Exosomal FMR1-AS1 facilitates maintaining cancer stem-like cell dynamic equilibrium via TLR7/NFκB/c-Myc signaling in female esophageal carcinoma

Abstract

Background: Though esophageal cancer is three to four times more common among males than females worldwide, this type of cancer still ranks in the top incidence among women, even more than the female specific cancer types. The occurrence is currently attributed to extrinsic factors, including tobacco use and alcohol consumption. However, limited attention has been given to gender-specific intrinsic genetic factors, especially in female.

Methods: We re-annotated a large cohort of microarrays on 179 ESCC patients and identified female-specific differently expressed lncRNAs. The associations between FMR1-AS1 and the risk and prognosis of ESCC were examined in 206 diagnosed patients from eastern China and validated in 188 additional patients from southern China. The effects of FMR1-AS1 on the malignant phenotypes on female ESCC cells were detected in vitro and in vivo. ChIRP-MS, reporter gene assays and EMSA were conducted to identify the interaction and regulation among FMR1-AS1, TLR7 and NFκB.

Results: We found FMR1-AS1 expression is exclusively altered and closely associated with the level of sXCI in female ESCC patients, and its overexpression may correlate to poor clinical outcome. ChIRP-MS data indicate that FMR1-AS1 could be packaged into exosomes and released into tumor microenvironment. Functional studies demonstrated that FMR1-AS1 could bind to endosomal toll-like receptor 7 (TLR7) and activate downstream TLR7-NFκB signaling, promoting the c-Myc expression, thus inducing ESCC cell proliferation, anti-apoptosis and invasion ability. Exosome incubation and co-xenograft assay indicate that FMR1-AS1 exosomes may secreted from ESCC CSCs, transferring stemness phenotypes to recipient non-CSCs in tumor microenvironment. Furthermore, we also found a correlation between the serum levels of FMR1-AS1 and the overall survival (OS) of the female ESCC patients.

Conclusions: Our results highlighted exosomal FMR1-AS1 in maintaining CSC dynamic interconversion state through the mechanism of activating TLR7-NFκB signaling, upregulating c-Myc level in recipient cells, which may be taken as an attractive target approach for advancing current precision cancer therapeutics in female patients.

Keywords: CSC; ESCC; Exosome; NFκB; TLR7; c-Myc; lncRNA.

Fig. 1

Female specific X-associated lncRNA screening and FMR1-AS1 expression patterns in female ESCC samples and cells. a The venn diagram in (A) depicts the number of gene probes that are differentially expressed in the female ESCC group versus male. b The distribution of those female differentially expressed genes on each chromosome after annotation. c The heat map shows all 142 differentially expressed genes (p < 0.001) specific in female ESCC microarray samples, red = upregulated genes; green = downregulated genes. d LncRNAs that differentially expressed in female ESCC microarray samples. e FMR1-AS1 expression in female ESCC and matched non-tumor tissues from Suzhou (n = 206) and Guangzhou (n = 188) (***p < 0.001). f Kaplan-Meier plots for the disease-free survival rate of female ESCC patients in groups of FMR1-AS1 high or low expression levels in the Suzhou cohort (n = 206, discovery set), Guangzhou cohort (n = 188, validation set), and pooled populations (n = 394, pooled analysis). g Northern-blot of FMR1-AS1 in two pairs of ESCC tissue samples

