Gastric cancer is biologically heterogeneous and it presents various genetic alterations. Most gastric cancer patients are diagnosed at an advanced stage, and conventional chemotherapy has shown limited efficacy. A recent report predicted the prognosis and response to adjuvant chemotherapy in gastric cancer patients by classifying individuals based on cancer-related genes [1
]. Although significant efforts have been made for targeted molecular therapies, only a few targeted therapies that extend survival are currently available for patients with gastric cancer. The majority of these patients exhibit resistance while the monoclonal anti-HER2 antibody Trastuzumab is effective in some HER
-positive gastric cancer patients [2
]. Ramucirumab, which is an antibody that targets VEGFR-2, has proven to be useful alone or in combination with paclitaxel as a second-line treatment for advanced gastric cancer [5
]. To date, the gastric cancer-related potential therapeutic targets include EGFR, VEGF, MET, FGFR, PI3K/mTOR, and HDAC [5
]. Unfortunately, clinical trials for inhibitors of these identified targets have failed to demonstrate significant clinical efficacy [5
]. Therefore, there is a critical need to identify novel therapeutic targets based on the molecular mechanisms of gastric cancer.
The Cancer Genome Atlas classifies gastric cancer into four types based on genetic changes [9
]. Somatic mutations, such as amplification, deletions, and structural abnormalities in chromosomal region, TP53 mutations, and amplification of the RTK/RAS/MAPK pathway characterize the chromosomal instability (CIN) subtype. Genomically stable (GS) subtype, which is associated with diffuse gastric cancer, has frequently mutations in RHOA and CDH1 gene or fusions involving Rho-family GTPase-activating proteins (GAP). The microsatellite instability (MSI) subtype is characterized by PI3KCA, ERBB3
, and MLH1
silencing, MHC class 1 gene alterations, and tumor-specific neoantigens. The Epstein-Barr virus (EBV) subtype has a high rate of PIK3CA
mutations and high PD-L1
mutation frequently occurs in 80% of EBV subtype tumors and 3% of CIN subtype tumors [11
]. The mutation or amplification of PIK3CA
promotes cell growth and drug resistance [12
) family that comprises VGLL1
is named after the Drosophila transcriptional coactivator Vestigial (Vg) [14
]. They contain the TOUDU domain to mediate interactions with TEA domain transcription factors (TEADs), which are essential in development [14
, which is a member of the Hippo signaling pathway, is overexpressed in various cancers and it requires coactivators, such as Yes-associated protein (YAP
) or transcription coactivator with a PDZ-binding motif (TAZ) to induce the expression of c-myc
, and VEGF-A
]. VGLL4 functions as a tumor suppressor in cancer by competing with YAP for TEAD binding [18
]. Interestingly, the structural similarities between VGLL1 and YAP or TAZ suggest the formation of the VGLL1–TEAD complex [21
expression is reported to be associated with reduced overall survival (OS) in triple-negative basal-like breast carcinoma [23
]. However, the molecular function of VGLL1
in cancer remains unclear.
Here, we investigated the clinical relevance and molecular function of VGLL1 in gastric cancer through in vitro experiments and in vivo mouse models. We further explored the underlying regulatory mechanisms for evaluating VGLL1 as a potential therapeutic target in gastric cancer
The Hippo signaling pathway suppresses the proliferation and metastasis of cancer cells by negatively controlling YAP and TAZ, which are cofactors of TEAD transcription factors. The structural similarities between VGLL1 and YAP or TAZ suggest the formation of VGLL1–TEAD complex for cancer malignancy [21
]. However, the molecular mechanism of VGLL1 in cancer is still poorly characterized. Here, we present the regulation mechanism of VGLL1
to induce MMP9 expression that promotes gastric cancer malignancy (Figure 6
Genetic alterations in the EGFR
, and the PI3K
pathway are often observed in gastric cancer. PIK3CA
is the most mutated PI3K isoform, with an 18% mutation and 5% amplification frequency in gastric cancer [26
]. Microarray analysis of gastric cancer patient tissues revealed that VGLL1
was a prognostic biomarker and its expression was highly correlated with that of PIK3CA
. Moreover, a high expression of VGLL1
predicted worse OS in gastric cancer patients.
We observed that VGLL1 promoted the proliferation and tumorigenesis of gastric cancer cells in in vitro cell culture and in vivo xenograft mouse models. Importantly, shVGLL1-expressing NUGC3 cells suppressed lung metastasis in a mouse model, implying a crucial role of VGLL1 in cancer metastasis.
