FOXP4 Is a Direct YAP1 Target That Promotes Gastric Cancer Stemness and Drives Metastasis

Abstract

The Hippo-YAP1 pathway is an evolutionally conserved signaling cascade that controls organ size and tissue regeneration. Dysregulation of Hippo-YAP1 signaling promotes initiation and progression of several types of cancer, including gastric cancer. As the Hippo-YAP1 pathway regulates expression of thousands of genes, it is important to establish which target genes contribute to the oncogenic program driven by YAP1 to identify strategies to circumvent it. In this study, we identified a vital role of forkhead box protein 4 (FOXP4) in YAP1-driven gastric carcinogenesis by maintaining stemness and promoting peritoneal metastasis. Loss of FOXP4 impaired gastric cancer spheroid formation and reduced stemness marker expression, whereas FOXP4 upregulation potentiated cancer cell stemness. RNA sequencing analysis revealed SOX12 as a downstream target of FOXP4, and functional studies established that SOX12 supports stemness in YAP1-induced carcinogenesis. A small-molecule screen identified 42-(2-tetrazolyl) rapamycin as a FOXP4 inhibitor, and targeting FOXP4 suppressed gastric cancer tumor growth and enhanced the efficacy of 5-fluorouracil chemotherapy in vivo. Collectively, these findings revealed that FOXP4 upregulation by YAP1 in gastric cancer regulates stemness and tumorigenesis by upregulating SOX12. Targeting the YAP1-FOXP4-SOX12 axis represents a potential therapeutic strategy for gastric cancer. Significance: Hippo-YAP1 signaling maintains stemness in gastric cancer by upregulating FOXP4, identifying FOXP4 as a stemness biomarker and therapeutic target that could help improve patient outcomes.

Figure 1.

FOXP4 is overexpressed in gastric cancer and is correlated with poor survival. A, Clinical cases with genetic or mRNA alterations in FOXPs in TCGA cohort. The genetic and mRNA alterations in FOXP1 to FOXP4 account for 4.9%, 5.2%, 6.9%, and 16.2% of gastric cancer cases, respectively. B, Copy number gain or amplification of FOXP4 in primary gastric cancer samples (n = 2) was detected by FISH analysis. C and D,FOXP4 mRNA levels in nonpaired and paired samples from TCGA-STAD dataset. E, IHC staining of the expression and cellular localization of FOXP4 in cancer cells and adjacent normal tissues (n = 3). F and G, Western blot analysis (n = 2) of FOXP4 protein expression in paired gastric cancer tissues and cell lines. N, adjacent nontumor; T, tumor. H and I, High FOXP4 expression was associated with poor prognosis in both Hong Kong and Beijing cohorts.

Figure 2.

FOXP4 depletion exerts antitumor effects in gastric cancer. A and B, siFOXP4 inhibited cancer cell proliferation and colony formation (n = 3). C, siFOXP4 suppressed gastric cancer cell invasiveness (n = 3). D and E, Western blot analysis of cell cycle–associated and apoptosis-associated proteins after FOXP4 knockdown. F, FOXP4 knockdown–induced apoptosis was confirmed by flow cytometry (n = 3). G, Representative patient-derived organoid images with shFOXP4-mediated knockdown. Scale bar, 50 μm. H, Subcutaneous injection of FOXP4-depleted gastric cancer cells formed smaller xenografts than the control group mice. I, According to IC₅₀ displayed, siFOXP4 increased the 5-FU sensitivity. J, FOXP4 deletion and 5-FU combination suppressed peritoneal metastasis and prolonged survival time of mice. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 3.

FOXP4 is a direct downstream of YAP1. A, RNA-seq revealed that among the FOXP family, FOXP4 exhibits significant downregulation after the knockdown of YAP1. B, The Eukaryotic Promoter Database showed a putative YAP1/TEAD4-binding site on the FOXP4 promoter (−647 bp; P < 0.001). C, ChIP-qPCR assay (n = 3) confirmed the direct binding of YAP1/TEAD4 complex to the FOXP4 promoter. TSS, transcription start site. D, Luciferase reporter assays (n = 3) verified that YAP1 can bind with the FOXP4 promoter (wild-type binding motif). E, Western blot analysis showed that siRNA-mediated YAP1 depletion led to a significant decrease in FOXP4 protein in both gastric cancer cell lines. F, Western blot analysis revealed that the overexpression of wild-type YAP1 or constitutively active YAP (YAP^5SA) increased FOXP4 expression. G and H, Administration of CA3 or VT107 inhibited the expression of YAP1 and FOXP4 dose-dependently. I, Left, workflow for generating an MNNG (N-methyl-N’-nitro-N-nitrosoguanidine)-induced gastric cancer model. d.w., drinking water. Middle and right, IHC staining showed that Yap1/Taz double-knockout mice (Yap1^−/−Taz^−/−) exhibited low FOXP4 expression in the MNNG-induced gastric cancer model. Scale bar, 50 μm. J and K, IHC staining confirmed a significant correlation between YAP1 and FOXP4 in both intestinal and diffuse gastric cancer types. Scale bar, 50 μm. L and M, Patient-derived organoid models further depicted that FOXP4 expression is regulated by YAP1. Scale bar, 50 μm. ***, P < 0.001.

