Background
Esophageal cancer is one of the major malignancies threatening human health. Esophageal squamous cell carcinoma (ESCC) accounts for more than 90% of esophageal cancer cases in China. Most patients with ESCC are diagnosed at an advanced stage, and the overall 5-year survival rate is only approximately 30% [
1] due to invasive growth and distal metastasis. However, the molecular mechanisms underlying the invasion and metastasis of ESCC are still not fully understood, and there are no effective targeted drugs for clinical treatment to date. Therefore, there is an urgent need to identify the key molecules affecting the invasion and metastasis of ESCC.
Insulin-like growth factor 2 mRNA binding protein 1 (IGF2BP1) is a highly conserved RNA binding protein that mainly binds mRNA and thereby affects RNA transcription, processing, translation and metabolism. IGF2BP1 overexpression is often correlated with poor prognosis in a variety of cancer types, including melanoma [
2], breast [
3], ovarian [
4‐
6], colon [
7,
8], liver [
9,
10], and lung [
11,
12] cancers. It has been reported that IGF2BP1 promotes cell proliferation or invasion by stabilizing several mRNA targets such as CD44 and c-myc [
13], which have been confirmed as oncogenes [
14,
15]. Moreover, IGF2BP1 has been proven to be a N6-methyladenosine (m
6A) reader that recognizes and binds m
6A-modified mRNA and thus enhances its stability [
16‐
19]. Additionally, IGF2BP1 has been designated an oncofetal protein due to its space-time specific expression pattern: it is predominantly expressed in embryonic development and suppressed in most adult tissues but re-expressed in multiple tumor types [
20].
We found remarkable upregulation of IGF2BP1 in ESCC tissues by immunohistochemistry (IHC). However, there is no information available on the role of IGF2BP1 in ESCC. In this study, we focused on the roles of IGF2BP1 overexpression in malignant phenotypes and the underlying mechanisms in ESCC cells, aiming to explore the possibility of IGF2BP1 as a biomarker and therapeutic target for the disease.
Materials and methods
Tissue specimens and cell lines
ESCC and operative margin tissues were procured from surgical resection specimens. All of the patients received no treatment prior to surgery and signed separate informed consent forms for sample collection and molecular analysis. The study was approved by the Ethics Committee of the Cancer Institute (Hospital), Chinese Academy of Medical Sciences (CAMS) & Peking Union Medical College (PUMC) (No. 16–171/1250).
ESCC cell lines KYSE30, KYSE70, KYSE150, KYSE180, KYSE450 and KYSE510 were generously provided by Prof. Shimada (Kyoto University, Japan); TE1, TE4 and TE10 were purchased from ATCC, and Eca109 was purchased from Cell Resource Center, Institute of Basic Medicine, Chinese Academy of Medical Sciences. All of the cell lines were authenticated by short tandem repeat (STR) profiling and cultured in RPMI-1640 media with 10% fetal bovine serum (FBS500-S, AusGeneX) in a humidified incubator at 37 °C and 5% CO2.
Plasmid constructs, transfection and lentiviral transduction
The materials and methods are provided in Additional file 1: Table
S1-3.
Immunohistochemistry (IHC)
IHC assays were conducted as reported previously [
21]. Slides were incubated with primary antibody at 4 °C overnight. The tissues were incubated with a Mouse/Rabbit Enhanced Polymer Detection System (PV-9000, ZSGB-BIO) and then chromogenic substrate DAB (ZLI-9017, ZSGB-BIO). The tissue microarrays were scanned with a Nano Zoomer digital pathology biopsy scanner (HAMAMATSU, Japan). Immunoreactive scores were calculated by multiplying the scores of staining signal intensity and the percentage of positive cells. The intensity was scored as follows: 0 (negative), 1 (weak), 2 (moderate), and 3 (strong); the proportion of positive cells was scored as follows: 0 (negative), 1 (1-20%), 2 (21-50%), and 3 (51-100%). Antibodies used for IHC were listed in Additional file 1: Table
S4.
RNA in situ hybridization (RISH)
INHBA mRNA in situ hybridization was performed on 6 μm thick tissue microarrays (TMAs) with RNAscope 2.5 HD Reagent Kit-BROWN (322,300, ACD) following the manufacturer’s instructions.
