Background
Non-small cell lung cancer (NSCLC) is a highly malignant tumor, with high clinical incidence and mortality that annually increase globally [
1]. Even when individuals with NSCLC are treated with a combination of surgery and chemotherapy, the survival rate is low because of cancer cell metastasis and invasion [
2]. Therefore, finding an effective target to inhibit cancer cell growth and invasion in NSCLC is urgently needed.
More than 100 chemical modifications of cellular RNA are known. Reversible RNA modification constitutes a new level of post-transcriptional regulation of gene expression, involved in many physiological processes and with multiple biological roles [
3]. N
6-methyladenine (m
6A) is a dynamic reversible chemical modification. It is the most abundant internal modification of mRNA and a new transcription marker. In mammals, plants, and some prokaryotes, m
6A is widely involved in mRNA metabolism, affecting mRNA stability and splicing, RNA nucleation, RNA-protein interaction, and mRNA translation [
4,
5]. The enzymes m
6A demethylases are also known as “erasers”. Two eukaryotic m
6A demethylases have been identified to date: fat mass and obesity-associated protein (FTO) and AlkB homolog 5 (ALKHB5). ALKHB5 is a member of the AlkB family, which plays an important role in regulation of many biological processes, such as mRNA processing [
6]. According to recent studies, ALKBH5 plays an important role in the occurrence and development of cancer in human [
6,
7]. For example, ALKBH5 holds prognostic value and inhibits the metastasis of colon cancer [
8]; ALKBH5 controls trophoblast invasion at the maternal-fetal interface by regulating the stability of
Cyr61 mRNA [
9]; METTL3 and ALKBH5 oppositely regulate m
6A modification of
TFEB mRNA, dictating the fate of hypoxia/reoxygenation-treated cardiomyocyte [
10]; ALKBH5 inhibits pancreatic cancer cell motility by decreasing methylation of the long non-coding RNA KCNK15-AS1 [
11]. Moreover, HuR restrains translation inhibition mediated by some miRNAs by directly binding and sequestering microRNAs (miRNAs). In addition, studies have shown that m
6A indirectly impacts transcript stability, by affecting HuR binding and microRNA targeting [
12,
13]. However, the mechanism through which ALKBH5 regulates NSCLC tumor growth and metastasis is not clear.
A group of YTH domain-containing proteins (YTHDFs) have been identified as m
6A “readers” that recognize m
6A marks and mediate m
6A function [
14]. The human YTH domain family consists of three members: YTHDF1–3. Each member contains a highly conserved single-stranded RNA-binding domain, located at their carboxyl termini (the YTH domain) and a relatively less conserved amino-terminal region [
15]. YTHDF1 improves the translation efficiency by binding to m
6A-modified mRNA [
16], whereas YTHDF2 reduces the stability of mRNA by recruiting an mRNA degradation system [
17]. YTHDF3 serves as a hub to fine-tune the accessibility of RNA to YTHDF1 and YTHDF2. YTHDFs have many important biological functions [
18]. For instance, YTHDF3 suppresses interferon-dependent antiviral responses by promoting FOXO3 translation in HREpiC cells [
19] and YTHDF2 promotes lung cancer cell growth by facilitating translation of 6-phosphogluconate dehydrogenase mRNA [
20]. However, the manner in which YTHDF3 cooperates with YTHDF1 and YTHDF2 to promote the translation or decay of m
6A-modified YAP mRNA in NSCLC remains to be elucidated.
MicroRNAs (miRNAs) are a group of non-coding single-stranded RNA molecules 20–24 nucleotides-long, encoded by endogenous genes. miRNA interacts with a specific mRNA, triggering its degradation, inhibiting translation and widely participating in the organism growth, development, differentiation, metabolism, defenses, and other processes [
21]. Significant differences in the expression of various miRNAs in healthy cells and tumor cells have been recently reported. These miRNAs play a role similar to that of proto-oncogenes or tumor suppressor genes, by regulating different target genes, and are closely related to the occurrence, development, treatment, and prognosis of tumors in human [
22].
