mannose enhances the sensitivity of cancer cells to chemotherapy, radiotherapy, and immunotherapy^{13, 14}, and that D-mannose ameliorates ulcerative colitis in mice by inhibiting macrophage activation through the inhibition of IL-1-β production¹⁵. Recently, D-mannose was found to inhibit oxidative reactions and block phagocytosis in experimental neuroinflammation¹⁶. However, the role of D-mannose in angiogenesis and in the treatment of CRC is unclear.

In this study, we found that D-mannose significantly decreased the protein level of VEGFR2 and effectively inhibited angiogenesis and tumor growth in CRC. Mechanistically, D-mannose promotes VEGFR2 degradation by enhancing TFE3-mediated lysosomal biogenesis. Importantly, oral administration of D-mannose strongly inhibited angiogenesis and tumor growth of CRC in mice. Collectively, D-mannose may be useful for clinical treatment of CRC.

Cell Culture and reagents

HUVEC and HCT116 cells were cultured in DMEM supplemented with 10% FBS and 100µg/mL of penicillin and streptomycin. 786-O cells were cultured in RPMI-1640 supplemented with 10% FBS and 100µg/mL of penicillin and streptomycin. The cells were cultured in an environment containing 5% carbon dioxide at 37℃. The drugs used in this study include D-mannose (Sigma-Aldrich, 3458-28-4), L-mannose (Acmec, L82450), Fructose (Sangon Biotech, A100226), Galactose (Sangon Biotech, A600215), Fucose (Sangon Biotech, A606132), MG132 (MCE, HY-13259), NH4Cl (Sangon Biotech, A100621) 3-MA (TagertMol, T1879), Cycloheximide (Selleck, S7418), Rapamycin (Meilunbio, MB1197), and VEGF (MCE, HY-P73556).

siRNA Transfection

After transient transfection of siRNAs (siRNAs against Control, MITF, TFE3 and TFEB were commercially synthesized in Shanghai GenePharma), using Lipofectamine RNAiMAX Transfection Reagent (Invitrogen, 13778-150), the cells were cultured in serum-free cell culture medium for 4–6 h, and then replaced with complete culture medium. siRNA#1 sequences were previously reported¹⁷. siRNA#2 was purchased from Santa (siMITF#2, sc-35934; siTFEB#2, sc-38509; siTFE3#2, sc-38507).

Western blot (WB)

The total proteins from the cell lines were extracted using RIPA lysis buffer with 1% PMSF (Beyotime). The proteins were separated by SDS-PAGE gel electrophoresis and transferred onto a nitrocellulose membrane. The membrane was blocked with 5% skimmed milk powder at room temperature for 1.5 hour, and then incubated with primary antibody overnight at 4℃. After washing with TBST for three times, the secondary antibody was incubated at room temperature for 1 h. Then it was washed with TBST again for three times and visualized with the Chemiluminescent HRP Substrate (Millipore) on the Chemiluminescent Imaging System (Tanon Science and Technology Co, Ltd). All antibodies used for western blot are commercially available and shown in Table S1.

Quantitative Real-time PCR (qRT-PCR)

Total RNA was extracted from cells using the EZ-press RNA Purification Kit (EZBioscience), and RNA was synthesised into cDNA using a gDNA remover mixed with a reverse transcription master mix (EZBioscience). qRT-PCR was performed on the Applied Biosystem 7300 plus Sequence Detection System (Applied Biosystems). The Ct values were analyzed using the 2^−ΔΔCt method and the final results were presented as relative fold change. The mRNA expression of genes was normalized to the expression of β-actin gene. The primer sequences used in this study were listed in Table S2.

Separation of Nuclear and Cytoplasmic Proteins

The separation of nuclear and cytoplasmic proteins was conducted using the nuclear protein and cytoplasmic protein extraction kit (Beyotime, P0027) according to the instructions. It is required to add 1% phase phosphatase inhibitor and 1% protease inhibitor when employing reagents A and B. WB was used to examine the acquired protein samples.

LysoTracker Red staining

Cells were incubated at 37°C for 60 min in medium supplemented with Lysosomal Tracking Red DND-99 dye (50 nM, Yeason, 40739ES50) for specific staining of lysosomes. Cell nuclei were then labelled with Hoechst dye. After rinsing with PBS, observation and photographs were taken by using a fluorescence microscope (OLYMPUS, CKX53).