We revealed that PI3K/AKT signaling was involved in the regulation of VGLL1
transcription. Promoter analysis demonstrated that the β-catenin/p300/TCF4/LEF1 complex induces the transcription of VGLL1
. MMP9 was explored as a potential candidate target genes of VGLL1, because it has been reported as a molecular marker of metastasis in gastric cancer [24
]. VGLL1 interacted with TEAD4 at the −571 TEA site in the MMP9
promoter. The MMP9 levels were elevated in metastasized cancer cells in the lungs and liver in a VGLL1
-overexpressing NUGC3 xenograft model. The MMPs, a family of zinc containing enzymes, degrade components of the extracellular matrix and regulate extracellular matrix turnover, as well as cancer processes, such as proliferation, angiogenesis, and tumor metastasis [27
]. Interestingly, MMP9 mainly promotes tumor invasion and metastasis, while TIMP-1 inhibits the functions of MMP9 in gastric cancer, which suggests that the imbalance between MMP9 and TIMP-1 expression may occur in tumor progression [25
]. We will further investigate the relationship between VGLL1 and TIMP.
Reportedly, the amino acid sequences SVIFT/HQIVHV of β2 at interface I and the VxxHF/LxxLF motif at interface II of VGLL1/YAP interact with TEAD4 to drive the expression of target genes [21
]. Interestingly, we observed that VGLL1 regulated only MMP9
mRNA expression, whereas YAP only regulated CTGF
mRNA expression in NUGC3 cells. Moreover, cell proliferation promoted by VGLL1
overexpression was not affected by YAP
overexpression or knockdown (Supplementary Figure S4
), which indicated that VGLL1 and YAP1 independently induced transcription of their target genes. Therefore, it is likely that the TEAD4–VGLL1 and TEAD4–YAP complexes use their distinctive transcriptional machineries.
Several inhibitors of the YAP–TEAD4 complex binding have been reported. Flufenamic acid, which binds the central pocket of the YAP-binding domain of TEAD2, inhibits the proliferation and migration of cancer cell [22
]. Verteporfin, which disrupts the YAP–TEAD complex, is found to increase the sensitivity to paclitaxel in HCT-8/T cells [30
], as well as sensitivity to erlotinib in lung cancer cells [31
]. Likewise, the disruption of TEAD-VGLL1 interactions might be of use in development of anticancer drugs.
In this study, we discovered VGLL1 as a novel prognostic biomarker correlated with PIK3CA or PIK3CB in gastric cancer. We elucidated the molecular mechanism underlying the regulation of VGLL1 transcription by the PI3K/AKT/β-catenin. The formation of the VGLL1–TEAD4 complex activates the transcription of MMP9, which then promotes proliferation and metastasis in gastric cancer cells. Taken together, we clearly elucidated the molecular mechanism that involves VGLL1 that promotes malignancy in gastric cancer, thus suggesting VGLL1 as a therapeutic target in treatment of gastric cancer.
4. Materials and Methods
U0126, LY294002, KN93, SP600125, were purchased from Sigma-Aldrich (St. Louis, MO, USA). TGF-β was purchased from Peprotech (Seoul, Korea).
4.2. Cell Culture
Human gastric cancer cells (NUGC3, AGS, NCI-N87, SNU1, SNU5, SNU16, SNU216, SNU484, SNU668, Hs746T, and MKN28 cells) were cultured in RPMI-1640 medium that contained 10% fetal bovine serum (FBS). NUGC3 cells constitutively expressing VGLL1 (NUGC3-VGLL1 cells) were selected with 500 μg/mL geneticin (Thermo Scientific, Logan, UT, USA). HEK293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% FBS. All the cells were cultured in an atmosphere of 5% CO2 at 37 °C. STR profiling by Korea Cell Line Bank authenticated all of the cell lines (Seoul, Korea).
A retrospective review of a gastric cancer cohort database prospectively maintained at Yonsei University College of Medicine (Seoul, South Korea) was conducted to identify all the gastric adenocarcinoma patients who underwent curative D2 gastrectomy between 2000 and 2010. Demographic and clinicopathological information and tumor tissue samples were obtained from 556 patients. The institutional review board of Severance Hospital approved this study (Seoul, Korea; 2015-3104-001).