The Hippo signaling pathway is an inhibitory pathway that hinders cell growth and controls cell proliferation, organ size, and homeostasis [
23]. This pathway is highly evolutionarily conserved. The main components of the mammalian pathway are Mst1/2, LATS1/2, and Yap/TAZ. After activation of the Hippo signaling pathway, Mst1/2, as the core component of this kinase chain, is activated and phosphorylates a downstream component of LATS1/2. LATS1/2 mainly inhibits the proliferation and migration of tumor cells by blocking cell cycle progression and plays an important regulatory role in cell apoptosis. LATS1/2 phosphorylates Yap/TAZ, which inhibits YAP activity; then, cytoplasmic 14–3-3 protein binds phosphorylated Yap to promote YAP degradation. Dephosphorylated Yap/TAZ translocate to the nucleus, where it binds tea domain family members (TEADs) to promotes target gene transcription. The target genes include
Ctgf,
Cyr61,
Oct4,
p73, and
ZEB1, and control organ size, tumor cell proliferation, and metastasis [
24]. However, the transcriptional regulation of the Hippo signaling pathway, especially following
YAP transcription modified by m
6A, has not yet been delineated.
We here aimed to explore the mechanism whereby ALKBH5 inhibits tumor growth and metastasis by regulating the expression and activity of YAP in NSCLC. Using lung healthy cells and tumor cells, we demonstrated the following: (1) ALKBH5 decreases the level of m6A YAP mRNA modification; (2) YTHDF1 and YTHDF2 competitively bind YTHDF3 in an m6A-independent manner to regulate YAP expression; (3) YTHDF2 facilitates the decay of YAP mRNA, which is mediated by Argonaute 2 (AGO2) system and regulated by m6A modification; (4) YTHDF1 promotes m6A modification-mediated YAP mRNA translation via interaction with eIF3a; (5) ALKBH5 decreases the activity of YAP by regulating the miR-107/LATS2 axis in an HuR-dependent manner. These findings clarify the role of m6A signaling in the control of YAP expression and activity and suggest novel prognostic factors for NSCLC tumor growth and metastasis.
Methods
Molecular biology
Myc-tagged YAP, YTHDF1, YTHDF2, YTHDF3 and Flag-tagged ALKBH5, YTHDF1, YTHDF2, YTHDF3 constructs were made using the pcDNA 3.1 vector (Invitrogen, Carlsbad, CA, USA). Sequences encoding the Myc epitope (EQKLISEEDL) and Flag epitope (DYKDDDDK) were added by PCR through replacement of the first Met-encoding codon in the respective cDNA clones.
Cell lines and culture
Human lung normal cell line BEAS-2B and NSCLC cell lines A549, H1299, Calu6 and H520 were purchased from American Type Culture Collections (Manassas, VA). Cell lines were cultivated in RPMI-1640 medium supplemented with 10% FBS (Hyclone, USA), penicillin/streptomycin (100 mg/mL). Culture flasks were kept at 37 °C in a humid incubator with 5% CO2.
RNA isolation and reverse transcription (RT)-PCR assay
We used TRIzol reagent (TransGen Biotech, Beijing, China) to isolate total RNA from the samples. RNA was reverse transcribed into first-strand cDNA using a TransScript All-in-One First-Strand cDNA Synthesis Kit (TransGen Biotech). cDNAs were used in RT-PCR and quantitative real-time PCR assay with the human GAPDH gene as an internal control. The final quantitative real-time PCR reaction mix contained 10 μL Bestar® SYBR Green qPCR Master Mix, Amplification was performed as follows: a denaturation step at 94 °C for 5 min, followed by 40 cycles of amplification at 94 °C for 30 s, 58 °C for 30 s and 72 °C for 30 s. The reaction was stopped at 25 °C for 5 min. The relative expression levels were detected and analyzed by ABI Prism 7900HT/FAST (Applied Biosystems, USA) based on the formula of 2-ΔΔct. RT-PCR analysis of miR-107 was conducted with One step miRNA RT kit (D1801, HaiGene, China) for reverse-transcription. The PCR was performed with the Dream taq Green master mix (Fermentas, K1082) following the manufacturer’s protocols, then used the 4% agarose gel at 120 V for 70 min. We got the images of RT-PCR by Image Lab™ Software (ChemiDocTM XRS+, BiO-RAD) and these images were TIF with reversal color format. Primers for qPCR:
ALKBH5 forward primer: 5′-GCCTATTCGGGTGTCGGAAC-3′.
