Cancer Cell, Volume 25

Supplemental Information

A Peptide Mimicking VGLL4 Function Acts

as a YAP Antagonist Therapy against Gastric Cancer Shi Jiao, Huizhen Wang, Zhubing Shi, Aimei Dong, Wenjing Zhang, Xiaomin Song, Feng He, Yicui Wang, Zhenzhen Zhang, Wenjia Wang, Xin Wang, Tong Guo, Peixue Li, Yun Zhao, Hongbin Ji, Lei Zhang, and Zhaocai Zhou

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Supplemental Data

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Figure S1, related to Figure 1. (A) 24 human gastric tumors and their paired adjacent normal tissues were tested. Expression of VGLL4 and YAP in the tissue extracts was determined using specific antibodies (upper). Scatter plot of YAP levels at different tumor grades of GC (below). The horizontal lines in the scatter plot represent group medians. (B) Representative cores of VGLL4 staining on tissue microarray (upper). YAP staining in normal mucosa, gastric dysplasia and gastric cancer were calculated (below). YAP expression levels: negative (-), weak (+), moderate (++), strong (+++). (C) The transcriptional levels of YAP in GC. (D) Box plot of YAP target genes by VGLL4 mRNA levels in GC sample of Stage I & II (left) or Stage II& IV (right). (E) Box plot of VGLL4, YAP and its target genes mentioned above according to TNM stage. The relative mRNA levels of VGLL4, YAP and its target genes were compared between Stage I & II and Stage III and IV. (F) Box plot of YAP target genes by YAP to VGLL4 ratio at the mRNA levels. (G) Box plot of YAP target genes according to VGLL4 levels. The relative mRNA levels of CTGF, CYR61 and CDX2 in GC samples were analyzed by VGLL4 mRNA levels.

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Note: The horizontal lines in the box plots represent the median, the boxes represent the interquartile range, and the whiskers represent the 2.5th and 97.5th percentiles. Student’s t-test was used to compare the difference between the two groups. n=3, *vs control group, p<0.05; NS no significance.

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Table S1, related to Figure 1. Clinical Significance of VGLL4 Expression in GC

Groups VGLL4 expression

n Positive (%) p value

(Fisher’s test) − + + + + + + Sex Male 23 17 6 5 51 54.9

0.5553 Female 18 14 7 1 40 55.0 Age <60 17 17 7 3 44 61.4

0.6719 >=60 24 14 6 3 47 48.9

Tumor Size pT1+pT2 10 13 8 4 33 75.8

0.0322* pT3+pT4 31 18 5 2 58 43.1

Lymph node metastasis N0+N1 9 19 8 3 39 76.9

0.0021* N2+N3 32 12 5 3 52 38.5

Distant metastasis M0 31 29 12 5 77 59.7

0.1675 M1 10 2 1 1 14 28.5

Tumor stage I+II 10 12 9 4 35 71.5

0.0125* III+IV 31 19 4 2 56 44.6

Total 41 31 13 6 91 Note: Fisher’s exact test was used to test the association between two categorical variables; * represents statistically significant, p<0.05.

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Table S2, related to Figure 1. Univariate and Cox Multivariate Analyses on IHC Microarray for Overall Survival

Groups Univariate analysis Cox multivariate analysis

HR 95% CI p value HR 95% CI p value Age (years) < 60 vs≧60 1.59 0.82-3.12 0.1262 1.43 0.89-3.26 0.0951

Gender Male vs Female 1.47 0.74-2.08 0.2437 1.13 0.84-1.47 0.1137

Tumor Size (pT1+T2) vs (pT3+T4)

2.48 1.81-4.02 0.0037* 2.25 1.81-3.24 0.0029*

Lymph node metastasis (N0+N1) vs (N2+N3) 2.64 1.23-4.40 0.0053* 2.93 1.39-5.15 0.0008*

Distant metastasis M0 vs M1 2.18 1.33-2.83 0.0120* 1.80 1.02-2.54 0.0072*

Tumor stage Stage I&II vs III&IV 5.02 2.53-7.46 <0.0001* 3.87 1.40-6.33 <0.0001* VGLL4 expression 0.21 0.12-0.63 0.0215* 0.44 0.31-0.88 0.0710 YAP expression 1.72 0.87-2.42 0.0322* 1.50 0.85-2.05 0.2182

Note: * Statistically significant, p<0.05.

