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. 2023 Sep 1;42(1):222.
doi: 10.1186/s13046-023-02758-2.

FAT4 overexpression promotes antitumor immunity by regulating the β-catenin/STT3/PD-L1 axis in cervical cancer

Dongying Wang  1 , Shuying Wu  1 , Jiaxing He  1 , Luguo Sun  2 , Hongming Zhu  1 , Yuxuan Zhang  1 , Shanshan Liu  1 , Xuefeng Duan  1 , Yanhong Wang  2   3 , Tianmin Xu  4
Affiliations

FAT4 overexpression promotes antitumor immunity by regulating the β-catenin/STT3/PD-L1 axis in cervical cancer

Dongying Wang et al. J Exp Clin Cancer Res. .

Abstract

Background: FAT4 (FAT Atypical Cadherin 4) is a member of the cadherin-associated protein family, which has been shown to function as a tumor suppressor by inhibiting proliferation and metastasis. The Wnt/β-catenin pathway activation is highly associated with PD-L1-associated tumor immune escape. Here, we report the mechanism by which FAT4 overexpression regulates anti-tumor immunity in cervical cancer by inhibiting PD-L1 N-glycosylation and cell membrane localization in a β-catenin-dependent manner.

Methods: FAT4 expression was first detected in cervical cancer tissues and cell lines. Cell proliferation, clone formation, and immunofluorescence were used to determine the tumor suppressive impact of FAT4 overexpression in vitro, and the findings were confirmed in immunodeficient and immunocomplete mice xenografts. Through functional and mechanistic experiments in vivo and in vitro, we investigated how FAT4 overexpression affects the antitumor immunity via the β-catenin/STT3/PD-L1 axis.

Results: FAT4 is downregulated in cervical cancer tissues and cell lines. We determined that FAT4 binds to β-catenin and antagonizes its nuclear localization, promotes phosphorylation and degradation of β-catenin by the degradation complexes (AXIN1, APC, GSK3β, CK1). FAT4 overexpression decreases programmed death-ligand 1 (PD-L1) mRNA expression at the transcriptional level, and causes aberrant glycosylation of PD-L1 via STT3A at the post-translational modifications (PTMs) level, leading to its endoplasmic reticulum (ER) accumulation and polyubiquitination-dependent degradation. We found that FAT4 overexpression promotes aberrant PD-L1 glycosylation and degradation in a β-catenin-dependent manner, thereby increasing cytotoxic T lymphocyte (CTL) activity in immunoreactive mouse models.

Conclusions: These findings address the basis of Wnt/β-catenin pathway activation in cervical cancer and provide combination immunotherapy options for targeting the FAT4/β-catenin/STT3/PD-L1 axis. Schematic cartoons showing the antitumor immunity mechanism of FAT4. (left) when Wnts bind to their receptors, which are made up of Frizzled proteins and LRP5/6, the cytoplasmic protein DVL is activated, inducing the aggregation of degradation complexes (AXIN, GSK3β, CK1, APC) to the receptor. Subsequently, stable β-catenin translocates into the nucleus and binds to TCF/LEF and TCF7L2 transcription factors, leading to target genes transcription. The catalytically active subunit of oligosaccharyltransferase, STT3A, enhances PD-L1 glycosylation, and N-glycosylated PD-L1 translocates to the cell membrane via the ER-to-Golgi pathway, resulting in immune evasion. (Right) FAT4 exerts antitumor immunity mainly through following mechanisms: (i) FAT4 binds to β-catenin and antagonizes its nuclear localization, promotes phosphorylation and degradation of β-catenin by the degradation complexes (AXIN1, APC, GSK3β, CK1); (ii) FAT4 inhibits PD-L1 and STT3A transcription in a β-catenin-dependent manner and induces aberrant PD-L1 glycosylation and ubiquitination-dependent degradation; (iii) Promotes activation of cytotoxic T lymphocytes (CTL) and infiltration into the tumor microenvironment.