1 × 103 cells were seeded into each well of a 96-well plate (with 3 replicates in each group), and the cell viability was quantified every 24 h using Cell Counting Kit-8 (CK-04, Dojindo, Japan) according to the manufacturer’s instructions. Absorbance at 450 nm was measured by an Elx 808 Microplate Reader (BioTek, USA). For the colony formation assay, 1 × 103 cells were seeded into each well of a 6-well plate and treated with the indicated dose of BTYNB (with 3 replicates in each group) for 7 ~ 14 days. The colonies of cells were fixed with 100% methanol, stained using 0.1% crystal violet, and then counted.
Wound-healing assay
Cells were seeded in six-well plates and grown until they reached full confluence. Cells were scratched a wound vertically and washed with PBS. The scratches were observed and photographed at indicated time points. The wound areas were measured using ImageJ (Ver. 1.51j8, NIH, USA).
Cellular invasion and migration assays
Invasion and migration assays were performed in Transwell plates as described previously [
22]. After incubation for 36 h (KYSE30) or 24 h (TE1), the membranes with stained cells were placed on slide and mounted with coverslip, followed by scanning and imaging with a Nano Zoomer digital pathology biopsy scanner (HAMAMATSU, Japan). The areas covered by stained cells in three random fields were measured by ImageJ. More details are provided in Additional file
1: Supplementary_Materials and Methods.
Cell apoptosis analysis
Cells treated with 10 µM BTYNB for 48 h were digested, collected and stained with fluorescently labeled Annexin V and PI using an Annexin V FITC Apoptosis Detection Kit (AD10, Dojindo). Flow cytometry was adopted to detect the percentage of apoptotic cells.
Xenograft assay
Four-week-old female BALB/c nude mice (HFK Bioscience Co., LTD, Beijing, China) were purchased and randomly divided into two groups by body weight (10 per group). The mice were injected with 1 × 106 KYSE30 cells stably expressing shIGF2BP1 or shNon-silencing (shNS) via the tail vein. Eight weeks later, the mice were sacrificed, and the whole lung tissues were separated and fixed in Bouin’s Fixative Solution (PH0976, Phygene). Then, the number of lung metastases was counted, and the lung tissues were embedded in paraffin, cut into 3 μm sections, and stained with hematoxylin and eosin (H&E). All animal experiments were approved by the Animal Center of the Institute of National Cancer Center/Cancer Hospital, CAMS & PUMC (NCC2019A016).
Western blotting
Total protein was isolated using RIPA buffer (C1053, Applygen) supplemented with protease inhibitors (B14001, Bimake) and phosphatase inhibitors (B15001, Bimake) and quantified with a Pierce BCA Protein Assay Kit (23,225, Thermo). Antibodies for immunoblotting were listed in Additional file 1: Table
S4.
Reverse transcription PCR (RT-PCR) and quantitative real-time RT-PCR (qRT-PCR)
Total RNA was isolated using an RNApure Tissue & Cell Kit (CW0506, Cwbiotech) following the manufacturer’s instructions, and cDNA was synthesized using a HiFiScript cDNA Synthesis Kit (CW2569M, Cwbiotech). Then, RT-PCR was conducted with TaKaRa Ex Taq (RR001A, TaKaRa) on a SimpliAmp Thermal Cycler (ABI, USA). qRTPCR was performed using a TB Green™ Premix Ex Taq Kit (RR420A, TaKaRa) on an ABI QuantStudio DX real-time PCR system (ABI, USA). The relative expression levels of mRNA were assessed through the comparative threshold cycle method (2
−ΔΔCt) with GAPDH as an internal control. All primers used in this study are listed in Additional file 1: Table
S5.
RNA coimmunoprecipitation combined with high-throughput sequencing (RIP-seq)
RIP was performed using an EZ-Magna RIP Kit (17–701, Millipore). RNA was finally purified with TRIzol reagent (Invitrogen) and analyzed by RT-PCR or RNA-seq (Wuhan Seqhealth Tech Co. Ltd.). The sequences of primers for RT-PCR are described in Additional file 1: Table
S5.