Fig. 2

Biological characterization of FMR1-AS1. a Ribosome occupancy at the FMR1 and FMR1-AS1 locus. The green peaks indicate reads density that mapped at the region. b RNA fluorescence in situ hybridization to localize FMR1-AS1. c Relative abundance of FMR1-AS1 transcript in cytoplasm, nucleus and chromatin in female ESCC cells. GAPDH, XIST and LincRNA-p21 were used as controls respectively. d Expression of FMR1-AS1 in ESCC cells induced by TNF-α with or without NF-kB inhibition by sc-3060 or JSH-23 (mean ± SD, n = 5, *p < 0.01). e Chromatin immunoprecipitation showing p65, p50 and RNAP II occupancy at the FMR1-AS1 locus in ESCC cells. Locations of amplicons (C1–C4) are indicated in the scheme above. Values represent the enrichment of bound protein fractions relative to input (mean ± SD, n = 3). f Luciferase reporter assay in ESCC cells induced by TNF-α with or without NF-kB inhibition by sc-3060 or JSH-23, and the reporter constructs expressing the luciferase gene under FMR1-AS1 promoter segment (mean ± SD, n = 5, *p < 0.01). g, h sXCI detection assays, based on HpaII digested genomic DNA PCR on the highly polymorphic CAG repeat-region of androgen receptor (AR), which amplifies the undigested inactive X chromosome only. i sXCI frequency in the FMR1-AS1 high/low groups of female ESCC tissues (P_χ² < 0.001). j FMR1-AS1 expression in female ESCC patients with or without sXCI (CR ≥ 3). k, l Correlation analysis on the expression level of FMR1-AS1, XIST (R² = 0.012, P < 0.4585) and TSIX (R² = 0.3353, P < 0.001)

Fig. 3

Effects of ectopic FMR1-AS1 expression on female ESCC cells. a Cell proliferation assay on FMR1-AS1-upregulated, FMR1-AS1-downregulated and respective control ESCC cells using the Cell Counting Kit-8 (mean ± SD, n = 6, *p < 0.05). b Apoptosis rates on ESCC cells transfected with FMR1-AS1, Control, sh-Control, sh-FMR1-AS1 #1 and sh-FMR1-AS1 #2 lentiviruses, using flow cytometry (mean ± SD, n = 6, *p < 0.05). c, d Migration and invasion of ESCC cells transfected with FMR1-AS1, Control, sh-Control, sh-FMR1-AS1 #1 and sh-FMR1-AS1 #2 lentiviruses. Right panel is the quantification of migrated/invaded cells (mean ± SD, n = 6, *p < 0.05). e Xenograft mice subcutaneously implanted FMR1-AS1-upregulated, FMR1-AS1-downregulated and respective control ESCC cells (mean ± SD, n = 6, *p < 0.05). f Ki67 immunostaining in female ESCC samples and FMR1-AS1 expression in Ki67 strong and weak samples. g Ki67 immunostaining in xenograft tumors of FMR1-AS1-upregulated, FMR1-AS1-downregulated and respective control ESCC cells. h TUNEL staining in xenograft tumors of FMR1-AS1-upregulated, FMR1-AS1-downregulated and respective control ESCC cells

Fig. 4

FMR1-AS1 could be packaged into exosomes and activates TLR7- NFκB-c-Myc signaling. a Outline of the ChIRP-MS workflow. Briefly, cells are crosslinked by 3% formaldehyde for 30 min and solubilized by sonication. FMR1-AS1 are pulled out by biotinylated antisense oligos, and associated proteins are eluted with free biotin. Each size fraction is subjected to LC/MS-MS identification. b Over 60% of FMR1-AS1 was retrieved from the cell by ChIRP, while no Gapdh was detected. RNase treatment eliminates FMR1-AS1 transcripts prior to pull-down. c FMR1-AS1 associated proteins that were identified as exosomal proteins by ExoCarta database. d Functional classification of FMR1-AS1 ChIRP-retrived proteins. e, f Identification of PRDX2 and TLR7 in both even/odd probe group retrieved proteins using Mass spectrometry. g Validation of ChIRP-enriched proteins by immunoblotting using TLR7, hnRNPK, PRDX1, PRDX2, ECM1 and β-actin antibodies. h Western-blotting validation of TLR7 in FMR1-AS1 pulldown protein extractions. i, j RNA-immunoprecipitation (RIP) experiments were performed using TLR2/3/4/7/8 and IgG antibodies to immunoprecipitate and a primer to detect FMR1-AS1 in ECA-109 and KYSE-150 cells. k NFκB activities in FMR1-AS1-upregulated, FMR1-AS1-downregulated and respective control ESCC cells, examined by EMSA. l NFκB activity FMR1-AS1-upregulated, FMR1-AS1-downregulated and respective control ESCC cells, examined by luciferase reporter plasmid with FMR1-AS1 promoter (mean ± SD, n = 6, *p < 0.05). m Western blotting showing nuclear p65, p50 and c-Myc levels in FMR1-AS1-upregulated, FMR1-AS1-downregulated and respective control ESCC cells. n NFκB activity in FMR1-AS1-upregulated, FMR1-AS1-downregulated and respective control ESCC cells, examined by luciferase reporter plasmid with MYC gene promoter (mean ± SD, n = 6, *p < 0.05)

