Elevated expression of the RNA-binding protein IGF2BP1 enhances the mRNA stability of INHBA to promote the invasion and migration of esophageal squamous cancer cells

Abstract

Background: The mechanisms underlying the occurrence and development of esophageal squamous cell carcinoma (ESCC) remains to be elucidated. The present study aims to investigate the roles and implications of IGF2BP1 overexpression in ESCC.

Methods: IGF2BP1 protein expression in ESCC samples was assessed by immunohistochemistry (IHC), and the mRNA abundance of IGF2BP1 and INHBA was analyzed with TCGA datasets and by RNA in situ hybridization (RISH). The methylation level of the IGF2BP1 promoter region was detected by methylation-specific PCR (MSP-PCR). Cell viability, migration, invasion and in vivo metastasis assays were performed to explore the roles of IGF2BP1 overexpression in ESCC. RNA immunoprecipitation sequencing (RIP-seq) and mass spectrometry were applied to identify the target RNAs and interacting proteins of IGF2BP1, respectively. RIP-PCR, RNA pulldown, immunofluorescence (IF), gene-specific m⁶A PCR and RNA stability assays were used to uncover the molecular mechanisms underlying the malignant phenotypes of ESCC cells caused by IGF2BP1 dysregulation. BTYNB, a small molecular inhibitor of IGF2BP1, was evaluated for its inhibitory effect on the malignant phenotypes of ESCC cells.

Results: IGF2BP1 overexpression was detected in ESCC tissues and associated with the depth of tumor invasion. In addition, IGF2BP1 mRNA expression in ESCC cells was negatively correlated with the level of its promoter methylation. Knockdown of IGF2BP1 inhibited ESCC cell invasion and migration as well as tumor metastasis. Mechanistically, we observed that IGF2BP1 bound and stabilized INHBA mRNA and then resulted in higher protein expression of INHBA, leading to the activation of Smad2/3 signaling, thus promoting malignant phenotypes. The mRNA level of INHBA was upregulated in ESCC tissues as well. Furthermore, IGF2BP1 interacted with G3BP stress granule assembly factor 1 (G3BP1). Knockdown of G3BP1 also down-regulated the INHBA-Smad2/3 signaling. BTYNB abolished this activated signaling and significantly attenuated the malignant phenotypes of ESCC cells.

Conclusions: Elevated expression of IGF2BP1 is a frequent event in ESCC tissues and might be a candidate biomarker for the disease. IGF2BP1 overexpression promotes the invasion and migration of ESCC cells by activating the INHBA-Smad2/3 pathway, providing a potential therapeutic target for ESCC patients with high expression of IGF2BP1.

Keywords: Esophageal squamous cell carcinoma; IGF2BP1; INHBA; Invasion; Migration; RNA binding protein.

Fig. 1

IGF2BP1 is overexpressed in SCC tissues. (A) Representative IHC staining of IGF2BP1 in ESCC and adjacent nonmalignant tissues. Scale bar = 200 μm (100×); scale bar = 50 μm (400×). (B) IGF2BP1 mRNA levels in ESCC patients and normal tissues in TCGA database. **P < 0.01. (C) IGF2BP1 mRNA levels in HNSCC, LUSC, CESC patients and normal tissues in TCGA database. (D) The expression of IGF2BP1 mRNA in all major tissues and organs in the human body was analyzed in the HPA database. (E) IGF2BP1 protein and mRNA levels of ESCC cell lines were analyzed using RT-PCR and Western blotting, respectively. (F) The methylation level of the first intron of the IGF2BP1 gene in the ESCC cell genome was assessed by MSP-PCR. Three pairs of methylated (M) and unmethylated (U) primers targeting three CG sites in the first intron were designed to amplify DNA converted by bisulfite