Biotin RNA pull-down assay
RNA pull-down assays were performed as previously described [
23]. The RNA-protein complex was immunoprecipitated with streptavidin magnetic beads (HY-K0208, MedChemExpress). The complex was divided into two equal portions for RT-PCR and WB analysis. The sequences of biotin-labeled DNA probes are provided in Additional file 1: Table
S6.
RNA stability assay
Cells were treated with actinomycin D (ActD, 5 µg/mL) for 0, 2, or 4 h. Total RNA was extracted, and the relative level of mRNA at each time point was analyzed by quantitative real-time PCR with GAPDH as an internal control. The mRNA half-life was estimated according to a previous description[
24]. Primers for qPCR are listed in Additional file 1: Table
S5.
Gene-specific m6A qPCR
The methylated mRNAs were immuno-precipitated as previously reported [
25], eluted with elution buffer (10 mL of 0.1 M DTT, 0.44 g of NaCl, 2.5 mL of pH 7.5 1 M Tris-HCl, 0.1 mL of 0.5 M EDTA, 0.5 mL of 10% SDS, 10 µL of RNase inhibitor, ddH
2O up to 50 mL) and recovered with the RNeasy Micro Kit (74,004, Qiagen), further analyzed by RT–PCR along with input control. m
6A antibody was described in Additional file 1: Table
S4.
Methylation-specific PCR (MSP-PCR)
Genomic DNA of ESCC cells was extracted using the QIAamp DNA Mini Kit (QIAGEN) and transformed with the Epitect Fast DNA Bisulfite Kit (QIAGEN). The sequences of primer pairs against the first intron are provided in Additional file 1: Table
S7.
Coimmunoprecipitation-based mass spectrometry (Co-IP-MS)
Protein A/G magnetic beads (HY-K0202, MedChemExpress) were used to perform coimmunoprecipitation. Protein samples were then subjected to WB assay or SDS–PAGE followed by Coomassie staining. Gel pieces were cut off and sent to Shanghai Applied Protein Technology Co. Ltd. for mass spectrometry analysis. Antibodies used for Co-IP were listed in Additional file 1: Table
S4. More details are provided in Additional file
1: Supplementary _Materials and Methods.
Immunofluorescence (IF) staining
Immunofluorescence was performed conventionally and the antibodies were described in Additional file 1: Table
S4. Immunofluorescence was detected by confocal microscopy (PE double spinning disk confocal, USA).
Statistical analysis
IBM SPSS Statistics 23.0 software was applied for data analysis, and P < 0.05 was considered statistically significant. Fisher’s exact test was used to assess the IHC score difference between ESCC tissues and adjacent noncancerous specimens. The correlation between the protein expression level and clinicopathological parameters was analyzed by Pearson’s chi-square test. Comparisons between two groups were performed by independent samples T tests, and one-way ANOVA was used for multiple comparisons. Rstudio software (1.1463) was used for Gene Ontology (GO) and pathway enrichment analysis.
Discussion
The insulin-like growth factor-2 mRNA-binding protein family (IGF2BPs), composed of IGF2BP1, IGF2BP2 and IGF2BP3, has a crucial role in early embryonic development. IGF2BP1 and IGF2BP3 are oncofetal proteins because they are mostly silent in adult organs, except in the reproductive system[
13,
20,
34]. In contrast, IGF2BP2 is the only expressive IGF2BP in most adult tissues. IGF2BP1 and IGF2BP3 are re-expressed in many types of tumors, and IGF2BP2 was also found to be excessively expressed in malignancies due to genomic amplification according to pan-cancer analysis with TCGA data. Growing evidence supports the pro-oncogenic roles of these RNA-binding proteins in cancer progression by influencing their RNA target fate [
35].
