Tumor-secreted exosomal miR-141 activates tumor-stroma interactions and controls premetastatic niche formation in ovarian cancer metastasis

Abstract

Background: Metastatic colonization is one of the critical steps in tumor metastasis. A pre-metastatic niche is required for metastatic colonization and is determined by tumor-stroma interactions, yet the mechanistic underpinnings remain incompletely understood.

Methods: PCR-based miRNome profiling, qPCR, immunofluorescent analyses evaluated the expression of exosomal miR-141 and cell-to-cell communication. LC-MS/MS proteomic profiling and Dual-Luciferase analyses identified YAP1 as the direct target of miR-141. Human cytokine profiling, ChIP, luciferase reporter assays, and subcellular fractionation analyses confirmed YAP1 in modulating GROα production. A series of in vitro tumorigenic assays, an ex vivo model and Yap1 stromal conditional knockout (cKO) mouse model demonstrated the roles of miR-141/YAP1/GROα/CXCR1/2 signaling cascade. RNAi, CRISPR/Cas9 and CRISPRi systems were used for gene silencing. Blood sera, OvCa tumor tissue samples, and tissue array were included for clinical correlations.

Results: Hsa-miR-141-3p (miR-141), an exosomal miRNA, is highly secreted by ovarian cancer cells and reprograms stromal fibroblasts into proinflammatory cancer-associated fibroblasts (CAFs), facilitating metastatic colonization. A mechanistic study showed that miR-141 targeted YAP1, a critical effector of the Hippo pathway, reducing the nuclear YAP1/TAZ ratio and enhancing GROα production from stromal fibroblasts. Stromal-specific knockout (cKO) of Yap1 in murine models shaped the GROα-enriched microenvironment, facilitating in vivo tumor colonization, but this effect was reversed after Cxcr1/2 depletion in OvCa cells. The YAP1/GROα correlation was demonstrated in clinical samples, highlighting the clinical relevance of this research and providing a potential therapeutic intervention for impeding premetastatic niche formation and metastatic progression of ovarian cancers.

Conclusions: This study uncovers miR-141 as an OvCa-derived exosomal microRNA mediating the tumor-stroma interactions and the formation of tumor-promoting stromal niche through activating YAP1/GROα/CXCRs signaling cascade, providing new insight into therapy for OvCa patients with peritoneal metastases.

Keywords: Hippo/YAP1/pathway; Ovarian cancer; Peritoneal metastases; Tumor-stroma interactions; cancer-associated fibroblasts; miR-141.

Fig. 1

Identification of miR-141 as a secretary miRNA from ovarian cancers. (A) The heatmap indicated the changes in secretary miRNA expression in ovarian cancer patients and normal donors using a miRCURY LNA miRNA PCR Array. N = 2 independent experiments (left). The graph showed the expression of secretory miRNAs compared between ovarian cancer patients and normal donors in the miRCURY LNA miRNA Cancer Focus PCR Panel (right). N = 2 independent experiments. (B) The graphical chart showed the elevated level of exosomal miR-141 in the serum of OvCa patients (n = 62) compared with normal women (n = 24) by qPCR analysis (mean ± SEM, t-test). (C) The graphical chart showed the secretory miR-141 in a panel of ovarian cancer cell lines by qPCR analysis. N = 1 independent experiment. (D) Knockdown of n-SMase2 by a lentiviral shRNAi approach led to the complete suppression of miR-141 exosome production in the conditioned medium of CAOV3 and OVCA433 cells. β-actin and U6 were the internal controls. (n = 3, mean ± SEM, t-test, **P < 0.01) (E) The immunofluorescence microscope showed PHK67 labeled exosomal miR-141 derived from OVCA433 conditioned medium, localized in the cytoplasm of WI-38 and T HESC stromal cells. Representative images are shown in color. Blue, DAPI staining in the nucleus. Green, staining of the exosomes with PKH67. Scale bar = 20 μm