NAG Activity Assay

Cells were washed with PBS, extracts were added and cells were scraped off with a cell scraper. Cells were broken by ultrasound and then centrifuged to remove the supernatant. NAG assay was performed using Solarbio's kit (BC4290). Finally, the absorbance at 400nm was measured.

Colony Formation Assay

One thousand cells were seeded in a 6-well plate. The medium was changed every three days and the culture was stopped after two weeks when visible colonies were formed. After washing with PBS, cells were fixed with 4% methanol for 20 minutes and stained with crystal violet for 30 minutes. The crystal violet was discarded, washed with PBS and photographed.

Wound Healing Assay

When the cell density has grown to 90–100%, scratch the cells with a sterile pipette tip. Remove the medium, wash with PBS for three times to remove floating cells, and then treated with D-mannose and VEGF for 24h. Photos were taken under a microscope at 0 and 24 h and the migration rate was measured by Image J.

Cell Migration Assays

Cell migration was evaluated using a 24-well Transwell plate with polycarbonate membrane inserts (Corning). Add homogenous single-cell suspension (200 µL, 5 × 10⁴ cells/well) into the serum-free medium, and add 500 µL complete medium into the lower chambers. After 24 hours of incubation in a CO₂ incubator at 37°C, the migrated cells were fixed with methanol and stained with crystal violet for 30 minutes. Take pictures of different visual fields under the microscope, calculate the number of cells and conduct statistical analysis.

Tube formation assay

After spreading 50 µl of Matrigel per well, the 96-well plate was placed in a humidified incubator at 37°C for 30 min to allow the Matrigel to cure. Well-grown HUVEC were resuspended with the appropriate drugs to make a cell suspension of 1 × 10⁵ cells/ml. The prepared cells were added to a 96-well plate coated with Matrigel (200 µl per well) and cultured. The number of tubes formed was counted under an inverted microscope.

Immunohistochemical (IHC)

The obtained tissues were fixed using 4% paraformaldehyde, after which the tissues were embedded in paraffin and sectioned. After dewaxing of tissue sections, antigen repair was performed using citrate buffer at a concentration of 10 mM, pH 6.0 at 100°C. Sections were washed using PBS and the tissue was circled using an immunohistochemical pen to create a hydrophobic isolation zone. The sections were blocked using 3% BSA for 1 h. Overnight incubation was performed using a primary antibody, and after washing, incubation was performed for 30 min at room temperature using a secondary antibody. The sections were then stained using 3, 3′-diaminobenzidine (DAB) and counterstained with hematoxylin. Then the sections were observed and photographed under a light microscope and analyzed using ImageJ software.

Animal Experiments

The animal experiments in this study conform to the ethical review approved by the Department of Experimental Animal Science of Tongji University. The experiment was conducted as previously reported¹⁸. Briefly, male nude BALB/c mice (6- to 8-wk-old) were purchased from GemPharmatech Laboratory Animals. Mice were fed under SPF conditions and can freely choose food and water. Light/dark cycle was conducted every 12 hours, and the temperature was controlled (22–23℃). CT26 cells (1 × 10⁶) were injected into the right back of mice. 10% D-mannose was added to the drinking water of each mouse, and 200 µL of 20% D-mannose solution was fed to the mice by gavage every three days. At the end of the experiment, tumor volume and weight were recorded, tumor volume was calculated using the formula: length/2 × width².

Statistical analysis

Graphpad Prism 8.0.2 software was used to statistically analyze the experimental data. Two independent samples t-tests were used for comparisons between two groups, and one-way analysis of variance (ANOVA) was used for comparisons between multiple groups. *P < 0.05 was considered statistically significant.