4.4. Microarray Experiments and Data Processing
The total RNA extracted from 556 gastric cancer tissues was used for labeling and hybridization, according to the manufacturer’s protocols (Illumina HumanWG-6 BeadChip, version 2, Illumina, San Diego, CA, USA). The arrays were scanned with an Illumina Bead Array Reader confocal scanner (BeadStation 500GXDW; Illumina, Inc., San Diego, CA, USA), as per the manufacturer’s instructions. After scanning, the microarray data were log2 transformed, median centered across genes and samples, and normalized while using quantile normalization in the R language environment (version 3.2.5, The R Foundation for Statistical Computing, Vienna, Austria). The microarray data set of gastric cancer samples from patients is available in the NCBI Database of GEO datasets under the data series accession numbers GSE13861 and GSE84437. The microarray analysis of VGLL1 siRNA-treated NUGC3 cells while using same procedure and microarray data set is available in the NCBI Database of GEO datasets under accession numbers GSE130071. We applied the FDR approach for analysis of microarray data. After FDR correction, we selected genes (p value < 0.05 and log2FC > 1) for GO analysis.
4.5. Statistical Analysis of Microarray
Pearson correlation coefficients were calculated for evaluating the association between genes. We obtained an optimal cut-off for gene expression from ROC analysis to classify patients into two subgroups by single gene expression, in which the best cut-off was determined by the expression with the highest multiply of sensitivity and specificity. Statistical analyses was carried out while using Medcalc version 18.11.6 (MedCalc software, Ostend, Belgium). The Kaplan-Meier method was used to calculate the time before death or recurrence, and difference between the times was assessed using logrank test (MedCalc software, Ostend, Belgium). A gene set enrichment analysis was carried out to identify the most significant gene sets associated with molecular and cellular functions. Fisher’s exact test estimated the significance of over-represented gene sets. Gene set enrichment analyses were performed using the DAVID bioinformatics resources (ver. 6.8, Laboratory of Human Retrovirology and Immunoinformatics, Frederick, MD, USA).
4.6. Plasmids Construction
pOBT7-VGLL1 and pCMV-SPORT6-TEAD4 were obtained from Korean UniGene Information (KUGI). VGLL1 mRNA was amplified via PCR and then cloned into the HindIII/BamHI sites of pcDNA3.1 with Myc-tag. TEAD4 was inserted in the HindIII/BamHI sites of pEGFP-N1. YAP was amplified via PCR and then cloned into the EcoRV/XbaI sites of pcDNA3.1 with Myc-tag. Fragments of the MMP9 promoter (−657/+25, −557/+25) were PCR-amplified and subsequently inserted into the KpnI/XhoI site of pGL4.17 (Luc2/neo). The MMP9 promoter, which contained mutation in TEA binding site (5′-CATTCC-3′→5′-CAGGGC-3′), was generated while using the Quikchange Site-Directed Mutagenesis kit. The fragment of VGLL1 promoter was PCR-amplified and cloned into the XhoI/HindIII site of pGL2-basic luciferase plasmid. pCMV3-β-catenin, pCMV3-p300, pCMV3-TCF4, and pCMV3-LEF1 were obtained from Sino Biological (Wayne, PA, USA).
4.7. Live cell Assay for Cell Proliferation and Migration
Proliferation rates, which were based on cell confluence, were determined by live-cell imaging (IncuCyte ZOOM System, Essen BioScience, Ann Arbor, MI, USA), as described previously [32
]. To analyze cell migration, the cells were cultured in 96-well ImageLock Plates (Essen BioScience) to reach confluence prior to wound creation. A scratch was made in confluent monolayers while using a 96-pin WoundMaker (Essen BioScience, Ann Arbor, MI, USA). The cells were washed with PBS and then incubated using the IncuCyte ZOOM system. Cell migration was analyzed at 2 h intervals throughout the duration of the experiment.
4.8. Spheroid Formation
Three-dimensional (3D)-Spheroid culture was induced, as described previously [33
]. In brief, cells were trypsinized, counted, and diluted to 5 × 104
cells in a 20 μL droplet. The droplets of cell suspension were placed on the lid of a sterile non-adherent polystyrene petri dish that was filled with DPBS, and then cultured at 37 °C in a 5 % CO2
incubator for 48 h.
4.9. Mice Experiments
The bioethics committee of the Korea Research Institute of Bioscience and Biotechnology approved all animal experiments (KRIBB-ACE-16101, KRIBB-ACE-17051, and KRIBB-ACE-18209). In vivo xenografts were performed, as described previously [32
]. NUGC3 cells (5 × 106
) that were infected with a lentiviral shVGLL1
vector were subcutaneously injected into five-week-old female BALB/c nude mice. According to the protocol that was published by Le A. [34
], stable VGLL1
-expressing HEK293T cells (1 × 107
) were subcutaneously injected into five-week-old female BALB/c nude mice.