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Table S3, related to Figure 1. Up-regulation of YAP Correlates with Gastric Cancer Progression

Groups YAP mRNA

n Increased

(%) p value

(Fisher’s test) Non-increased Increased Age (years) <60 17 20 37 59.4

0.5060 >=60 17 30 47 61.7

Gender Male 19 31 50 68.0

0.6530 Female 15 19 34 55.0 Helicobacter Pylori Positive 12 33 45 68.9

0.0007* Negative 22 17 39 51.2

Lauren Intestinal 27 39 66 66.1

1.0000 Non-intestinal 7 11 18 55.6

Differentiation Low 23 27 50 58.0

0.0373* Moderate or High 11 23 34 64.7

Lymphatic invasion Ly0-1 7 17 24 45.4

0.2234 Ly2-3 27 33 60 66.3

Tumor Size pT1+pT2(<=5 cm) 11 6 17 35.3

0.0289* pT3(>5 cm)+pT4 23 44 67 67.2

Lymph node metastasis N0+N1 10 24 34 44.1

0.1144 N2+N3 24 26 50 72.0

Distant metastasis M0 29 45 74 60.1

0.5166 M1 5 5 10 60.0

Tumor stage Stage I + Stage II 8 25 33 45.4

0.0223* Stage III + Stage IV 25 26 51 70.6 Total 34 50 84

Note: Fisher’s exact test was used to test the association between two categorical variables; * represents statistically significant, p<0.05.

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Table S4, related to Figure 1. Ratio of YAP to VGLL4 (Protein Level) in Different Tumor Grades of Gastric Cancers

Tumor grade Ratio of YAP to VGLL4 p value (vs Grade I) I 2.7 ± 0.4 - II 4.1 ± 0.7 0.1341 III 6.3 ± 0.5 0.0475* IV 9.1 ± 1.6 0.0255*

Note: Student t test was used to test the difference between the two groups; * represents statistically significant, p<0.05.

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Table S5, related to Figure 1. YAP/VGLL4 Ratio (mRNA) Correlates with GC Progression

Groups YAP/VGLL4 mRNA ratio

n Increased

(%) p value

(Fisher’s test) Non-increaseda Increasedb Age (years) <60 14 23 37 62.2

0.6592 >=60 22 27 47 57.4

Gender Male 22 28 50 58.0

0.8258 Female 14 20 34 58.8 Helicobacter Pylori Positive 14 31 45 68.9

0.0271* Negative 22 17 39 43.6

Lauren Intestinal 26 40 66 60.6

0.2848 Non-intestinal 10 8 18 44.4

Differentiation Low 19 31 50 62.0

0.3693 Moderate or High 17 17 34 50.0

Lymphatic invasion Ly0-1 18 12 30 45.4

0.0225* Ly2-3 18 36 54 66.3

Tumor Size pT1+pT2(<=5 cm) 13 4 17 23.5

0.0056* pT3(>5 cm)+pT4 25 42 67 62.7

Lymph node metastasis N0+N1 18 16 34 47.1

0.1776 N2+N3 18 32 50 64.0

Distant metastasis M0 29 40 69 40.1

0.7794 M1 7 8 15 53.3

Tumor stage Stage I + Stage II 20 14 34 41.2

0.0240* Stage III + Stage IV 16 34 50 68.0 Total 36 48 84

Note: a Increased: ratio YAP/VGLL4 mRNA >5.2; b Non-increased: ratio YAP/VGLL4 mRNA: 0-5.2; Fisher’s exact test was used to test the association between two categorical variables; * represents statistically significant, p<0.05.

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Figure S2, related to Figure 2. (A) Protein levels of YAP and VGLL4 were determined by western blotting against special antibody. Relative gray values of protein bands are calculated. (B) Cell apoptosis was detected in several GC cells. BGC-823, MGC-803, HGC-27 and MKN-45 cells after transfection with VGLL4. (C-F) Cell growth (C), colony formation (D), TEAD4 luciferase activity (E) and YAP target genes’ expression (F) were detected in several GC cells transfected with different doses of

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VGLL4 in the absence or presence of YAP. (G) Cell viability was analyzed in cells mentioned above after transfection with special VGLL4 shRNA. (H) Rescue assay of VGLL4 and VGLL4 (HF4A) mutant on TEAD4 trans-activation (I) Relative mRNA levels of YAP target genes in cell after transfection with special shVGLL4. Note: Data were expressed as Means ± S.D. One-way analysis of variance (ANOVA) and Student’s t-test were used. n=3, #vs vector-transfected group, p<0.05; *vs YAP-transfected group, p<0.05, **p<0.01, ***p<0.001; NS, no significance.