Keywords: CTL; Cervical cancer; FAT4; PD-L1; Wnt/β-catenin pathway.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
FAT4 downregulated in cervical cancer and is associated with poor prognosis. A Schematic diagram of the functional structural domains of FAT4 protein. Red lines indicate possible β-catenin binding regions. (TM, transmembrane domain; LAMG: laminin G-like domain; EGFCA: EGF-like repeat). B Using the cBioPortal online analysis tools, data from the TCGA database were extracted and analyzed for cross-cancer FAT4 gene alterations. The frequency of FAT4 gene mutation, amplification, and profound deletion across cancers was shown by histograms. The results showed that FAT4 is frequently mutated or deleted in numerous types of human cancer. C Western blot showing FAT4 protein levels in 12 pairs of pericarcinomatous tissues and cervical cancers, GAPDH was loaded as a control. D Representative IHC images showing FAT4 expression in normal cervical (NC) tissues, pericarcinomatous tissue (PT), and cervical cancer (CC) tissues. Note that in normal cervical tissue, FAT4 is highly expressed in the cytomembrane of stratum superficiale and stratum spinosum cells (inserts show ×2 magnification). Scale bar = 100 μm. E The FAT4 mRNA expression data were obtained from TCGA and compared with normal tissues. F Immunoreactivity score of pericarcinomatous tissue and cervical cancer tissues. G Kaplan-Meier survival curves in 50 cervical cancer patients based on high (red) or low (black) FAT4 immunological scoring (P = 0.0036). H&I FAT4 protein expression was detected by (H) immunoblotting and (I) immunofluorescence in ME180, Caski, C33A, Hela, SiHa and U14 cervical cancer cell lines. FAT4 protein expression was relatively low in human-derived C33A and ME180 cell lines, and murine-derived U14 cell lines. Note that even in the cell lines with high FAT4 expression (SiHa, Hela, and Caski), the cell membrane localization of FAT4 was not evident, nuclei stained with Hoechst (blue)
Fig. 2
Fig. 2
FAT4 overexpression inhibited cervical cancer proliferation both in vitro and in vivo. A&B Human-derived ME180 cell lines, and murine-derived U14 cell lines were transfected with sgRNA targeting the FAT4/Fat4 or scrambled negative control (CTRL) sgRNA, and overexpression efficiency was analyzed by (A) immunoblotting and (B) RT-qPCR. C The CCK-8 assay was used to detect the proliferation of FAT4 overexpression cervical cancer cells and CTRL at the indicated time points. All error bars are expressed as mean ± SD, **P < 0.01, ***P < 0.001, and ****P < 0.0001. D Immunofluorescence detection of the subcellular localization of FAT4, note that cell membrane localization of FAT4 was significantly increased after overexpression (white arrow). E&F (E) Representative images and (F) the quantification results of colony formation assay in the indicated cervical cancer cells. Results are shown as mean ± SD. G-I Gross images of (G) negative control (CTRL) and sgFAT4-transfected ME180 xenografts in BALB/c nude mice, (H) tumor growth curves, and (I) Kaplan-Meier survival curves of these mice. J-L Gross images of (J) negative control and sgFat4-transfected U14 xenografts in BALB/c nude mice, (K) tumor growth curves, and (L) Kaplan-Meier survival curves of these mice
Fig. 3
Fig. 3
FAT4 overexpression promotes antitumor immunity in vivo. A-C Gross images of (A) negative control (CTRL) and sgFat4-transfected U14 xenografts in C57BL/6 mice, (B) tumor growth curves, and (C) Kaplan-Meier survival curves of these mice. D Four weeks after inoculation of sgRNA-Fat4 cells, regressed tumors were seen at the intersection of tumor neovascularization. E Ki67 (green) was visualized by IF, and nuclei were stained with Hoechst (dim gray) to calculate the Ki67 positive rate. F 5 × 105 U14 cells were injected into immunocompetent C57BL/6 mice on day 0 and analyzed at the indicated time. sc, subcutaneous. FC, flow cytometry. IF, immunofluorescence. IHC, immunohistochemistry. G-I Co-stained of FAT4 and granzyme B (activity of T cell) in the U14 frozen tumor sections (G), nuclei were stained with Hoechst (blue) to calculate the FAT4 positive rate (H), and quantification of GZMB+ regions (4 tissue slides per tumor, 3 mice per group, n = 12). Results are presented as mean ± SD, Scale bar = 100 μm (I). J&K Co-stained of FAT4 and cleaved caspase-3 (CCA3) in the U14 frozen tumor sections (J), nuclei were stained with Hoechst (blue) to calculate the CCA3 positive rate (K). Scale bar = 100 μm
Fig. 4
Fig. 4
