IGF2BP3 Triggers STAT3 Pathway by Stabilizing SRC RNA in an m6A-Dependent Manner to Promote Lymphatic Metastasis in LUAD

Abstract

Lymph node metastasis significantly affects the NSCLC patients' staging, treatment strategy, and prognosis. Studies have shown that IGF2BP3, an oncofetal protein and an m6A reader, overexpresses and correlates to lymph node metastasis and worse overall survival in histopathological studies including NSCLC, but its mechanism needs further study. This study explored IGF2BP3's function and mechanism in LUAD lymphatic metastasis using public databases, a human LUAD tissue microarray, human LUAD cells, and a lymphatic metastasis model in male BALB/c nude mice. Firstly, we proved that IGF2BP3 overexpression was positively correlated to patients' lymph node metastasis and worse overall survival in bioinformatics and a human LUAD tissue microarray analysis. IGF2BP3 was knocked out or overexpressed in human LUAD cell lines. Functionally, IGF2BP3 facilitated NCI-H1299, NCI-H358, and A549 cell growth, migration, invasion, and EMT in vitro, and promoted tumorigenesis, lymphangiogenesis, and lymphatic metastasis of NCI-H1299 cells in BALB/c nude mice. Mechanically, RIP, RNA pull-down assay, MeRIP, mRNA stability assays, rescue experiments, and immunohistochemical assays were conducted. IGF2BP3 was demonstrated to bind to the m6A site of the 3'UTR region of SRC, stabilizing its mRNA and activating the downstream STAT3 signaling pathway and lymphatic growth factors such as VEGF-C, therefore affecting lymphatic metastasis. The cell migration and EMT function of IGF2BP3 were partially rescued by utilizing SRC siRNA or AZD0530, an SRC inhibitor. This study demonstrated that IGF2BP3 promotes lymphatic metastasis in LUAD via activating the m6A-SRC-STAT3-VEGFC signaling axis, indicating that IGF2BP3 is a potential therapeutic target to overcome metastasis in LUAD patients.

Keywords: IGF2BP3; LUAD; N6‐methyladenosine; SRC; lymphatic metastasis.

FIGURE 1

IGF2BP3 overexpression was correlated to lymph node metastasis in human LUAD tissues. (A) The boxplot shows that IGF2BP3 mRNA expression is correlated to N stages across various types of tumors including LUAD based on TCGA data. (B) The point plot shows the mRNA expression between 515 LUAD and 59 normal tissues based on TCGA data. (C) The boxplots show a relationship between IGF2BP3 mRNA expression and type‐3 EMT in LUAD TCGA data. (D) Overall and disease‐specific survival with different IGF2BP3 mRNA expression levels based on LUAD TCGA data. (E, F) Representative images (E) and quantitative statistical bar graphs (F) of immunohistochemistry on a human lung adenocarcinoma tissue microarray. NAT, Normal adjacent tissue. (G, H) Kaplan–Meier survival curves showing the overall survival categorized by patients' nodal status (G) or IGF2BP3 expression levels (H), via analyzing the clinicopathological characteristics of patients from the lung adenocarcinoma tissue microarray. *p < 0.05, **p  < 0.01, ***p < 0.001, ****p < 0.0001, ns: not statistically significant.

FIGURE 2

IGF2BP3 promotes cell migration, invasion, and EMT in human LUAD cells. (A–C) Western blot analysis showing that IGF2BP3 was knocked out in NCI‐H1299 and A549 cells and was overexpressed in NCI‐H1299 and NCI‐H358 cells at protein levels. (D) The GFP fluorescence imaging and bright imaging showing the transduction efficiency of lentiviral vectors in NCI‐H299 and NCI‐H358 cell lines. (E) RT‐qPCR analysis showing IGF2BP3 overexpression in NCI‐H1299 and NCI‐H358 cells at mRNA levels. (F–I) The ability of proliferation of LUAD cells was evaluated by the colony formation assay and CCK‐8 assay. (J–O) The wound‐healing assays (J–L) and transwell experiments (M–O) show the migratory and invasive abilities of LUAD cells after IGF2BP3 knockout or overexpression. (P–R) Western blot analysis showing the expression of E‐Cadherin, N‐Cadherin, and vimentin in the IGF2BP3 knockout LUAD cells or IGF2BP3‐overexpressed LUAD cells and their control cells. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 3