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Figure S3, related to Figure 3. (A) Gel filtration of VGLL4 and TEAD4. Recombinant proteins of VGLL4, TEAD4, and a mixture of VGLL4 and TEAD4 with a molar ratio of 1:2 were loaded to Superdex 75 column. (B) Purified proteins of TEAD4, VGLL4 and VGLL4-TEAD4 mixture with a molar ratio of 1:2 were concentrated to 2 mg/ml. The weighted distribution and homogeneity of the three samples were determined at 25°C by dynamic light scattering. Scattered light was measured at a 90° angle using a 658 nm wavelength. (C-E) The TDU2 of VGLL4 forms an intermolecular beta-sheet with two TEAD4 molecules. Two TEAD4 molecules are colored green and cyan, respectively. The 2Fo-Fc electron density map of VGLL4 in the VGLL4-TEAD4 complex at 2.9-Å resolution was contoured at 1.0 σ. (F) Sequence alignment of TEAD4 interacting domains from human and mouse, including

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VGLL4, VGLL1 and YAP. The conserved TEAD-binding motif shared by YAP and VGLL4 proteins was highlighted in red. (G) Structural comparison of TEAD4 interacting domains from VGLL4, VGLL1 and YAP. The TDU2 of VGLL4 has an extra short helix, while YAP has an additional C-terminal coil region. (H) Structural comparison of TEAD4 and its interacting domains complexes. The individual interfaces were labeled.

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Table S6, related to Figure 3. Data Collection and Refinement Statistics

Data collection Resolution (Å) 50.0 - 2.90 (2.95 - 2.90) Space group P 32 2 1

Unit cell (Å, °) 94.817, 94.817, 135.172 90, 90, 120

Total reflections 85101 Unique reflections 15590 Multiplicity 5.5 (5.5) Completeness (%) 96.54 (97.61) I/σ(I) 15.86 (5.28) Wilson B-factor (Å2) 43.21 R-merge 0.164 (0.942) Refinement R-work 0.186 R-free 0.247 Number of atoms Protein 3665 Ligands 33 Water 7 RMS bonds (Å) 0.009 RMS angles (°) 1.261 Ramachandran Favored (%) 96 Outliers (%) 0 Average B-factor (Å2) 25.70

Statistics for the highest-resolution shell are shown in parentheses.

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Figure S4, related to Figure 5. (A) Evaluation of Super-TDU on cell growth of GC cell lines. Several GC cell lines including BGC-823, HGC-27 and MKN-45, were treated with Super-TDU at different concentrations, and then ATP cell viability assay was performed after treatment with 24h. (B) YAP target genes’ levels after Super-TDU treatment in cell lines. Relative mRNA levels of YAP target genes CTGF, CYR61 and CDX2 in cells treated with Super-TDU were analyzed. (C) MBP-TEAD4 or MBP coupled on amylose resin were mixed with wild-type (WT), mutation MF2A or HFMF4A for 1 hr at 4°, and then washed 3 times. The input and output samples were loaded to SDS-PAGE and detected by Coomassie blue staining. (D) MGC-803 cells were treated with Super-TDU or its mutant peptides, MF2A and HF4A at different doses, and then cell viability were determined by ATP assay. (E) Evaluation of Super-TDU on tumor growth of several GC cell lines. Mice were photographed after sacrificing. Tumors harvested from each mouse photographed before further processing (left). Tumor volume for each group (eight mice) was plotted (right) as indicated. (F) Relative mRNA levels of YAP target genes in sample from tumor formation assay. (G) Tumor weight in MGC-803 nude mice model after injection with different doses of Super-TDU. (H) β2-microglobulin levels in nude mice serum by ELISA assay. (I) Cell growth assay in different types of cancer cell lines, as well as HEK293 cells, after treatment with Super-TDU. (J) Protein levels of YAP and VGLL4 in different cell lines. (K) The absolute transcript copy numbers of YAP (left) and VGLL4 (middle) as well as the ratio of YAP to VGLL4 (right) in several cell lines. Note: Bar graphs represent Means ± S.D. One-way analysis of variance (ANOVA) and Student’s t-test were used. n=3, *vs control-treated group, p<0.05; **, p<0.01; NS, no significance.

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Figure S5, related to Figure 5. (A) Relative mRNA levels of YAP (left) and VGLL4 (middle) as well as the ratio of YAP to VGLL4 (right) in primary gastric cancer (GC) cell lines. (B) Protein levels of YAP and VGLL4 in primary gastric cancer cell lines. (C) GC primary cells were treated with Super-TDU or 5-FU for 48 h, and then ATP cell viability assay was performed and presented as Means ± S.D. * vs control group, p<0.05; ** p<0.01. (D) Cells were treated with different concentrations of 5-FU for 48 h, and then ATP cell viability assay was performed and expressed as Means ± S.D. (T1 and T2 represent 2 primary gastric cancer cells, N1 and N2 represent paired control cells).