D-mannose significantly decreases VEGFR2 protein level

VEGFR2 is critical for angiogenesis and development of tumor ¹⁹. To explore whether the metabolites affect the expression level of VEGFR2, we evaluated the effect of different hexoses on VEGFR2 in human umbilical vein endothelial cells (HUVEC) and colon cancer cell line HCT116. We observed that D-mannose, but not other hexoses, significantly reduced VEGFR2 protein level in both the cell lines (Fig. 1A-B). Similar phenomenon was observed in human renal clear cell adenocarcinoma line 786-O (Fig. 1C). In addition, we examined the changes in VEGFR2 at the transcriptional level and found that the above hexoses did not affect the mRNA level of VEGFR2 (Fig. 1D-F). We next treated the cells with D-mannose for different times and showed a time-dependent decrease in VEGFR2 protein levels (Fig. 2G-I). Furthermore, we found that D-mannose treatment reduced VEGFR2 protein level in a dose-dependent manner (Fig. 1J-L). To clarify whether the effect of D-mannose on VEGFR2 is isoform-specific, we treated cells with D-mannose or L-mannose and found that only D-mannose significantly reduced the protein level of VEGFR2, which suggests that D-mannose has an isoform-specific regulatory function on VEGFR2 protein level (Fig. 1M-O). Together, these results suggest that D-mannose downregulated VEGFR2 protein level.

D-mannose mediates VEGFR2 degradation via the lysosomal pathway

Next, to determine whether D-mannose reduces the expression of VEGFR2 by affecting its stability. We treated cells with the protein synthesis inhibitor cycloheximide (CHX) and found that VEGFR2 was degraded faster in cells treated with D-mannose compared with controls (Fig. 2A-C), indicating that D-mannose reduced the protein stability of VEGFR2. Given that the proteins in eukaryotes are mainly degraded via the proteasome²⁰ or lysosomal²¹ pathway, we then investigated the potential contribution of the proteasome or lysosomal pathway to D-mannose-induced degradation of VEGFR2 protein. We treated cells with NH₄Cl (an inhibitor of lysosome acidification), MG132 (an inhibitor of proteasome degradation) and 3-MA (an inhibitor of autophagy pathway) in the presence of D-mannose. Interestingly, results displayed that only NH₄Cl could partially restored D-mannose induced degradation of VEGFR2, but not MG132 or 3-MA (Fig. 2D-F), indicating that D-mannose promotes VEGFR2 degradation via lysosome.

D-mannose increases the biogenesis of lysosome and promotes TFE3-dependent degradation of VEGFR2

To further explore how D-mannose promotes lysosomal degradation of VEGFR2, we first examined whether D-mannose treatment affects lysosomal activity using LysoTracker Red. Interestingly, similar to rapamycin, D-mannose significantly increased lysosomal activity (Fig. 3A). In addition, we found that D-mannose significantly up-regulated the expression of many lysosomal biogenesis-related genes, including Atg9b, Map1lc3, and Vps11 etc. (Fig. 3B). We next examined N-Acetyl-β-D-Glucosidase (NAG) activity under D-mannose treatment and found that D-mannose was able to up-regulate NAG activity (Fig. 3C), suggesting that D-mannose can increase lysosomal protease activity. Lysosomal biogenesis is triggered primarily by the MiT/TFE family, including TFEB, TFE3, and MITF, thereby increasing lysosome numbers and promoting protein degradation^{17, 22}. We knocked down TFEB, TFE3, and MITF by siRNA in HUVEC, respectively, and found that D-mannose-induced VEGFR2 degradation was significantly reversed when silencing TFE3, but not TFEB or MITF (Fig. 3D-E), suggesting that D-mannose-induced VEGFR2 degradation is dependent on TFE3. Consistently, D-mannose treatment promoted TFE3 expression and increased its nuclear translocation (Fig. 3F). In addition, knockdown of TFE3 abolished the upregulation of NAG enzyme activity induced by D-mannose (Fig. 3G). In conclusion, the above results indicated that D-mannose could promote the nuclear translocation of TFE3 and lysosomal biogenesis to degrade VEGFR2.

Next, we explored the molecular mechanism of how D-mannose promotes TFE3 nuclear translocation. It was previously reported that glycogen synthase kinase 3 beta (GSK3β)-mediated phosphorylation of TFE3 promotes its subsequent nuclear export and inactivation^{23, 24}. By WB analysis, we found that D-mannose treatment significantly increased GSK3β phosphorylation at Ser9 in HUVEC (Fig. 3H), suggesting that D-mannose inhibited GSK3β activity. Taken together, these data suggest that D-mannose inactivates GSK3β by inducing its phosphorylation (Ser9), which promotes the nuclear translocation of TFE3, thereby enhancing lysosomal biogenesis and subsequent VEGFR2 degradation.