ALKBH5 reverse primer: 5′-CTGAGGCCGTATGCAGTGAG-3′.
YTHDF1 forward primer: 5′-GCACACAACCTCCATCTTCG-3′.
YTHDF1 reverse primer: 5′-AACTGGTTCGCCCTCATTGT-3′.
YTHDF2 forward primer: 5′-TCTGGAAAAGGCTAAGCAGG-3′.
YTHDF2 reverse primer: 5′-CTTTTATTTCCCACGACCTTGAC-3′.
YTHDF3 forward primer: 5′-TGACAACAAACCGGTTACCA-3′.
YTHDF3 reverse primer: 5′-TGTTTCTATTTCTCTCCCTACGC-3′.
YAP forward primer: 5′-GGATTTCTGCCTTCCCTGAA-3′.
YAP reverse primer: 5′-GATAGCAGGGCGTGAGGAAC-3′.
Cyr61 forward primer: 5′-GGTCAAAGTTACCGGGCAGT-3′.
Cyr61 reverse primer: 5′-GGAGGCATCGAATCCCAGC-3′.
CTGF forward primer: 5′-ACCGACTGGAAGACACGTTTG-3′.
CTGF reverse primer: 5′-CCAGGTCAGCTTCGCAAGG-3′.
HuR forward primer: 5′-CGGAATTCAATACAATGTCTAATGG-3′.
HuR reverse primer: 5′-GGGGTACCATTGGCGCAAAATGAG-3′.
LATS2 forward primer: 5′-CAGGATGCGACCAGGAGATG-3′.
LATS2 reverse primer: 5′-CCGCACAATCTGCTCATTC-3′.
E-cadherin forward primer: 5′-ACCATTAACAGGAACACAGG − 3′.
E-cadherin reverse primer: 5′-CAGTCACTTTCAGTGTGGTG-3′.
Vimentin forward primer: 5′- CGCCAACTACATCGACAAGGTGC-3′.
Vimentin reverse primer: 5′-CTGGTCCACCTGCCGGCGCAG-3′.
GAPDH forward primer: 5′-CTCCTCCTGTTCGACAGTCAGC-3′.
GAPDH reverse primer: 5′-CCCAATACGACCAAATCCGTT-3′.
miR-107 forward primer: 5′- GCCGAATTCAAAGCGAGATTCCATCAGCA-3′.
miR-107 reverse primer: 5′- GCCGGATCCTGTCAACCCAGAACTCAAAGG-3′.
Western blot analysis
Human lung cancer cells were transfected with the relevant plasmids and cultured for 48 h. For western blot analysis, cells were lysed in NP-40 buffer (10 mM Tris pH 7.4, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA pH 8.0, 1 mM EGTA pH 8.0, 1 mM PMSF, and 0.5% NP-40) at 25 °C for 40 min. The lysates were added to 5× loading dye and then separated by electrophoresis. The primary antibodies used in this study were 1:1000 rabbit anti-Flag (sc-166,384, Santa Cruz, Dallas, TX, USA) and 1:1000 Abcam (Cambridge, UK) antibody of Myc (ab32072), ALKBH5 (ab234528), YAP (ab56701), YTHDF1 (ab220162), YTHDF2 (ab220163), YTHDF3 (ab220161), HuR (ab200342), LATS2 (ab135794), CTGF (ab6992), Cyr61 (ab24448), AGO2 (ab32381), eIF3a (ab86146), Vimentin (ab45939), E-cadherin (ab1416), cleaved Capase-3 (ab32042) and Tubulin (ab6046).
Immunohistochemical analysis
Tumor tissues were fixed in 4% paraformaldehyde overnight and then embedded in paraffin wax. Four-micrometer thick sections were stained using hematoxylin and eosin (H&E) for histological analysis.