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Table S7, related to Figure 5. Biochemical Analysis for Blood in Mice Treated with Super-TDU

Index Saline (10 ml/kg)

Super-TDU (1 mg/Kg)

5-FU (50 mg/Kg)

Total white blood cell (×109/L) 8.5±1.3 8.9±1.9 3.8±1.1* Neutrophils (%) 17.8±1.2 17.5±1.3 17.2±0.9 Lymphocytes (%) 65.5±2.4 68.5±2.0 31.2±1.3* Red blood cell (×1012/L) 7.8±0.7 8.0±0.3 5.2±0.7 Mean corpuscular volume (fL) 36.3±1.6 35.2±0.5 32.6±1.5 Platelet (×109/L) 616.2±68.4 620.3±60.9 41.6±2.6* SGOT (AST)(U/L) 111.8±6.6 138.5±4.5 173.2±7.6* SGPT (ALT) (U/L) 54.5±0.7 61.2±0.9 81.5±1.6*

Note: Student t test was used to test the difference between the two groups; * vs saline group, p<0.05.

Table S8, related to Figure 5. Pharmacokinetic Data of the Super-TDU in Mice by Intravenous Injection

PK parameter 250 μg/kg 500 μg/kg

AUC (ng∙h /mL) 24.1±5.32 46.3±11.2 Cmax (ng/mL) 6.12±0.85 13.3±3.64 CL (ml/min/kg) 7.41±1.22 7.72±0.85 t1/2α (hours) 0.78±0.36 0.82±0.31 t1/2β (hours) 2.71±0.64 2.52±0.37 Vss (ml/kg) 6.55±0.81 6.39±0.78

The correlation coefficient for two-compartment model fit was 0.994 ± 0.005 for mice individual serum concentration versus time curves. (PK, pharmaco-kinetic; AUC, area under the serum concentration versus time curve; Cmax, maximum serum concentration; CL, mean systemic clearance; t1/2α, terminal distribution half-life; t1/2β, terminal elimination half-life; Vss, apparent volume of distribution.) Table S9, related to Figure 5. IC50 of Super-TDU for Different Cell Lines and Primary Gastric Cancer Cells

Cell lines HEK293 MCF-7 Jurkat Raji IC50 (ng/ml) >320.0 99.8±1.4 274.8±2.3 302.0±2.6 Cell lines A549 U2OS MGC-803 HeLa

IC50 (ng/ml) 73.0±1.2 129.1±1.6 57.9±0.8 56.2±0.7 Cell lines HCT116 T1 T2

IC50 (ng/ml) 65.2±0.8 131.6±0.8 240.0±0.6

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Supplemental Experimental Procedures Collection of Human Gastric Cancer Specimens Tissue specimens were collected from 84 patients with gastric cancer who underwent gastrectomy between 2008 and 2012. All cases of gastric cancer and adjacent non-tumor tissues were diagnosed clinically and pathologically. Data on clinicopathological features and prognoses of the patients were collected and analyzed retrospectively. The disease stage of each patient was classified or reclassified according to the 2009 AJCC staging system (Balch et al., 2009). A total of 84 patients were followed up until the end of the year 2012 and twenty-eight of them were lost during the follow up period. Other research specimens included fast frozen tissue for RNA isolation, protein extraction and paraffin-embedded tissue for continued histological observation. Correlation of VGLL4 and YAP Target Genes in GC Sample We investigated at mRNA levels the relationship between VGLL4 and YAP target genes in GC. However, our analysis using all patient samples failed to find any statistically significant correlations due to individual variations of the limited GC samples. To minimize the interference caused by individual variations, patients were divided by their TNM stage, and then clustered into three subgroups according to their VGLL4 mRNA levels. Hierarchical cluster analysis showed that during the early-stage of GC (stage I&II), the mRNA levels of YAP target genes CTGF and CYR61 in high VGLL4 expression group was obviously lower than those in normal and low VGLL4 expression group, whereas this difference was diminished in the late-stage of GC (stage III&IV) (Figures S1D). Real-time PCR Real-time PCR was performed on Applied Biosystems Step Two Real-Time PCR System (Applied Biosystems) using the comparative Ct quantization method. Real-time PCR Master Mix (Toyobo) was used to detect and quantify the expression level of target gene. GAPDH was as internal control. The primers are as follows: hVGLL4: 5'-AACTGCAACCTCTCGCACTG-3’ (F),

5'-GAGTGGGTGTCGCTGTTGAA-3’ (R); hYAP: 5'-GCATGATCTGCCCTAAGGC-3’ (F),

5'-TGACCGCCGAGTACACCAT-3’ (R); hCTGF: 5'-AAAAGTGCATCCGTACTCCCA-3’ (F),

5'-CCGTCGGTACATACTCCACAG-3’ (R); hCYR61: 5'- GGTCAAAGTTACCGGGCAGT-3’ (F),

5'- GGAGGCATCGAATCCCAGC-3’ (R); hCDX2: 5'-GACGTGAGCATGTACCCTAGC-3’ (F),

5'-GCGTAGCCATTCCAGTCCT-3’ (R); hTAZ: 5'- TCCCAGCCAAATCTCGTGATG-3’ (F),

5'- AGCGCATTGGGCATACTCAT-3’ (R); hGAPDH: 5’-GGCATCCTGGGCTACACTGA-3’(F), 5’-GAGTGGGTGTCGCTGTTGAA-3’(R).