We established an in vivo xenograft mouse model to study the effect of VGLL1 on gastric cancer metastasis. Five-week-old female nude mice (six mice per group) were subcutaneously inoculated with NUGC3-EV or NUGC3-VGLL1
cells (5 × 106
) in the right flank. Surgical resection of the primary tumor was performe when the average tumor volume reached 500 mm3
. After four weeks, the mice were sacrificed by cervical dislocation under isoflurane anesthesia, and lung and liver tissue was also removed for subsequent experiments. For a tail vein-injection assay of cancer metastasis, NUGC3 cells (1 × 106
) cells that were infected with a lentivirus expressing shControl or shVGLL1
were injected into the tail vein of mice (four mice per group) in 100 µL of phosphate-buffered saline (PBS). After 16 weeks, the lungs were removed and fixed. Hematoxylin and eosin (H&E) assessed tumor metastasis to the lungs and liver. Photos of random fields were obtained at a magnification of 40× (3 fields/mouse) and analyzed while using NIH Image software for the quantitation of metastasis (ver. 1.48, Wayne Rasband, Bethesda, Maryland, USA) [35
4.10. Western Blot Analysis
The cells were lysed with RIPA buffer (Millipore, Billerica, MA, USA) containing protease inhibitor cocktail (Roche), and the lysates were quantified with a protein assay kit (Bio-Rad, Hercules, CA, USA). The cell lysates were separated using SDS-PAGE and then transferred to PVDF membranes. The proteins were identified while using appropriate antibodies. Anti-VGLL1 (10124-2-AP) was purchased from Proteintech (Rosemont, IL, USA). Anti-TEAD4 (ab58310) and anti-MMP9 (ab76003) were purchased from Abcam (Cambrige, MA, USA). Anti-GFP (NB600-308) was purchased from Novus Biologicals (Centennial, CO, USA). Anti-Myc (sc-789) and anti-HA (sc-805) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-GAPDH (LF-PA0212) was purchased from AbFrontier (Seoul, Korea). Anti-β-tubulin (2128), anti-pSer473-AKT (9271), anti-AKT (9272), anti-p-β-catenin (9561), and anti-β-catenin (9562) were purchased from Cell Signaling (Danvers, MA, USA). Anti-Flag (F1804) was purchased from Sigma-Aldrich (St. Louis, MO, USA). The protein signal was detected while using an enhanced chemiluminescence kit (Millipore, Burlington, MA, USA).
4.11. Reverse Transcriptase Polymerase Chain Reaction and Quantitative Real-Time PCR
The total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and cDNA was synthesized using TOPscript™ RT DryMIX (Enzynomics, Daejeon, Korea). RT-PCR was performed using Dr. Taq MasterMix (Doctor Protein, Daejeon, Korea). Quantitative real-time PCR was performed using a SYBR Green master mix kit (Qiagen, Valencia, CA, USA). The following sequences of primers were used: VGLL1 (F) 5′-GAGCTGTGGCATTTCTCCTC-3′, (R) 5′-AAGTGGGTGTGAGCAGCTTT-3′, MMP9 (F) 5′-TCTATGGTCCTCGCCCTGAA-3′, (R) 5′-CATCGTCCACCGGACTCAAA-3′, TEAD4 (F) 5′-GAACGGGGACCCTCCAATG-3′, (R) 5′-GCGAGCATACTCTGTCTCAAC-3′, YAP (F) 5′-CGCTCTTCAACGCCGTCA-3′, (R) 5′-AGTACTGGCCTGTCGGGAGT-3′, CTGF (F) 5′-CTTGCGAAGCTGACCTGGAA-3′, (R) 5′-AAAGCTCAAACTTGATAGGCTTGGA-3′, PIK3CA (F) 5′-TGCAGCCATTGACCTGTTTA-3′, (R) 5′-GTCAAAACAAATGGCACACG-3′, GAPDH (F) 5′-TCATGACCACAGTCCATGCC-3′, (R) 5′-TCCACCACCCTGTTGCTGTA-3′, RPL13A (F) 5′-CATCGTGGCTAAACAGGTAC-3′, and (R) 5′-GCACGACCTTGAGGGCAGC-3′. The primers of PIK3CB (P144505) was purchased from Bioneer (Daejeon, Korea).