However, few studies on IGF2BP1 have been reported in ESCC. Herein, we discovered remarkably high expression of IGF2BP1 at both the mRNA and protein levels, indicating transcriptional dysregulation in ESCC. More importantly, our functional and mechanistic investigations revealed that IGF2BP1 facilitated the migration, invasion and metastasis of ESCC cells by activating the INHBA-Smad2/3 cascade. INHBA, a member of the TGF-β superfamily, has been reported to be overexpressed in multiple types of cancers, including ESCC, and associated with poor prognosis [
36‐
41]. Consistently, our analyses of TCGA data and RISH on TMAs demonstrated the upregulation of INHBA at the transcriptional level in ESCC tissues. In addition, we found that INHBA mRNA was frequently overexpressed in ESCC and invasive breast cancer. More importantly, the downstream molecule of INHBA-Smad2 and Smad3 were also upregulated in ESCC tissues. Combined with the spatial distribution of INHBA mRNA in ESCC tissues, we speculate that INHBA may play an important role in cell invasion and migration. Although the roles of INHBA in cancer are controversial, the majority favor its oncogenic effects. Seder et al. reported that INHBA promoted cell proliferation and was regulated by promoter demethylation in ESCC cells [
42]. Another study suggested that INHBA affects cell migration and positively correlated with genes involved in extracellular structure organization [
43]. In the present study, we identified INHBA as a direct target of IGF2BP1 with a functional role in tumor invasion induced by IGF2BP1. Mechanistically, IGF2BP1 bound and stabilized INHBA mRNA, consequently leading to an increase in INHBA protein. Moreover, as an m
6A reader proven by recent research, IGF2BP1 preferentially recognizes m
6A-modified mRNAs and promotes their stability in an m
6A-dependent manner [
17]. We indeed observed that INHBA mRNA was m
6A modified and that the turnover of INHBA was m
6A-dependent. Therefore, it is likely that mRNA methylation is required in the regulation of INHBA by IGF2BP1.
RNA-binding proteins participate in forming ribonucleoprotein (RNP) granules that regulate mRNA translation, localization, and turnover [
44]. Our Co-IP-MS results confirmed that IGF2BP1 functions by interacting with other RBPs. G3BP1 was validated as a partner of IGF2BP1 and contributed to positive regulation of INHBA-Smad2/3 signaling. G3BP1 contains two C-terminal motifs (associated with RNA binding) and an RNA recognition motif (RRM). It has been demonstrated that G3BP1 promotes stress-induced RNA granule interactions to preserve polyadenylated mRNA [
45]. Meanwhile, G3BP1 is involved in protein degradation by stably associating with USP10 deubiquitinase [
46]. Our experimental results showed that INHBA protein was significantly decreased and IGF2BP1 was slightly downregulated after G3BP1 knockdown. Future studies will be needed to clarify the specific details regarding whether and how the interaction between IGF2BP1 and G3BP1 activate INHBA-Smad2/3 signaling.
Several studies have demonstrated that BTYNB, a structure-specific inhibitor, could block the binding of IGF2BP1 to its oncogenic target mRNA, thus disrupting their interaction [
32,
33]. We evaluated the efficacy of BTYNB in vitro and found that the typical malignant phenotypes of ESCC cell lines with high IGF2BP1 expression were sharply repressed, and the IGF2BP1-INHBA interaction was disturbed by BTYNB. Our results implied that IGF2BP1 could be a potential target of ESCC.
As mentioned above, IGF2BP1 has long been considered an oncofetal protein. In fact, according to the HPA database, IGF2BP1 mRNA is expressed only in the testis and placenta and weakly in the kidney, and IGF2BP1 protein is expressed only in the adult testis, ovary, and bronchial tissues, supporting an expression pattern in few adult tissues. In the present study, our IHC results showed that IGF2BP1 is highly expressed in nearly 50% of ESCC tissues but not expressed or only weakly expressed in all surgical margin specimens. Based on these published data and our observations, IGF2BP1 could be a very promising target for ESCC, making it possible to specifically target tumor cells without disturbing noncancerous tissues. These data imply that IGF2BP1 is a potential molecular target for ESCC therapy.
Little information is available describing how the expression of IGF2BP1 is modulated at the transcriptional and posttranscriptional levels. It has been proposed that IGF2BP1 transcription is induced by β-catenin [
47] and c-Myc [
48]. In addition, let-7 could regulate IGF2BP1 posttranscriptionally [
49]. Our observation linked genomic hypomethylation in the first intron to high IGF2BP1 expression, suggesting a new perspective on aberrant transcription of this gene.
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.