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mCTGF: 5'- GGACACCTAAAATCGCCAAGC-3’ (F), 5'-ACTTAGCCCTGTATGTCTTCACA-3’ (R);

mCYR61: 5'- TAAGGTCTGCGCTAAACAACTC-3’ (F), 5'- CAGATCCCTTTCAGAGCGGT-3’ (R);

mCDX2: 5'- TACCCGGACTACGGTGGTTAC-3’ (F), 5'- GTGATGGTGCGCGTGGTAT-3’ (R);

mGAPDH: 5’-AATGGATTTGGACGCATTGGT-3’(F), 5’- TTTGCACTGGTACGTGTTGAT -3’(R).

(F, forward; R, reverse). Analysis of VGLL4 in GC Microarray To strengthen our main conclusion and further investigate the clinical significance of VGLL4 in GC, we performed tissue microarray analysis. We classified as VGLL4 positive all of the tumors that presented more than 5% of cells over the threshold. As shown in Figures 1I and 1J, impaired expression of VGLL4 was present in 12 (13%) of the 91 normal tissues. GC had the highest percentage of impaired VGLL4 expression (45%, 41/91). The difference between normal and GC was statistically significant (p<0.001). Compared to GC, a smaller proportion of cases of dysplasia showed impaired VGLL4 expression (32%, 10/31). The difference between normal and dysplasia was also significant (p<0.01). Impairment of VGLL4 expression in GC was most common in the form of absent staining (32/46); 6 cases did not meet our quantitative criteria for positivity while 4 cases showed exclusively cytoplasmic delocalization. Immunoblotting and Immunoprecipitation Immunoblotting and immunoprecipitation were performed as described previously (Shi et al., 2013). HA, Flag, β-actin and VGLL4 antibodies (WB) were bought from Sigma (St. Louis, MO). VGLL4 antibody used in IHC was purchased from Abcam (Cambridge, England). Antibody against YAP was from Cell Signaling Technology (Boston, MA). Tissue Microarray and Immunohistochemical Staining The expression levels of VGLL4 and YAP were assessed by IHC on tissue microarray containing 91 GC patients. For immunohistochemistry, TMA sections were incubated with anti-VGLL4 antibody (1:25 dilution; Abcam) and anti-YAP antibody (1:25 dilution; Cell Signaling Technology). VGLL4 and YAP staining were scored by two independent pathologists, blinded to the clinical characteristics of the patients. The scoring system was based on the staining intensity and extent. Staining intensity was classified as 0 (negative), 1 (weak), 2 (moderate) and 3 (strong). Staining extent was dependent on the percentage of positive cells (examined in 200 cells) were divided into 0 (< 5%), 1 (5%-25%), 2 (26%-50%), 3 (51%-75%) and 4 (> 75%). According to the staining intensity and the staining extent scores, the IHC result was classified as 0-1, negative (-); 2-4, weakly positive (+); 5-8, moderately positive (++) and 9-12, strongly positive (+++). Cell Culture HEK 293, HEK293T, HeLa, MCF-7, SW480 and HCT116, cells were grown in DMEM