Over-expression and knockdown of genes
Overexpressing plasmid (2 μg), siRNA (1.5 μg) and shRNA (1.5 μg) of indicated genes were transfected into cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) for over-expression and knockdown of indicated genes, followed by analysis 48–72 h later. The selected sequences for knockdown as follow:
shALKBH5–1: 5′-CCTCATAGTCGCTGCGCTCG-3′.
shALKBH5–2: 5′-ATAGTTGTCCCGGGACGTCA-3′.
siYAP-1: 5′- AAGGTGATACTATCAACCAAA-3′.
siYAP-2: 5′- AAGACATCTTCTGGTCAGAGA-3′.
siALKBH5–1: 5′-ACAAGTACTTCTTCGGCGA-3′.
siALKBH5–2: 5′-GCGCCGTCATCAACGACTA-3′.
siYTHDF1–1: 5′-CCGCGTCTAGTTGTTCATGAA-3′.
siYTHDF1–2: 5′-CCTCCACCCATAAAGCATA-3′.
siYTHDF2–1: 5′-CTGCCATGTCAGATTCCTA-3′.
siYTHDF2–2: 5′-GCTCCAGGCATG AATACTATA-3′.
siYTHDF3–1: 5′-GGACGTGTGTTTATAATTA-3′.
siYTHDF3–2: 5′-GACTAGCATTGCAACCAAT-3′.
siAGO2: 5′-GCACGGAAGTCCATCTGAA-3′.
sieIF3a: 5′-CAGTTGATGGCAAATTACT-3′.
siHuR: 5′-TGTCAAACCGGATAAACGC-3′.
sicontrol: 5′-TTCTCCGAACGTGTCACGA-3′.
shcontrol: 5′-ACGTGACACGTTCGGAGAATT-3′.
CCK8 assays
Cell viability and growth was determined using CCK8 assays in 96-well plates. Cells were transfected with the relevant plasmids culturing for 48 h, followed by incubation with 10 μL CCK8 for 4 h. Absorbance was read at 450 nm using a spectrophotometer (Tecan, Männedorf, Switzerland).
Immunofluorescent staining
To examine the protein expression and location by immunofluorescent staining, NSCLC cells were seeded onto coverslips in a 24-well plate and left overnight. Cells were then fixed using 4% formaldehyde for 30 min at 25 °C and treated with 3% bovine serum albumin (BSA) in phosphate buffered saline (PBS) for 30 min. The coverslips were incubated with rabbit anti-ALKBH5, YTHDF1, YTHDF2, YTHDF3, YAP, CTGF, Cyr61, Ki67, Annexin V, Ki67, Edu, Vimentin and mouse anti-E-cadherin monoclonal antibody (Abcam) at 1:200 dilution in 3% BSA. Alexa-Fluor 488 (green, 1:500, A-11029; Invitrogen, USA) and 594 (red, 1:500, A-11032; Invitrogen, USA) tagged anti-rabbit or -mouse monoclonal secondary antibody at 1:1000 dilution in 3% BSA. Hoechst (3 μg/mL, cat. no. E607328; Sangon Biotech Co., Ltd.) was added for nuclear counterstaining. Six pictures were obtained with a Zeiss Axio Imager Z1 Fluorescent Microscope (Zeiss, Oberkochen, Germany). The results were presented as the Mean ± SD.
Subcellular fraction
Transfected A549 and H1299 cells were harvest in PBS and resuspended for 10 min on ice in 500 μL CLB Buffer (10 mM Hepes, 10 mM NaCl, 1 mM KH2PO4, 5 mM NaHCO3, 5 mM EDTA, 1 mM CaCl2, 0.5 mM MgCl2). Thereafter, 50 μL of 2.5 M sucrose was added to restore isotonic conditions. The first round of centrifugation was performed at 6300 g for 5 min at 4 °C. The pellet washed with TSE buffer (10 mM Tris, 300 mM sucrose, 1 mM EDTA, 0.1% NP40, PH 7.5) at 1000 g for 5 min at 4 °C until the supernatant was clear. The resulting pellets were nucleus. The resulting supernatant from the first round was transferred and subjected to differential centrifugation at 14000 rpm for 30 min. The resulting pellets were membranes and the supernatant were cytoplasm.
RNA immunoprecipitation assay
RNA immunoprecipitation (RIP) was performed using Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore) according to the manufacturer′s instructions. Briefly, cells were collected and lysed in complete RIPA buffer containing a protease inhibitor cocktail and RNase inhibitor. Next, the cell lysates were incubated with RIP buffer containing magnetic bead conjugated with indicated antibody (Abcam) or control normal human IgG. The samples were digested with proteinase K to isolate the immunoprecipitated RNA. The purified RNA was finally subjected to qPCR to demonstrate the presence of the binding targets.