Fig. 2

IGF2BP1 promotes the invasion and migration of ESCC cells in vitro as well as lung metastasis in vivo. (A) Cell viability was quantified using a CCK-8 assay. (B) Cell invasion and migration abilities were examined using Transwell assays. Representative results (left) and statistical plots (right) are shown. (C) Cell motility was assessed by the wound-healing assay. Representative results (left) and statistical plots (right) are shown. (D) Cell metastatic potential was evaluated using an in vivo pulmonary metastasis assay. Representative images of fixed lung tissues (top) and the results of H&E staining (bottom) are shown. The arrows indicate the lung metastatic nodules. The number of metastatic nodules was plotted (right). **P < 0.01; ***P < 0.001; ****P < 0.0001. NC: Negative Control

Fig. 3

IGF2BP1 enhances ESCC cell invasion and migration by activating INHBA-Smad2/3 signaling. (A) The mRNA levels of IGF2BP1 and INHBA in KYSE30 and TE1 cells after IGF2BP1 knockdown were determined by qRT-PCR. (B-C) The interaction between IGF2BP1 protein and INHBA mRNA in ESCC cells was validated with RIP-PCR (B) and RNA pull-down assay (C). (D) Western blotting analysis of the indicated proteins in ESCC cells transfected with IGF2BP1-specific siRNA or NC siRNA. (E) The decay rate of INHBA mRNA after IGF2BP1 depletion was evaluated by RNA stability assay. (F) Cell lysates were immunoblotted for the indicated proteins after METTL3/14 transient knockdown. (G) m⁶A modification in INHBA mRNA was tested by gene-specific m⁶A PCR. (H-I) Cell invasion and migration abilities were examined with Transwell assays. (J) Western blotting analysis of INHBA and Smad2/3 in ESCC cells transfected with INHBA siRNA (above) and in ESCC cells stably expressing shIGF2BP1 transfected with pcDNA3.1-INHBA or empty vector (below). *P < 0.05; ***P < 0.001

Fig. 4

G3BP1 interacts with IGF2BP1. (A) Identification of interacting proteins by Co-IP-MS. The red boxes indicate the differential proteins that were cut off and identified using MS. The whole lane of the IgG group served as a negative control. The table below shows the top eight candidate interacting proteins. (B) Bubble plot of GO enrichment based on mass spectrometry results. (C) The interaction between IGF2BP1 and G3BP1 in ESCC cells was detected with an endogenous immunoprecipitation assay. (D) Cellular localization of endogenously expressed IGF2BP1 (red) or G3BP1 (green) was detected by immunofluorescence staining using laser confocal microscopy. DAPI was used to stain nuclei (blue). Scale bar = 30 μm. (E) Western blotting analysis of the indicated proteins in ESCC cells transfected with NC-siRNA or G3BP1 siRNA. GAPDH was used as a loading control

Fig. 5

INHBA is upregulated in ESCC and invasive breast cancer. (A) INHBA mRNA levels in ESCC and normal tissues analyzed using TCGA datasets. (B) RISH scores of INHBA in ESCC and normal tissues. (C) Representative RISH staining of INHBA mRNA in ESCC and adjacent normal tissues. Scale bar = 200 μm (100×); scale bar = 100 μm (200×). (D) Analysis of Smad2 and Smad3 mRNA levels in ESCC and normal tissues in TCGA datasets. (E) INHBA mRNA levels in invasive breast cancer and normal tissues from TCGA database. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001

Fig. 6

BTYNB inhibits the invasion, migration and proliferation of ESCC cells in vitro. (A) The effects of BTYNB on cell invasion and migration were examined by Transwell assay. 20 µM BTYNB was added to the lower compartment for 24-36 h. (B) Colony formation of KYSE30 and TE1 cells exposed to different concentrations of BTYNB. (C) Cell viability of ESCC cells exposed to BTYNB was determined by CCK-8 assay. KYSE30 and TE1 cells were treated with 5 µM and 10 µM BTYNB for 72 h. (D) Cell apoptosis was determined by flow cytometry. ESCC cells were treated with 10 µM BTYNB for 48 h. (E) Western blotting analysis of the indicated proteins in cells treated with BTYNB. (F) The IGF2BP1-INHBA interaction upon BTYNB treatment (20 µM BTYNB for 24 h) was assessed by RIP-PCR. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001