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medium (Invitrogen, Carlsbad, CA), A549, U2OS, Jurkat, Raji, BGC-823, HGC-27, MGC-803, MKN-45 cells and primary gastric cancer cells were cultured in RPMI 1640 medium (Invitrogen, Carlsbad, CA). All the cell lines were maintained in culture supplemented with 10% heat-inactivated fetal calf serum,100 u/mL of penicillin, and 100 µg/mL of streptomycin (FCS) at 37°C with 5% CO2 in a humidified incubator (Thermo, Waltham, MA). Eukaryotic Plasmid Construction VGLL4 was subcloned into the pCDNA3.1-Flag vector. YAP1 and TEAD4 cDNAs were subcloned into the pCDNA3.0-HA vector to allow expression of these cDNAs under the control of a CMV promoter. Two pairs of short hairpin RNA (shRNA) oligos of VGLL4 were designed and synthesized. The complementary oligonucleotides encoded a hairpin structure with a 21-mer stem derived from the coding sequences or the 5-UTR sequence of VGLL4, respectively. For oligo-1 (from the coding sequence of VGLL4), forward oligo: 5' CCGGGAGCCTGGGCAAGAATTACAACTCGAGTTGTAATTCTTGCCCAGGCTCTTTTTG-3'; reverse oligo: 5' AATTCAAAAAGAGCCTGGGCAAGAATTACAACTCGAGTTGTAATTCT TGCCCAGGCTC-3'. For oligo-2 (from the 5’-UTR sequence of VGLL4), forward oligo: 5'-CCGGAGGAGCTACTCAGCAACAATTGACTCGAGTCAATTGTTGCTGAGTAGCTCCTTTTTTG-3'; reverse oligo: 5'- AATTCAAAAAAGGAGCTACTCAGCAACAATTGACTCGAGTC AATTGTTGCTGAGTAGCTCCT -3'. A scramble DNA duplex was also designed as control. For annealing to form DNA duplexes, 0.01 M each of forward and reverse oligos were used. The duplexes were diluted and then ligated with pLKO.1-GFP vector. The products were transformed into DH5α-competent cells. Ampicillin-resistant colonies were chosen, identified by restriction digestion, and further confirmed by DNA sequencing. Transfection and Luciferase Assay Transient transfection of the cells was performed using Lipofectamine 2000 from Invitrogen (San Diego, CA) according to the manufacturer’s instructions. To select stable transfectants, the cells were transfected and incubated overnight, and then switched to medium containing G418 (600 µg/ml) for further incubation. The medium that contained G418 was changed every 2-3 days. After 2 weeks, isolated colonies began to appear. In 3 weeks, a pool of G418-resistant cells was obtained for further studies. Luciferase activities were determined using the dual-Luciferase Assay System (Promega). Relative luciferase activity was calculated as the ratio of luciferase/renilla activity. Apoptosis Analysis Apoptotic cells in early and late stages were detected using an annexin V-FITC Apoptosis Detection Kit from Beyotime (Haimeng, Zhejiang, China). In brief, the cells were transfected with siRNA. At 96 hr post-transfection, culture media and cells were collected and centrifuged. After washing, cells were resuspended in 490 μl annexin V binding buffer, followed by the addition of 5 μl annexin V-FITC and 5 μl propidium iodide. The samples were incubated in the dark for 5 min at room temperature and analyzed using flow cytometry (BD Bioscience). Soft Agar Colony Formation Assay

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Cells were transfected with indicated plasmids. A total of 104 cells were seeded on soft agar in 6-well plates, and colonies with a diameter of >0.05 mm were counted 14 days after seeding. Cloning, Protein expression and Purification Mouse TEAD4 YAP-binding domain (residues 210-427) and the tandem TDU domains of VGLL4 (residues 203-256) were cloned into vector HT-pET-28a and expressed in E.coli BL21 (DE3) cells. TEAD4 and VGLL4 were purified by Ni affinity chromatography and size exclusive chromatography (Superdex 75, GE healthcare). Purified proteins of VGLL4 and TEAD4 were mixed with 1:2 molar ratios and applied to Superdex 75 again. MBP, SUMO and GST-fused proteins were purified by affinity chromatography and size exclusive chromatography. Dynamic Light Scattering Proteins of VGLL4 and TEAD4 were mixed with 1:2 molar ratios at 4 °C for 30 minutes, and then concentrated to 2 mg/ml. The weighted distribution and homogeneity of the three samples were determined at 25°C by dynamic light scattering. Scattered light was measured at a 90° angle using a 658 nm wavelength. Crystallization, Structure Determination, and Refinement The protein of VGLL4-TEAD4 complex was concentrated to 7.2 mg/ml and then performed crystal screening. Crystals were grown in reservoir solution consisted of 0.2 M L-proline, 0.1 M HEPES pH 7.5, 24% w/v polyethylene glycol 1500. Diffraction data were collected at beamline BL17U, Shanghai Synchrotron Radiation Facility (SSRF) of China, and processed using HKL2000 (Otwinowski and Minor, 1997). The structure of VGLL4-TEAD4 complex was solved by molecular replacement with program Phaser-MR in the Phenix using TEAD4 (PDB code 3JUA) as a search model (Adams et al., 2010; Emsley et al., 2010). A solution with two TEAD4 molecules in the asymmetric unit was found. The structure was refined using phenix.refine and model building was performed in Coot (Emsley et al., 2010; McCoy et al., 2007). Structural Comparison of VGLLs/YAP-TEAD Complex The structures of the TEAD4 molecules are essentially the same with those previously reported. The values of the root-mean-square deviation are 0.399 Å and 0.409 Å for 161 Cα of molecule A and 153 Cα of molecule B, respectively, when overlapped with the structure of TEAD4 previously determined in a complex with YAP (Chen et al., 2010). The antiparallel β sheet sandwiching VGLL4 is mediated by the main chains of residues 227-231 from the β1 strand of TDU2 and residues 334-338 from the β7 strands of TEAD4 molecules A and B (Figures 3 and S3C-E).