RNA pulldown
Harvested cells were rinsed and sonicated in NET-2 buffer (50 mM Tris-HCl, pH 7.4, 150–300 mM NaCl, 0.05% NP40, PMSF, Benzamidine). Cell lysates were incubated with biotin-labeled probes synthesized from Sangon (Shanghai, China), and then pulled down with streptavidin beads (Sigma-Aldrich). Precipitated RNA and proteins were subsequently subjected to RT-PCR and western blot analyses, respectively.
MS2 coat protein system to enrich mRNA
The MS2 coat protein system was performed as described previously [
25]. Briefly, stably expressed pcDNA3.1-YAP-MS2–12X (YAP-MS2) NSCLC cells were co-transfected with pcDNA3.1-MS2-GFP and relevant pcDNA3.1-YTHDFs then cultivated in RPMI-1640 medium supplemented with 10% FBS (Hyclone, USA), penicillin/streptomycin (100 mg/mL). Culture flasks were kept at 37 °C for 48 h in a humid incubator with 5% CO
2. The cell lysate from these transfected cells were immunoprecipitated by GFP antibody to enrich YAP mRNA then performed the following experiment.
Total m6A measurement
The total m6A content of Total RNA was determined using an m6A methylation quantification kit (EpiGentek, USA). Briefly, after total RNA was isolated and purified, the bind RNA was planted to the assay wells and cultured with the capture antibody. After that, the wells were washed, and the detection antibody and enhancer solution were added. The m6A level was detected according to the fluorescence after the wells were incubated with the fluoro developer solution.
RNA m6A quantification using HPLC–tandem mass spectrometry
mRNA was isolated from total RNA by using a Dynabeads mRNA Purification Kit (Thermo Fisher Scientific), and rRNA contaminants were removed by using a RiboMinus Eukaryote Kit (Thermo Fisher Scientific). Subsequently, mRNA was digested into nucleosides by using nuclease P1 and alkaline phosphatase and was then filtered with a 0.22 mm filter. The amount of m
6A was measured according to HPLC–tandem mass spectrometry, following the published procedure [
26]. Quantification was performed by using the standard curve obtained from pure nucleoside standards that were run with the same batch of samples. The ratio of m
6A to A was calculated based on the calibrated concentrations.
m6A mRNA immunoprecipitation
m6A Ribonucleoprotein Immunoprecipitation reactions were performed by first isolating PolyA+ RNA from treated NSCLC cells. Protein G Dynabeads (Thermo Fisher Scientific, Baltics UAB) were washed 3× in 1 mL of IPP buffer (10 mM Tris-HCL pH 7.4, 150 mM NaCl, 0.1% NP-40). 25 μl of beads required per IP. Anti-N6-methyladenosine human monoclonal antibody (EMD Millipore, Temecula, CA, MABE1006) was added to the beads (5 μg/IP) and brought up to 1 mL with IPP buffer. Bead mixture was tumbled for 16 h at 4 °C. Beads were washed 5× with IPP buffer and 100 ng of PolyA+ RNA was added to the beads along with 1 mM DTT and RNase out. The mixture was brought up to 500 μl with IPP buffer. Bead mixture was tumbled at 4 °C for 4 h. Beads were washed 2× in IPP buffer, placed into a fresh tube, and washed 3× more in IPP buffer. m6A RNA was eluted off the beads by tumbling with 125 μl of 2.5 mg/mL N6-Methyladenosine-5′-monophosphate sodium salt (CHEM-IMPEX INT’L INC., Wood Dale, IL). Supernatant was added to Trizol-LS followed by RNA isolation as manufacture’s protocol. Final RNA sample was brought up in 10 μl of water.