FAT4 overexpression activates CTL and inhibits PD-L1 expression in immunoreactive mice models. A&D Fluorescence-activated cell sorting (FACS) plots and quantification of CD8+ GZMB+ and CD8+ IFN-γ+ in CD3+ tumor infiltrating lymphocyte (TILs) derived from sgFat4 or CTRL group. All error bars are expressed as mean ± SD, ***P < 0.001 and ****P < 0.0001. B Fluorescence-activated cell sorting (FACS) quantification of CD8+ /CD3+ in CD45+ cells. C&E FACS plots and quantification of CD4+ GZMB+, CD4+ IFN-γ+ in CD3+ TILs derived from sgFat4 or CTRL group. F&G Co-stained of FAT4 and PD-L1 in the U14 frozen tumor sections (F), nuclei were stained with Hoechst (blue) to calculate the PD-L1 positive rate (G). Scale bar = 100 μm. H&I Immunohistochemical images (H) and immunoreactivity score (I) of FAT4, cleaved caspase-3, and PD-L1 expression in U14 C57BL/6 xenografts. All error bars are expressed as mean ± SD, **P < 0.01, ***P < 0.001 and ****P < 0.0001. J.Cd274 mRNA levels from the indicated U14 xenografts. Results are presented as mean ± SD, n = 5
Fig. 5
Fig. 5
FAT4 overexpression inhibits the Wnt/β-catenin pathway in cervical cancer cells. A KEGG pathway enrichment analysis. Note that FAT4 overexpression is highly relevant for the Wnt signaling pathway, N-Glycan biosynthesis, and immune editing-related pathways and these findings are consistent in the C33A and ME180 cell lines. B Immunofluorescence staining for FAT4 (red) and total-β-catenin (green) in ME180 cells. In CTRL cells, the total-β-catenin runs throughout the cytoplasm, and sgFAT4 is enriched in the cell membrane, with significant co-localization with FAT4. C The co-immunoprecipitation assay detects increased binding of overexpressed FAT4 to endogenous β-catenin. Immunoprecipitation with β-catenin antibody followed by immunoblotting with FAT4 antibody. D Interaction among the degradation complex (AXIN, GSK3β, CK1α, APC) and β-catenin in CTRL or FAT4 overexpression cells was determined by co-immunoprecipitation assay. Immunoprecipitation with β-catenin antibody followed by immunoblotting with AXIN, GSK3β, CK1α, and APC antibodies. E Immunoblotting was used to detect the expression of active-β-catenin, total-β-catenin, C-Myc, MMP9, and Cyclin D1, phospho-GSK-3β (Ser9) and total-GSK-3β in CTRL or FAT4 overexpression cells. F FAT4 inhibits β-catenin/TCF/TEF luciferase reporter activity. The TOPFlash/FOPFlash luciferase reporter assay was performed in CTRL or sgFAT4 ME180 cells. pSV40-Renilla was used as an internal control. G&H Immunohistochemical images (H) and Immunoreactivity score (G) of active-β-catenin and total-β-catenin expression in U14 C57BL/6 xenografts. ***P < 0.001 and ****P < 0.0001
Fig. 6
Fig. 6
FAT4 overexpression inhibits PD-L1 expression and cell membrane localization. A Immunoblotting was used to detect the expression of PD-L1 and STT3A in CTRL or FAT4 overexpression cells. B Immunofluorescence staining for PD-L1 expression in ME180 and U14 cells. CTRL is located mainly on the cell membrane (white arrow). In FAT4 overexpression cells, FAT4 was detected throughout the cytoplasm with the reduced signal at the membrane (red arrows). Data represent mean ± SD. Scale bar = 10 μm. C RT-qPCR analysis of human CD274 and STT3A mRNA in sgFAT4 and CTRL ME180 Cells. D RT-qPCR analysis of mouse Cd274 and Stt3a mRNA in sgFat4 and CTRL U14 Cells. E Flow cytometric analysis of PD-L1+ membrane expression in ME180 and U14 cells. F&G ME180 cells were transfected with control vector or Active β-catenin vector for 48 h. Immunoblot analysis (F) and RT-qPCR analysis (G) were performed. H&I (H) Representative images and (I) quantitation of green-fluorescent labeling recombinant human PD-1 Fc protein or recombinant mouse PD-1 Fc protein on CTRL or FAT4 overexpression cells. Scale bar = 10 μm. All error bars are expressed as mean ± SD, ***P < 0.001 and ****P < 0.0001
Fig. 7
Fig. 7
FAT4 overexpression induces abnormal PD-L1 glycosylation and prevents its ER-to-Golgi translocation. A Half-life analysis of PD-L1 in CTRL or sgFAT4 cells were treated with 50 µg/mL actinomycin for the indicated times. PD-L1 levels were semi-quantified using β-tubulin as a loading control. At time 0, the relative PD-L1 levels were set to 1. B Immunofluorescence analysis of CTRL or sgFAT4 ME180 cells for (left) co-localization of endogenous PD-L1 and endoplasmic reticulum maker (TGN38), (right) co-localization of endogenous PD-L1 and Golgi maker (Calreguli, CALR), nuclei stained with Hoechst (blue). C Intensity profiles showing signals from two fluorescent channels. D Ubiquitination of PD-L1 protein in CTRL or FAT4 overexpression cells. Immunoprecipitation was performed with PD-L1 antibody followed by immunoblotting with ubiquitin antibody. MG-132 (5 μM; 24 h) treated CTRL cells as a positive control. E Interaction among GSK-3β, STT3A, and PD-L1 in CTRL or FAT4 overexpression cells was determined by co-immunoprecipitation assay. Immunoprecipitation with PD-L1 antibody followed by immunoblotting with GSK-3β and STT3A antibodies
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