Both VGLL4 and YAP have the TEAD-interacting domain, named TDU and TBD, respectively. The structures of the individual TDUs of VGLL4 resemble those of VGLL1 TDU and YAP TBD determined in complex with TEAD4 (Chen et al., 2010; Li et al., 2010; Pobbati et al., 2012) (Figure S3G). All these TEAD-interacting domains possess a V/LxxH/LF motif, forming a short helix (Figure S3F). Compared with other TEAD-interacting domains, VGLL4

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TDU2 has an additional short helix following this motif, strengthening its interaction with TEAD (Figure S3H).

Previous studies have identified three interfaces (interfaces 1-3) for the YAP-TEAD complex, but only two interfaces (corresponding to interfaces 1 and 2) for the VGLL1-TEAD complex (Figure S3H) (Chen et al., 2010; Li et al., 2010; Pobbati et al., 2012). The VGLL4-TEAD complex has one interface (corresponding to interface 2) for TDU1-mediated subcomplex and two interfaces (corresponding to interfaces 1 and 2) for TDU2-mediated subcomplex (Figure S3H). Notably, the interface 3, which is missing in all TDUs of VGLL1-4, is critical for the interaction between YAP and TEAD (Chen et al., 2010; Li et al., 2010). VGLL4 has a histidine in the VxxHF motif instead of leucine in the LxxLF motif of YAP. The histidine forms two hydrogen bonds with TEAD, providing additional stabilization for interface 2. Our current data revealed that the second interfaces (interface 2) of both TDU1 and TDU2 play the most important role for TEAD binding (Figure 3). Consistent with this, interface 2 is also crucial for the VGLL1-TEAD complex as previously determined (Pobbati et al., 2012). Taken together, these observations suggest that VGLL4 and YAP have partially overlapped but different essential binding sites on TEADs, thus probably acting as an inhibitor of each other via occluding the binding site on TEADs. Design of the Super-TDU We first fused different fragments of TEADs binding regions from VGLL4 and YAP with polyGGS linker. Several factors including the fragment length, linker length and expression order were then tested. As a result, the interface 2 of VGLL4 TDU2 and the interface 3 of YAP were combined to obtain a peptide containing the sequence of “SVDDHFAKSLGDTWLQIGGSGNPKTANVPQTVPMRLRKLPDSFFKPPE”. Note that the alanine (Lys-Ala-Leu-Gly) was mutated to serine (Lys-Ser-Leu-Gly) for better solubility.

In order to facilitate the uptake of the Super-TDU by cells or mice, we also tried to add a membrane translocating sequence (“GSDAATATRGRSAASRPTERPRAPARSASRPRRPVE”) and a nuclear localization sequence (“MTYPRRRFRRRRHRPRSHLGQILRRRPWLVHPRHRYRWRRKNGIFNT”) to the N-terminus and C-terminus of the Super-TDU, respectively (Chen et al., 2011), which indeed slightly improved the delivery of current version of the Super-TDU. Production of the Super-TDU The Super-TDU was expressed and purified in E.coli. Briefly, the DNA sequence encoding the Super-TDU peptides was cloned into pET-28a vector with an N-terminal 6 × His tag. The recombinant plasmids were transformed into E. coli BL21(DE3) codon plus competent cells for expression. Cells were grown in Terrific Broth medium to A600 = 1.0 at 37 °C, and then 0.5 mM isopropyl β-D-1-thiogalactopyranoside was added to induce protein expression at 18 °C. After 15 h, cells were harvested by centrifugation using 5,000 g for 15 min at 4 °C. Cells were resuspended in lysis buffer containing 20 mM Hepes pH 7.5, 500 mM NaCl, 5% glycerol, 20 mM imidazole, 1 mM DTT and 1 mM PMSF and then lysed by a High Pressure Homogenizer. The debris was removed by centrifugation for 40 min at 20,000g at 4 °C. The supernatant was mixed with Ni Sepharose (GE healthcare) for 1 h, and then the beads were washed with lysis buffer without PMSF. The proteins were eluted with 300 mM imidazole in lysis buffer.