qPCR for MeRIP
Reverse transcription was performed on 10 μl m6A PolyA+ RNA from the MeRIP with the iScript cDNA synthesis kit (Bio-Rad Laboratories, Hercules, CA). After diluting cDNA two-fold, quantitative real-time PCR was performed using the ABI Prism 7900HT/FAST (Applied Biosystems, USA) and primers from Integrated DNA Technologies, Inc. (Coralville, Iowa). Primers used are listed in the follows. Primer efficiency was verified to be over 95% for all primer sets used. Quantification of mRNA from the MeRIP was carried out via 2-ΔΔct. analysis against non-immunoprecipitated input RNA. All real-time PCR primer sets were designed so the products would span at least one intron (> 1 kb when possible), and amplification of a single product was confirmed by agarose gel visualization and/or melting curve analysis. Primers for MeRIP:
YAP m6A peak1 Forward primer: 5′-TGCGCGTCGGGGGAGGCAGAAG-3′.
YAP m6A peak1 Reverse primer: 5′-GGAATGAGCTCGAACATGCTG-3′.
YAP m6A peak2 Forward primer: 5′-TGAACCAGAGAATCAGTCAGAG-3′.
YAP m6A peak2 Reverse primer: 5′-GTACTCTCATCTCGAGAGTG-3′.
YAP m6A peak3 Forward primer: 5′-CCAGTGTCTTCTCCCGGGATG-3′.
YAP m6A peak3 Reverse primer: 5′-TATCTAGCTTGGTGGCAGCC-3′.
Enzyme linked immunosorbent assay (ELISA) analysis
A549 and H1299 cells were transfected with relevant plasmids and cultured for indicated times. For ELISA analysis, the YAP (Human) Cell-Based ELISA Kit (KA3582, Abnova, Taiwan, China) was used to detect the YAP protein expression following the manufacturer’s protocols. Briefly, fixing Solution, Quenching Buffer, Blocking Buffer, 1x primary antibodies were orderly added into the fixed cells and incubate overnight at 4 °C then add 50 μl of HRP-conjugated secondary antibodies and incubate for 1.5 h at room temperature and add 50 μl of TMB One-Step Reagent and incubate for 30 min at room temperature. Absorbance was read at 450 nm using a spectrophotometer (Tecan, Männedorf, Switzerland).
Analysis of publicly available datasets
To analyze correlation between ALKBH5, YAP, YTHDF1, YTHDF2 and YTHDF3 expression level and prognostic outcome of patients, Kaplan-Meier survival curves of NSCLC patients with low and high expression of ALKBH5, YAP, YTHDF1, YTHDF2 and YTHDF3 were generated using Kaplan-Meier Plotter (
www.kmplot.com/analysis and
www.oncolnc.org) [
27].
Human lung cancer specimen collection
All the human lung cancer and normal lung specimens were collected in Affiliated Hospital of Binzhou Medical College with written consents of patients and the approval from the Institute Research Ethics Committee.
In vivo experiments
To assess the in vivo effects of ALKBH5, 3 to 5-week old female BALB/c athymic (NU/NU) nude mice were housed in a level 2 biosafety laboratory and raised according to the institutional animal guidelines of Binzhou Medical University. All animal experiments were carried out with the prior approval of the Binzhou Medical University Committee on Animal Care. For the experiments, mice were injected with 5 × 106 lung cancer cells with stably expression of relevant plasmids and randomly divided into indicated groups (five mice per group). To assess the in vivo effects of cycloleucine, the xenografted tumors had reached approximately 5 mm in diameter from mice and then these xenografted mice were feed with Vehicle or cycloleucine (25 mg/kg twice weekly) and tumor volume were measured every 3 day. Tumor volume was estimated as 0.5 × a2 × b (where a and b represent a tumors short and long diameter, respectively). Mice were euthanized after 7 weeks and the tumors were measured a final time. Tumor and organ tissue were then collected from xenograft mice and analyzed by immunohistochemistry.
Statistical analysis
Each experiment was repeated at least three times. The statistical analyses of the experiment data were performed by using a two-tailed Student’s paired T-test and one-way ANOVA. Statistical significance was assessed at least three independent experiments and significance was considered at either P-value < 0.05 was considered statistically significant and highlighted an asterisk in the figures, while P-values < 0.01 were highlighted using two asterisks and P-values < 0.001 highlighted using three asterisks in the figures.