D-mannose inhibits proliferation, migration and capillary tube formation of HUVEC in vitro

Vascular endothelial growth factor (VEGF) is a key angiogenic growth factor that stimulates proliferation, migration, and capillary tube formation of HUVEC, primarily through VEGFR2²⁵. Therefore, we further examined the effects of D-mannose on proliferation, migration and capillary tube formation of HUVEC. Through these experiments, we found that D-mannose could significantly inhibit VEGF-enhanced cell proliferation (Fig. 4A). In addition, both wound healing and transwell migration assays demonstrated that D-mannose was able to repress the migration of HUVEC (Fig. 4B-C). Moreover, we found that D-mannose dramatically suppressed the tube formation via capillary tube formation assay (Fig. 4D). We also investigated the role of D-mannose in VEGFR2 signaling in HUVEC. Result showed that D-mannose reduced VEGF-induced p-AKT and p-ERK1/2 levels without affecting their protein levels (Fig. 4E). Collectively, these results suggest that D-mannose can inhibit proliferation, migration and capillary tube formation of HUVEC via suppressing VEGFR2 signaling.

D-mannose significantly suppresses the growth and angiogenesis of xenograft tumors in vivo

Since our data showed that D-mannose inhibited VEGFR2-mediated angiogenesis in vitro, we then evaluated whether D-mannose could inhibit tumor growth of CRC by suppressing tumor angiogenesis in vivo. We established a mouse CT26 colon cancer model to clarify the effect of D-mannose on CRC in vivo. The results showed that oral administration of D-mannose reduced tumor growth and tumor weight in mice (Fig. 5A-C). In addition, immunohistochemical (IHC) staining of tumor tissues showed that D-mannose also reduced the protein levels of Ki67, VEGFR2, and the vascular marker CD31 (Fig. 5D-E). Together, these results demonstrate that D-mannose represses the tumor growth of CRC by suppressing tumor angiogenesis.

Many studies have shown that glucose, a major source of cellular energy, plays a crucial role in tumor immunity and therapy. D-mannose, an isomer of glucose, exists naturally in many fruits, yet its function and its use in tumor therapy remains largely unexplored. In this study, we explored an unknown mechanism of D-mannose in anti-tumor treatment. We found that D-mannose promotes the degradation of VEGFR2, a major mediator of angiogenic signaling. Further studies revealed that D-mannose inactivated GSK3β, enhanced the nuclear translocation of TFE3, up-regulated the transcription levels of lysosomal biogenesis-related genes, and promoted lysosomal-mediated degradation of VEGFR2, thereby inhibiting angiogenesis and tumor growth of CRC (Fig. 6).

Angiogenesis, the formation and maintenance of vascular structures based on pre-existing blood vessels, plays an important role in physiological and pathological settings such as diabetes, inflammation, and cancer⁴.VEGFR2 is a central regulator of angiogenesis and can be activated in response to VEGF stimulation, thereby promoting downstream activation of ERK1/2 and AKT to induce angiogenic responses¹¹. Currently, anti-angiogenic therapy is widely used in the treatment of various cancers. For example, Apatinib, an oral receptor tyrosine kinase inhibitor that selectively targets VEGFR2, can inhibit the downstream signaling of VEGFR2 and has good anti-tumor effects on a variety of tumors, including CRC^{8, 26, 27}. In present study, we found that D-mannose can significantly down-regulate the protein level of VEGFR2, suggesting that D-mannose may be used to treat CRC by inhibiting angiogenesis and thus be a potential option for the treatment of CRC.