Fig. 2

MiR-141 reprograms stromal fibroblasts to be oncogenic drivers, and GROα is a major proinflammatory cytokine (A) The XTT cell proliferation assay indicated that stromal cell-conditioned medium (SCCM) from miR-141-transfected WPMY-1 cells (SCCM miR-141) increased the cell proliferation of ES-2 and SKOV3 cells on Day 2 and Day 3, compared with scrambled control medium from WPMY-1 cells (SCCM Ctrl) (n = 5, Mean ± SD, t-test, *P < 0.05, **P < 0.01). N = 1 independent experiment. (B) Transwell cell migration/invasion assays demonstrated that SCCM miR-141 treatment had convincingly induced a higher capacity of migration at 16 h or invasion at 24 h in ES-2 and SKOV3 cells as compared with SCCM Ctrl treatment (n = 6, mean ± SEM, t test, **P < 0.01, ***P < 0.001). Scale bar =100 μm. (C) Human XL cytokine array analysis revealed the number of inflammatory cytokines such as GROα in the stromal cell-conditioned medium derived from WPMY-1 scrambled control cells (SCCM Ctrl) and miR-141 overexpressing cells (SCCM miR-141). N = 2 independent experiments. (D) The XTT cell proliferation assay showed a dose-dependent increase in cell proliferation of ES-2 and OVCA433 cells upon a 4-day incubation with recombinant GROα (50 ng/mL) as compared with the respective untreated control (n = 6, mean ± SEM, two-way ANOVA, **P < 0.01, ***P < 0.001, ****P < 0.0001). N = 3 independent experiments. (E) Transwell cell migration/invasion assays showed that the recombinant GROα (60 ng/mL) remarkably promoted cell migration/invasion capacity in ES-2 and OVCA433 cells after 14 h (migration) and 20 h (invasion) as compared with the respective untreated control (Ctrl) (n = 6, mean ± SEM, t-test, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). Scale bar = 100 μm

Fig. 3

YAP1 of Hippo signaling is the direct target of miR-141 (A) The schematic drawing showed YAP1 constructs with wild-type (YAP1-WT) and mutant (YAP1-MT) miR-141 binding sites paired with the miR141 sequence (upper). Dual-luciferase assay showed the relative luciferase activities that cotransfection of pmir-YAP1-WT or pmir-YAP1-MT and the miR-141 expressing plasmid pmR-141 (pmR-ZsGreen1 empty vector was used as negative control) in WPMY-1 and HEK293 cell lines (mean ± SEM, t-test, *P < 0.05, **P < 0.01). N = 3 independent experiments. (B) Confirmation of miR-141 as a target of YAP1 by dose-dependent transfection of pmR-Zsgreen1-miR141 (0, 0.5, 1.0, and 2.0 μg) into WPMY-1, T HESC and WI-38 cells by western blot analysis. The relative YAP1 expression (YAP1/β-actin) was quantified by ImageJ software. (C) Graphic charts compared the relative transcription level of GROα between WPMY-1 stromal cells with either shRNA-mediated knockdown of YAP1 (YAP1-KD), CRISPR/Cas9 system-mediated knockout of YAP1 (YAP1^low/−) or knockout of TAZ (TAZ^low/− #1 and TAZ^low/− #2) with the respective control (Ctrl) (upper). QPCR and ELISA analyses showed the relative expression of GROα in WPMY-1 YAP1^low/− cells transfected with the YAP1-expressing plasmid (0, 1 and 2 μg) (lower) (mean ± SEM, t-test, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). N = 3 independent experiments. (D) Western blot analysis showed the changes in YAP1/TAZ in the cytoplasm and nucleus in WPMY-1 cells with shRNA-mediated YAP1 knockdown approach (YAP1-KD). β-actin and Lamin A/C were used as the internal controls for the cytosol and nuclear proteins, respectively. (E) Immunofluorescence microscopy showed changes in YAP1/TAZ in the cytoplasm and nucleus in YAP1 knockdown and miR-141-overexpressing WPMY-1 cells, and parental cells were used as a control. Scale bar = 20 μm. (F) The graph showed the relative percent input of ChIP that indicated the interaction between TEAD and the predicted binding sites on GROα in T HESCs cells with TEAD overexpression and vector control (mean ± SEM, t-test, *P < 0.05). (G) Graphic charts show the relative luciferase activity of cotransfected TEAD1, TAZ and pGL3-GROα (N ~ III) in the WPMY-1 cells by dual-luciferase assay (mean ± SEM, one-way ANOVA, *P < 0.05, ***p < 0.001, ****p < 0.0001). N = 3 independent experiments