Discussion
Lung cancer is a common malignant tumor, classified into small cell lung cancer (SCLC) and NSCLC. NSCLC accounts for approximately 85% of lung cancer cases [
33]. In recent years, although the treatment of NSCLC has improved, the 5-year survival rate has not improved significantly. After the treatment of early NSCLC, approximately 20% of patients have distant metastasis [
34]. The specific mechanism of NSCLC tumor growth and distant metastasis remains unclear. In the current study, we clarified the molecular mechanism of m
6A modification and function of
YAP, regulated by ALKBH5 and YTHDFs proteins, in the regulation of NSCLC tumor growth and metastasis. The presented findings indicate that m
6A modification of
YAP is a novel target for a potential NSCLC therapy.
m
6A is a conservative post-transcriptional modification, accounting for more than 60% of all RNA modifications. Proteins responsible for the addition, removal, and recognition of m
6A can be divided into three categories: writers, erasers, and readers, respectively. The dynamic and reversible m
6A modification is regulated by methylase and demethylase. The m
6A methyltransferase complex, the “writer”, is primarily composed of methyltransferase-like 3 (mettl3) and 14 (mettl14), and Wilms tumor 1-binding protein (wtap) [
14]. Demethylase, the “eraser”, is mainly composed of FTO and ALKBH5. ALKBH5 co-localizes with nuclear speckles to regulate the assembly/modification of mRNA processing factors, demethylate m
6A mRNA, and modulate mRNA export and stability [
35]. Therefore, loss of function of ALKBH5 leads to the disorder of many biological functions, for example, in
alkbh5-deficient cells, because of accelerated nuclear RNA output, the cytoplasmic RNA levels increase significantly, the overall RNA stability decreases, and spermatocytes undergo apoptosis [
7]. In addition,
alkbh5 gene knockout increases exon hopping, resulting in rapid degradation of transcripts with abnormal splicing [
36]. We here showed that ectopic expression of
ALKBH5 inhibited NSCLC cell proliferation, invasion, migration, and EMT (Fig.
1). Importantly, ALKBH5 decreased the m
6A level of
YAP and inhibited
YAP expression in NSCLC in a m
6A dependent manner (Fig.
2). Furthermore, a pharmacological inhibitor of m
6A, cycloleucine, could inhibited tumor growth and metastasis (Fig.
7), which indicated m
6A modification plays an important role in the development and progression of human tumors.
The m
6A binding proteins (“readers”) are primarily the YTH domain protein family (YTHDF1/2/3), nuclear heterogeneous protein HNRNP family, and IGF2BP protein family [
15]. In the current study, we showed that the m
6A modification of
YAP pre-mRNA is first recognized by YTHDF3, which is followed by a competitive binding of YTHDF1 and YTHDF2 to YTHDF3, to decide the fate of
YAP pre-mRNA, i.e., decay or translation (Fig.
3a). Specifically, recognition and binding of m
6A mRNA sites by YTHDF3 and YTHDF2 led to mRNA transfer from a translatable pool to mRNA degradation site. After YTHDF2 binds YTHDF3, which carries
YAP pre-mRNA containing m
6A, YTHDF2 presents
YAP mRNA to AGO2, and then AGO2 recruits other molecules to form RISC system to facilitate
YAP mRNA decay, reducing YAP protein level. It has been recently shown that microRNAs, such as miR-195, miR-375, and others, decrease
YAP mRNA levels by binding to
YAP 3′UTR [
37,
38]. Conversely, on the mRNA translation level, recognition and binding of m
6A by YTHDF1 and YTHDF3 result in enhanced protein synthesis. As shown in the current study, following YTHDF1 binding of YTHDF3, which carries
YAP pre-mRNA with m
6A modification, YTHDF1 presents
YAP mRNA to eIF3a-contained translation initiation complex to promote
YAP mRNA translation, consistently with previous reports [
39], leading to increased YAP protein levels. These observations account for regulation of YAP expression by m
6A modification, i.e., by balancing the function of YTHDF1 and YTHDF2 via the YTHDF3 hub in NSCLC tumor and normal tissues. Specifically, the expression of YTHDF2 was higher in normal tissues than in tumor tissues (Fig.