The stability and clearance of VEGFR2 are regulated by proteasome or endocytosis/lysosome-dependent degradation^{28, 29}. In this study, we found that D-mannose promoted lysosomal degradation of VEGFR2 by enhancing lysosomal biogenesis. Lysosomes are acidic compartments containing a variety of different types of hydrolases and sealed by the plasma membrane, which mediate the degradation of extracellular particles and intracellular components³⁰. The enhancement of lysosomal biogenesis promotes the clearance of accumulated proteins and damaged cells³¹. Lysosomal biogenesis is mainly triggered by the MiT/TFE family of transcription factors, including TFEB, TFE3, and MITF, which can bind to the Coordinated Lysosomal Expression and Regulation (CLEAR) element to stimulate the expression of its downstream target genes and promote lysosomal biogenesis and protein degradation^{17, 22}. Our study showed that D-mannose induces TFE3, but not TFEB and MITF, dependent lysosomal biogenesis in HUVEC. D-mannose treatment efficiently induced nuclear translocation of TFE3 and an increase of lysosomal NAG activity, facilitating the degradation of VEGFR2. We further explored the upstream molecular basis of D-mannose-induced TFE3 nuclear translocation and found that GSK3β was involved in this process. D-mannose was able to induce phosphorylation and inactivation of GSK3β, which promoted the nuclear translocation of TFE3.

In order to adapt to the expansion and growth of new blood vessels, HUVEC need to proliferate, migrate, and invade the basement membrane in response to angiogenic cues³². In this study, we found that D-mannose inhibited HUVEC proliferation, migration, and capillary formation. Activation of VEGFR2 was able to promote proliferation, migration and activation of core angiogenic factors ERK1/2, AKT and eNOS¹¹. Our study also demonstrated that D-mannose can block the activation of ERK1/2 and AKT downstream of VEGFR2, thereby inhibiting angiogenesis.

In conclusion, our studies suggest that D-mannose treatment resulted in the inactivation of GSK3β, which enhanced the nuclear translocation of TFE3, thereby enhancing lysosomal biogenesis and the degradation of VEGFR2 through lysosomes. Thus, our study reveals a novel mechanism by which D-mannose controls TFE3 mediated activation of lysosomal function and VEGFR2 degradation to inhibit angiogenesis and tumor growth of CRC, providing an experimental basis for the potential application of D-mannose in the clinical treatment of CRC.

VEGFR2

Vascular endothelial growth factor receptor-2

VEGF

Vascular endothelial growth factor

CRC

Colorectal cancer

HUVEC

Human umbilical vein endothelial cells

Hexokinase

NAG

N-Acetyl-β-D-Glucosidase

GSK3β

Glycogen synthase kinase 3 beta

CHX

cycloheximide

CLEAR

Coordinated Lysosomal Expression and Regulation

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Acknowledgements

We would like to thank PETCC, the Global Pets' Cell Resource Center, for kindly providing us with cells for testing.

Funding

This work was supported by the National Natural Science Foundation of China (82372754).

Author information

Yu Du and Xinchao Zhang contributed equally to this work.

Authors and Affiliations

Nourse Centre for Pet Nutrition, Wuhu, 241200, China

Yu Du & Yixin Xu

Tongji Hospital, School of Life Sciences and Technology, Tongji University, Shanghai 200120, China.

Xinchao Zhang, Yixin Xu, Yuefan Zhou & Yanping Xu

Contributions

Y.D. and X.Z. played major roles in designing and doing the experiments, analyzing the results and writing the manuscript; Y.X. and Y.Z. helped to conduct the experiments; Y.X. conceived and designed the study; Y.X. supervised the study.

Corresponding author

Correspondence to Yanping Xu.

Ethics declarations

Ethics approval and consent to participate

The trials were approved by the Ethics Committee of Tongji University and were conducted in accordance with the Declaration of Helsinki.

Consent for publication

Not applicable.

Competing Interests

The authors declare that they have no conflict of interest.

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No competing interests reported.

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D-mannose suppresses the angiogenesis and progression of colorectal cancer

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Abstract

Figures

Introduction

Materials and Methods

Cell Culture and reagents

siRNA Transfection

Western blot (WB)

Quantitative Real-time PCR (qRT-PCR)

Separation of Nuclear and Cytoplasmic Proteins

LysoTracker Red staining

NAG Activity Assay

Colony Formation Assay

Wound Healing Assay

Cell Migration Assays

Tube formation assay

Immunohistochemical (IHC)

Animal Experiments

Statistical analysis

Results

D-mannose significantly decreases VEGFR2 protein level

D-mannose mediates VEGFR2 degradation via the lysosomal pathway

D-mannose increases the biogenesis of lysosome and promotes TFE3-dependent degradation of VEGFR2

Discussion

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

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