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The proteins were concentrated using a 3 kDa cutoff Amicon Ultra-15 (Millipore) and then applied to a HiLoad 16/60 Superdex 75 column (GE Healthcare). Pulldown Assay MBP or GST-fused proteins coupled on amylase or glutathione resin were mixed with different prey proteins at 4 °C for 1 hr in 20 mM HEPES, 200 mM NaCl, 1 mM DTT, pH 7.5, and then washed three times. The proteins bound on the resin were eluted by the same buffer with addition of 20 mM maltose or reduced glutathione. The input and output samples were loaded to SDS-PAGE and detected by Coomassie blue staining or western blot. Biolayer Interferometry Analysis Interaction analysis was performed using an Octet Red 96 instrument (ForteBio) (Shi et al., 2013). Wild-type TEAD4 was labeled by biotin in 20 mM Hepes pH 7.5, 100 mM NaCl, 1 mM DTT, and biotinylated proteins were immobilized on streptavidin (SA) biosensors and incubated with VGLL4 WT and mutants proteins in 1 × kinetics buffer. Data were analyzed using Octet Data Analysis Software 7.0 (ForteBio). ChIP Assay MGC-803 cells were cultured with or without Super-TDU for 12 h, then chromatin-immunoprecipitation (ChIP) by YAP antibody or IgG control was amplified by reverse-transcript PCR and analyzed by gel shift assay. Xenograft Tumor Formation Assay During the tumor formation assay of BALB/c-nu/nu, cancer cell lines labeled with GFP or firefly luciferase were injected at the flank of the mice. Serum β2-microglobulin levels were detected in duplicate with the enzyme-linked immunosorbent assay (ELISA) (Sigma).

At the moment of establishing the mouse xenotransplant models, we found that the levels of human β2 microglobulin in mouse serum were closely correlated with tumor mass (r = 0.761). The levels of human β2 microglobulin were significantly reduced by the Super-TDU peptide in the xenograft experiments compared with control (Figure S4I). Pharmacological Evaluation of the Super-TDU The acute toxicity of the Super-TDU was determined in mice by intravenous injection according to National Cancer Institution (NCI) Toxicity Criteria version 2.0 1999. ICR mice were administered with peptides at dose levels of 125, 250, 500 and 1000 μg/kg, with a dose volume of 10 ml/kg. The control mice were administered with 10 ml/kg of control peptide. Observations included mortality, clinical signs, total body-weight gains, food consumption, and gross necropsy findings.

To evaluate the efficacy of the Super-TDU peptide in different cellular contexts, we tested the cytotoxic effects of the Super-TDU on a range of tumor cells (A549, MGC-803, MCF-7, HeLa, HCT116, SW480, Jurkat, Raji, and U2OS), normal cells (HEK293) and primary stomach cells (N1, T1, N2, T2) (Figures 6A and S4I). The cytotoxicity (IC50) of the Super-TDU against the selected cell lines are summarized in Table S9. The Super-TDU peptide had a moderate cytotoxic effect on primary gastric cancer cells T1 (IC50 of 131.6

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ng/ml) and a relatively lower cytotoxic effect on T2 (IC50 of 240.0 ng/ml), but was non-toxic to normal cell lines (HEK293) and normal primary stomach cells (N1, N2).

For pharmacokinetic studies of the Super-TDU with samples collected at different time points in ICR mice (n = 10), the maximum concentration (Cmax) of the Super-TDU at the two doses used was 6.12 ± 0.85 and 13.3 ± 3.64 ng/ml, respectively. The volume of distribution was 6.55 ± 0.81 and 6.39 ± 0.78 ml/kg, respectively. The Super-TDU concentration data of individual serum were well fit by a two-compartment model with zero-order input. Pharmacokinetic parameter estimates for ICR mice treated with the Super-TDU at both dose levels were listed in Table S8. Cytotoxicity The cells were seeded in a 96-well plate at 2×104 cells/well (n = 3) and treated with the Super-TDU peptide for 24 hours. Representative concentrations of the Super-TDU were set at 10, 20, 40, 80, 160, 320 ng/ml. HEPES buffer, a solvent control, was used at 0.5% (v/v) in the culture medium. ATP cell viability assay was then performed and IC50 was determined by fitting a 4-parameter curve. Toxicity Analysis in mice ICR mice and C57BL/6 were fasted for 16 to 18 hr before peptides were administered; water was given ad libitum. Mice (6~8 weeks old) received an intravenous bolus dose (125, 250, 500, and 1000 μg/kg body weight) of peptide, and the control group received saline (10 ml/kg) injection. The mice were continuously observed for 14 days after the administration and sacrificed on day 14 for routine blood test, examination of the blood biochemistry and pathological examination. Pharmacokinetic Assay Blood samples (n = 6) were obtained at 0.05, 0.1, 0.15, 0.2, 0.25, 0.5, 1, 1.5, 2, 3, and 6 hr after peptide administration by intravenous injection. Plasma samples were separated by centrifugation at 3,000 g for 10 min at 4°C. Pharmacokinetic evaluation of the area under the time-plasma concentration curve (AUC, measured in ng∙h/mL), and the residence rate constant at steady state (t , measured in h), plasma clearance (CL, measured in ml/min/kg), distribution volume (Vss, measured in ml/kg) were performed by the NONLIN program.

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