4.12. Gene Knockdown Using siRNA
Introducing siRNA into the target gene, using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions, was undertaken to perform gene knockdown. The siRNA sequences were, as follows: siScramble (siSC) 5′-CCUACGCCACCAAUUUCGUdTdT-3′, siVGLL1 5′-AGCCUAUAAAGACGGAAUGGA AUdTdT-3′ and 5′-CCCGGUGUGUCCUUUUCACCUACdTdT-3′, siTEAD4 5′-GACACUACUCUUACCGCAUdTdT-3′, siYAP 5′-CCACCA AGCUAGAUAAAGAAAdTdT-3′, siMMP9 5′-CCACAACAUCACCUAUUGGAUdTdT-3′, sip300 5′-GAUGAAUGCGGGCAUGAAU dTdT-3′, siβ-catenin 5′-CGUUCUCCUCAGAUGGUGUdTdT-3′, siTCF4 5′-CAGACAAAGAAAGUUCGAAdTdT-3′, siLEF1 5′-GAACGACUCUGAAAUCAUCUU dTdT-3′, siPIK3CA 5′-CUGAGAAAAUGAAAGCUCACUCUdTdT-3′, and siPIK3CB 5′-CAGUACAAUUUGGUGUCAUdTdT-3′.
4.13. Lentivirus Infection
shControl and shVGLL1(Sigma, TRCN0000019618) were packaged into lentivirus via HEK293T cells, using Lipofectamine and PLUS Reagent (Invitrogen, Carlsbad, CA, USA), and then transduced into NUGC3 cells. Post-transduction 48 h, the NUGC3 cells were selected with puromycin (1 μg/mL).
4.14. Immunohistochemistry (IHC)
US Biomax supplied tissue array blocks of human gastric cancer and normal tissues (Rockville, MD, USA). IHC was performed, as previously described [32
]. The slides were incubated with anti-VGLL1 (Proteintech, 10124-2-AP, Rosemont, IL, USA), anti-Ki67 (abcam, ab15580), and anti-MMP9 (abcam, ab76003) antibodies. After washing with PBS, the slides were incubated with biotinylated anti-rabbit IgG (Vector Laboratories, Burlingame, CA, USA) and avidin-biotin peroxidase (Vector Laboratories) and visualized using diaminobenzidine tetrahydrochloride (Vector Laboratories, Burlingame, CA, USA). The sections were counterstained with hematoxylin.
4.15. Invasion Assay
For invasion assays, chambers with 8.0-μm-pore PET membrane in 24-well cell culture inserts (BD Biosciences, San Jose, CA, USA) were used. The cells in serum-free medium were seeded into the upper part of each chamber with Matrigel coating, whereas the lower compartments were filled with the above-mentioned medium. The cells were then allowed to invade, being subsequently fixed with 10 % formalin, and stained with sulforhodamine B (SRB), as previously described [36
4.16. Luciferase Assay
A dual-luciferase reporter system determined the promoter activity (Promega, Madison, WI, USA). The cells were transfected with pGL4.17-MMP9-luciferase (MMP9-luc), pGL2-VGLL1-luciferase (VGLL1-luc), and pRL-SV40 plasmid encoding firefly (Renilla)-luciferase, using PolyFect (Qiagen, Valencia, CA, USA). The luciferase activity was measured using a luminometer (VICTOR X Light; PerkinElmer, Waltham, MA, USA). The results were normalized to the activity of Renilla luciferase.
4.17. Chromatin Immunoprecipitation (ChIP) Assays
ChIP assays were performed, as previously described [36
]. Briefly, the cells were crosslinked using 1% formaldehyde and then lysed. The chromatin was then sheared via sonication on ice. The lysates were incubated with antibodies targeting β-catenin, VGLL1 or TEAD4, or with normal mouse immunoglobulin G (IgG) overnight at 4 °C. Thereafter, protein was digested using proteinase K (Millipore, Middlesex County, MA, USA). The ChIP-enriched DNA was subjected to PCR while using either of the following two primers: VGLL1-a (5′-GTA GAC AAA GAG AGG AGC-3′ and 5′-GGC TTC CAT TGG CCA AAG-3′), VGLL1-b (5′-TTT GTT GTT GAC TCT GTG T-3′ and 5′-AAG GCG TTT CCT GCT AGC-3′), MMP9-a (5′-TACTGTCCCCTTTACTGC-3′ and 5′-CTTCCTCTCCCTGCTTCA-3′), and MMP9-b (5′-TGGTGTAAGCCCTTTCTC-3′ and 5′-AGGAGGCGCTCCTGTGAC-3′).
4.18. Statistical Analyses
Student’s t-tests or Chi-square tests were used for statistical analyses. The bars indicate S.D., and the asterisks denote significant differences (*** p ≤ 0.005, ** p ≤ 0.01, * p ≤ 0.05) between the means of two groups.