4). This indicates that after YTHDF3 recognizes m
6A modification on
YAP mRNA, YTHDF2 was more likely to bind to YTHDF3, promoting
YAP pre-mRNA decay, and thus maintaining normal development and growth of an organism. By contrast, the expression of YTHDF1 was higher in tumor tissues than in normal tissues (Fig.
5). This indicates that after YTHDF3 recognizes m
6A modification on
YAP mRNA, YTHDF1 was more likely to bind to YTHDF3, promoting
YAP mRNA translation, and thus excessive cell growth and metastasis in NSCLC.
miRNAs interact with specific mRNA to degrade the corresponding target mRNA, thus inhibiting the translation of target mRNA and widely participating in the life processes of an organism, such as growth, development, differentiation, metabolism, and defense responses [
40,
41]. Further, significant differences in the expression of various miRNAs in normal and tumor cells have been reported [
23]. These miRNAs play a role similar to that of proto-oncogenes or tumor suppressor genes, by regulating different target genes, which are closely related to the occurrence, development, clinical treatment, and prognosis of many tumors [
24]. Furthermore, HuR inhibits translation inhibition mediated by some miRNAs by directly binding and sequestering microRNA. Recently, HuR was shown to interact with such miRNAs as mir-16, mir-1192, and mir-29, to regulate the expression of
COX-2,
HMGB1, and
A20 genes, respectively [
42‐
44]. In the case of miR-107, we here demonstrated that HuR binds to miR-107 as a miRNA sponge and blocks the translation inhibition of LATS2. Panneerdoss et al. (2018) showed that ALKBH5 increases the expression of HuR [
13], in agreement with the findings of the current study. It remains unclear as to how ALKBH5 regulates HuR; however, we here showed that ALKBH5 KD does not regulate the HuR protein levels, indicating that m
6A modification is possibly involved in the regulation of HuR. In other words, ALKBH5 enhances LATS2 expression by regulating the HuR/miR-107 axis. LATS2 phosphorylates YAP to decrease YAP activity and promote the interaction with 14–3-3 protein for YAP degradation. Collectively, ALKBH5 decreased the activity of YAP by regulating miR-107/LATS2 axis in an HuR-dependent manner (Fig.
6).
Most recent studies have shown that, the MST/Hippo signaling pathway is closely associated with the biological evolution and development, playing an important role in the regulation of cell proliferation, survival, cell signal transduction, organ generation, stem cell self-renewal, and organ volume [
22]. Therefore, this signaling pathway has attracted increasing interest in the scientific community [
45,
46]. Several key proteins in the MST/Hippo signaling pathway participate in tumor formation and apoptosis. The pathway contains multiple cell signal switches, and various signal molecules coordinate with each other to regulate cell proliferation and apoptosis [
47]. The MST/Hippo signal pathway might be used to inhibit the growth of tumor cells, by regulating the Yap protein as a tumor suppressor. Yap activation is coordinated by the TEAD protein, and drugs may be used to inhibit the formation of the YAP–TEAD complex to block cell proliferation, which eventually leads to tumor apoptosis and death. Using this mechanism for drug research and development might provide valuable reference for the development of anti-tumor treatment. Specific drugs that block this signaling pathway, such as verteporfin, can inhibit cell proliferation by blocking the interaction between YAP and TEAD [
48]. Therefore, in-depth study of the MST/Hippo signaling pathway could clarify the precise mechanism of tumor formation, further guiding and improving the clinical treatment, and providing a theoretical basis for the discovery of unknown tumor therapeutic targets.
Our study was not free of limitations: for example, the specific microRNA was involved in the AGO2-mediated YAP mRNA decay. Although we specified some microRNAs such as miR-195, miR-847-3p, et al.; a certain report indicated that YTHDF2 promotes cancer cell growth, which is contrary to our findings. One possible explanation is that when YTHDF2 interacts with tumor suppressor genes then promotes cell growth. While when YTHDF2 interacts with oncogenes then inhibits cell growth; unknown molecular mechanisms that higher expressions of YTHDF1 were in tumor tissues while higher expressions of YTHDF2 were in normal tissues of NSCLC; whether the regulatory mechanism mentioned above of ALKBH5 and YTHDFs applies to other genes or is limited to YAP need to be further explored. Therefore, these unresolved limitations need to be addressed in future studies.