Fig. 5

Intercellular transfer of FMR1-AS1 by exosomes disseminates ESCC stem-like phenotypes. a Size distributions and statistics graph of exosomes isolated from CM of ECA-109 and KYSE-150 cells (Upper) and serum of female ESCC patients (Lower). b FACS analysis of exosomes isolated from the CM of ECA-109 and KYSE-150 cells (Upper) and serum of female ESCC patients using CD63 and CD81(Lower). c qRT-PCR analysis of FMR1-AS1 in exosomes isolated from the CM of FMR1-AS1-upregulated, FMR1-AS1-downregulated and respective control ESCC cells (n = 3). d qRT-PCR analysis of FMR1-AS1 in ESCC cells after 48 h incubation with indicated exosomes (PBS as control, n = 3). e qRT-PCR analysis of FMR1-AS1 in serum exosomes from female ESCC patients (n = 48, ***P < 0.001), compared with that from healthy control. f qRT-PCR analysis of FMR1-AS1 in serum exosomes from female ESCC patients (first visit patients group versus recurrent patients group, n = 66, ***P < 0.001). g The consistency between FMR1-AS1 tumor expression and serum expression within individual ESCC patients (R² = 0.7529, P < 0.001). h Kaplan-Meier plots for the disease-free survival rate of female ESCC patients in groups of serum FMR1-AS1 high or low expression levels in the Suzhou cohort (n = 146, discovery set), Guangzhou cohort (n = 107, validation set), and the overall populations (n = 253, pooled analysis). i, j Flow cytometry assay using antibodies against CD44 cell surface markers to obtain two subtypes of tumor cells (CD44 high and CD44 low) from 12 female ESCC patients. The histograms showing expression levels of FMR1-AS1, c-Myc and TSIX in these two cell subtypes (*P < 0.05). k, l CD44 immunostaining in female ESCC samples and FMR1-AS1, c-Myc expression levels in CD44 strong, medium and weak samples (*P < 0.05). m Western blotting of the c-Myc protein level and TSIX expression level on ESCC cells treated with or without c-Myc inhibitor (*P < 0.05). n NFκB activities of ESCC cells after 48 h incubation with indicated exosomes, examined by EMSA. o Western blotting showing c-Myc levels in ESCC cells after 48 h incubation with indicated exosomes. p, q Subcutaneous xenograft assay of FMR1-AS1-upregulated, FMR1-AS1-downregulated and respective control ESCC cells in nude mice with intratumoral injection of indicated exosomes (n = 5 per group)

Fig. 6

TLR7-NFκB signaling pathway activation is responsible for FMR1-AS1-mediated cancer stem cell transition. a, b NFκB activities and c-Myc level of ESCC cells with TLR7 knockdown after 48 h incubation with indicated exosomes, examined by EMSA and western blotting. c Subcutaneous xenograft assay of TLR7-downregulated and respective control ESCC cells in nude mice with intratumoral injection of indicated exosomes (n = 5 per group). d, e NFκB activities and c-Myc level of ESCC cells with MyD88 knockdown after 48 h incubation with indicated exosomes, examined by EMSA and western blotting. f Subcutaneous xenograft assay of MyD88-downregulated and respective control ESCC cells in nude mice with intratumoral injection of indicated exosomes (n = 5 per group). g Subcutaneous xenograft assay co-injected with cell mixture of ECA-109_Luc and ECA-109_FMR1-AS1/ECA-109_Control cells (1:5), KYSE-150_Luc and KYS-150_FMR1-AS1/KYSE-150_Control cells (1:5)

Fig. 7

A schematic diagram of FMR1-AS1 mediated TLR7-NFκB signaling activation between cancer stem-like cells and cancer cells of female ESCC