Identification of SRC as the IGF2BP3 target in LUAD. (A) The volcano plot shows the differentially expressed genes between the IGF2BP3 knockout group and the control group in NCI‐H1299 cells based on RNA sequencing data. (B) GO function analysis of differentially expressed genes following IGF2BP3 knockout in NCI‐H1299 cells. (C) The JAK–STAT signaling pathway. We are grateful to KEGG and have obtained copyright written permission (240910) from the KEGG. (D) Pearson correlation analysis shows a relationship between IGF2BP3 RNA expression and SRC RNA expression in the LUAD TCGA database. (E, F) RT‐qPCR analysis showed IGF2BP3 knockout decreased SRC mRNA expression in NCI‐H1299 and A549 cells, while IGF2BP3 overexpression increased SRC mRNA expression in NCI‐H1299 and NCI‐H358 cells. (G–L) Western blot analysis showing the expression of SRC and p‐SRC in the IGF2BP3 knockout LUAD cells or IGF2BP3‐overexpressed LUAD cells and their control cells. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 4

IGF2BP3 regulates the stability of SRC mRNA through m6A modification. (A) RIP‐qPCR analysis and western blot analysis showing the enrichment of SRC mRNA in anti‐IGF2BP3 precipitates (left) and IGF2BP3 immunoprecipitation (right) compared to the IgG group in NCI‐H1299 and A549 cells, respectively. (B) Schematic of four putative IGF2BP3 binding nucleotide regions within SRC mRNA. (C, E, F) RNA pull‐down assays using different RNA probes. PC RNA, Positive RNA, NC RNA, Negative RNA. (D) Homo sapiens : De novo m6A motif of GSM1166139. (G) MeRIP‐qPCR analysis showing the levels of m6A enrichment of SRC mRNA utilizing anti‐m6A antibodies and anti‐IgG in NCI‐H1299 cells. (H) RT‐qPCR analysis showing the expression of SRC mRNA at the predetermined times after actinomycin D (2 μg/mL) treatment in IGF2BP3 knockout NCI‐H1299 cells and their corresponding control cells. ***p < 0.001, ****p < 0.0001.

FIGURE 5

IGF2BP3 activates the SRC‐mediated JAK‐STAT3 signaling pathway and regulates lymphatic growth factors in LUAD cells. (A. B) Western blot analysis showing the expression of STAT3, p‐STAT3, FAK, and p‐FAK in the IGF2BP3 knockout NCI‐H1299 cells and the control cells. (C, D) The differentially expressed lymphatic growth factors between the IGF2BP3 knockout NCI‐H1299 cells and control cells based on RNA‐sequencing analysis and RT‐qPCR. (E, F) After transfection with si‐NC or si‐SRC for 48 h, the expression of SRC, p‐SRC, STAT3, p‐STAT3, and EMT‐related proteins were evaluated in IGF2BP3‐overexpressed NCI‐H1299 cells and corresponding control cells through western blot analysis. (G–M) The CCK‐8 assay (G), colony formation assay (H–I), and wound‐healing assays (J–M) in IGF2BP3 overexpression NCI‐H1299 cells and their control counterparts after transfection with si‐NC or si‐SRC or after treatment with Saracatinib (AZD0530) for a period of 48 h. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns: not statistically significant.

FIGURE 6

IGF2BP3 facilitates tumor lymphangiogenesis and lymph node metastasis in a xenograft mouse model. (A–C) Images of footpad tumors, popliteal lymph nodes, and inguinal lymph nodes, using vivo near‐infrared fluorescent imaging (A, B) and after excision (C), in the IGF2BP3 knockout group and the control group (n = 6 per group). (D–I) The volume and weight of footpad tumors (D, E), popliteal lymph nodes (F, G), and inguinal lymph nodes (H, I) for each group (n = 6 per group). (J–L) The number of popliteal lymph nodes (J), inguinal lymph nodes (K), and total lymph nodes (L) for each group (n = 6 per group). (M) RT‐qPCR analysis showing the SRC mRNA expression levels of mice footpad tumor tissues in the IGF2BP3 knockout group and the control group. (N, O) Western blot analysis showing the expression of IGF2BP3, SRC, and p‐SRC of footpad tumor tissues in the IGF2BP3 knockout group and the control group. (P–R) Representative images of footpad tumor tissues immunostained with IGF2BP3, SRC, LYVE‐1, VEGF‐C, and CD31 antibodies in intratumoral with different IGF2BP3 expression levels. Scale bars: 50 μm. (S) Metastatic and negative popliteal lymph nodes were identified through hematoxylin and eosin staining. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 7

A mechanism graphic in this study. This study unveiled the mechanism wherein IGF2BP3 was overexpressed in human LUAD tissues, promoted cell migration and invasion, EMT, lymphangiogenesis, and lymph node metastasis by binding the m6A site of the 3′UTR region of SRC and maintaining its stability so as to activate STAT3 signaling pathway and promote VEGFC expression in LUAD.