Fig. 4

GROα enhances the tumor colonization of ovarian cancer cells (A) The graph showed the procedures for generating Yap1 stromal-specific cKO mice by crossing Yap1^flox/flox mice with FSP1-Cre^+/+ mice. Three genotypes of mice (Yap1^+/+,  Yap1^+/−, and Yap1^−/−), were generated. (B) Immunofluorescence microscopy showed the mouse stromal fibroblasts extracted from Yap1 stromal-specific cKO mice (Yap1^+/− and Yap1^−/−) stained by the stromal cell markers FAP (green) and vimentin (red). Scale bar = 100 μm. (C) Western blot analysis compared the expression of Yap1 in mouse stromal fibroblasts extracted from Yap1 stromal-specific cKO mice (Yap1^+/− and Yap1^−/−) with control of wild-type (WT) mice (Yap1^+/+). (D) Bar charts showed the relative expression level of GROα in mouse stromal fibroblasts, murine stromal conditioned medium, and serum extracted from Yap1^+/+, Yap1^+/−, and Yap1^−/− mice by qPCR analysis and ELISA (n = 3, mean ± SEM, t-test, **P < 0.01, ***P < 0.001, ****P < 0.0001). (E) qPCR analysis and ELISA showed the relative expression of GROα in mouse stromal fibroblasts and murine stromal conditioned medium from Yap1^−/− mice that were transiently transfected with YAP1-expressing plasmid (0 μg, 1 μg and 2 μg) (n = 3, mean ± SEM, t-test, **P < 0.01, ****P < 0.0001). (F) The XTT cell proliferation assay indicated the relative cell growth of OVCA433 and ES-2 cells co-cultured with mouse stromal conditioned medium derived from Yap1 stromal-specific cKO mice (Yap1^+/−-CM and Yap1^−/−-CM). Stromal conditioned medium derived from Yap1^+/+ mice was used as a control (n = 6, mean ± SEM, 2-way ANOVA, ****P < 0.0001). N = 3 independent experiments. (G) The Transwell migration assay showed that the murine stromal conditioned medium from Yap1 cKO mice (Yap1^+/−-CM and Yap1^−/−-CM) promoted OVCA433 and ES-2 migration capacities, as compared with the respective control treated with Yap1^+/+-CM (n = 3, mean ± SEM, t-test, *P < 0.05, **P < 0.01, ****P < 0.0001). N = 3 independent experiments. (H) The Transwell invasion assay showed that the murine stromal conditioned medium from Yap1 cKO mice (Yap1^+/−-CM and Yap1^−/−-CM) promoted OVCA433 and ES-2 invasion capacities, as compared with the respective control treated with Yap1^+/+-CM (n = 3, mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001). N = 3 independent experiments

Fig. 5

Conditional knockout (cKO) of Yap1 in the stroma promotes in vivo tumor dissemination (A) The images (left) and quantifications (right) of the bioluminescence signals among Yap1^+/+, Yap1^+/− and Yap1^−/−mice upon intraperitoneal injection of GFP/Luc-labelled ID8 cells from Day 7 to Day 49 (n = 3, mean ± SEM, 2-way ANOVA, **P < 0.01, ***P < 0.001). (B) Dot plots displayed the ascites volume among Yap1^+/+, Yap1^+/−, and Yap1^−/− mice with an intraperitoneal injection of GFP/Luc-labelled ID8 cells (n = 5, mean ± SEM). (C) Dot plots displayed the number of tumor nodules among Yap1^+/+, Yap1^+/−, and Yap1^−/− mice with an intraperitoneal injection of GFP/Luc-labelled ID8 cells (n = 5, mean ± SEM, t-test, **P < 0.01, ****P < 0.0001). (D) ELISA revealed the amount of GROα in the ascites of Yap1^+/+, Yap1^+/− and Yap1^−/− mice with an intraperitoneal injection of GFP/Luc-labelled ID8 cells (n = 3, mean ± SEM, t-test, **P < 0.01, ****P < 0.0001). (E) The images (left and right) and quantifications (middle) of the intraperitoneal epifluorescence signals among Yap1^+/+, Yap1^+/− and Yap1^−/− mice with an intraperitoneal injection of GFP/Luc-labelled ID8 cells (n = 3, mean ± SEM, t-test, * P < 0.05, **P < 0.01)

Fig. 6

Depletion of CXCR1/2 impairs the oncogenic potential of OvCa cells (A) XTT cell proliferation assay indicated the relative cell growth of ID8 and ID8 Cxcr1/2 CRISPRi cell lines when co-cultured with murine stromal conditioned medium from Yap1 stromal-specific cKO mice (Yap1^+/−-CM and Yap1^−/−-CM). Yap1^+/+-CM was used as a control (n = 6, mean ± SEM, 2-way ANOVA, ****P < 0.0001). N = 3 independent experiments. (B) The schematic diagram showed the workflow of how ID8 cells with Cxcr1/2 were silenced using CRISPR interference (CRISPRi) approach (ID8 Cxcr1/2 CRISPRi cells) were generated and intraperitoneally injected into Yap1^+/+, Yap1^+/− and Yap1^−/− mice to establish a mouse tumor model. (C) The images (left) and quantifications (right) of the bioluminescence signals among Yap1^+/+, Yap1^+/− and Yap1^−/− mice with an intraperitoneal injection of GFP/Luc-labelled ID8 Cxcr1/2 CRISPRi cells from Day 7 to Day 49 (n = 3, mean ± SEM). (D) The images (left and right) and quantifications (middle) of the intraperitoneal epifluorescence signals among Yap1^+/+, Yap1^+/− and Yap1^−/− mice with an intraperitoneal injection of GFP/Luc-labelled ID8 Cxcr1/2 CRISPRi cells (n = 3, mean ± SEM)