Figure 4.

FOXP4 overexpression promotes tumor growth and partially rescues the suppressive effects of YAP1 knockdown. A and B, FOXP4 overexpression enhanced the proliferation and colony formation ability of MKN7 cells. C, FOXP4-overexpressed MKN7 cells showed enhanced migration and invasion abilities. D and E, FOXP4 overexpression significantly increased the tumor formation ability of MKN7 cells. F, Western blot analysis revealed that the expression level of stemness markers was upregulated in the FOXP4 overexpression cells. G, Stronger peritoneal metastasis signals were detected in mice injected with FOXP4-overexpressed MKN7 cells compared with the empty vector group. H, Rescue experiments were performed by re-introducing FOXP4 into gastric cancer cells with YAP1 depletion. Re-overexpressing FOXP4 partially abolished the suppressive effects of YAP1 knockdown. I and J, The suppressed proliferation and colony formation ability from YAP1 knockdown were rescued by FOXP4 overexpression. K, The decreased spheroid-forming ability induced by siYAP1 was rescued by FOXP4 overexpression. L, FOXP4 overexpression partially rescued the suppressed cell migration and invasion induced by YAP1 deletion. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 5.

Maintaining cancer stemness is the major role of FOXP4. A and B, GSEA demonstrated a positive correlation between FOXP4 and stem cell proliferation and upregulation. C, FOXP4 depletion compromises tumorsphere formation. D, RNA-seq revealed that various stemness markers were downregulated after knocking down YAP1 and FOXP4. E, scRNA-seq analysis demonstrated that YAP1/FOXP4 was co-upregulated with biological processes correlated with stem cell maintenance and proliferation. F and G, FOXP4 directly regulates SOX12 expression in gastric cancer, which was confirmed by the ChIP-qPCR assay. H and I, qRT-PCR and Western blot analysis of SOX12 expression in siFOXP4 transfectants. J and K, Both TCGA and ACRG cohorts demonstrated a positive association between FOXP4 and SOX12. TPM, transcript per million. ***, P < 0.001.

Figure 6.

SOX12 is a novel stemness marker in gastric cancer progression. A, IHC staining showed that Yap1^−/−Taz^−/− mice exhibited low SOX12 expression in the MNNG  (N-methyl-N’-nitro-N-nitrosoguanidine)-induced gastric cancer model. B and C, Highly expressed SOX12 gastric cancer cases were associated with unfavorable outcomes compared with the low-expression group. D–H, SOX12 depletion by siRNA inhibited cell proliferation, colony formation, cell metastasis abilities, and tumorsphere formation. I and J, Flow cytometry and Western blot analysis confirmed that SOX12 knockdown induced cell apoptosis. K, SOX12 overexpression in MKN7 cells increased the expression levels of canonical stemness markers. L, Heatmap displays multiple stemness markers that were upregulated in SOX12 high expression cases (from TCGA dataset). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 7.

Targeting FOXP4 by high-content screened small molecule. A, High-content small molecule screening was performed from 4,511 anticancer drugs to select the potent FOXP4 inhibitors. Four modeling tools were used to screen the most potent small molecules from the library. B, Four small molecules were deduced to potentially inhibit FOXP4 activity. C, The small molecule 42-(2-tetrazolyl) rapamycin displayed an inhibitory effect in the FOXP4 highly expressed gastric cancer lines MKN28 and NCI-N87. D and E, 42-(2-Tetrazolyl) rapamycin inhibited FOXP4 expression and cell colony formation ability in a dose-dependent manner. F, Dose–response combination assays of two drugs confirmed synergy among all the combinations of 5-FU and 42-(2-tetrazolyl) rapamycin, and the red peak of 3D plots represent the average highest single-agent model synergy scores. HSA, highest single agent. G, 42-(2-Tetrazolyl) rapamycin treatment enhanced the sensitivity towards 5-FU–induced apoptosis. H and I, Coadministration of 5-FU and 42-(2-tetrazolyl) rapamycin resulted in robust antitumor activity in gastric cancer xenografts and restrained gastric cancer peritoneal metastasis. The combination administration exhibited better survival in the mice model. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 8.

Overall schematic presentation of the YAP1–FOXP4–SOX12 axis in gastric cancer. YAP1 directly regulates FOXP4 expression via binding its promoter. FOXP4 exerts oncogenic properties by upregulating SOX12. As a novel therapeutic target, depleting FOXP4 suppresses cancer cell stemness, reverses drug resistance, and reduces peritoneal metastasis. Small molecule targeting FOXP4 quenches YAP1-driven gastric tumor progression and enhances the 5-FU efficacy. (Created with BioRender.com.)