WWC3 downregulation correlates with poor prognosis and inhibition of Hippo signaling in human gastric cancer

Figures & data

Table 1 Primer sequences

Genes	Sequence 5′ to 3′
WWC3 forward	CACGCCCAACACTTACTGCT
WWC3 reverse	CGTTAAACACCAGCGGCACT
YAP forward	CGCTCTTCAACGCCGTCA
YAP reverse	AGTACTGGCCTGTCGGGAGT
Cyclin D1 forward	TGGAGGTCTGCGAGGAACA
Cyclin D1 reverse	TTCATCTTAGAGGCCACGAACAT
Cyclin E forward	AGCCAGCCTTGGGACAATAAT
Cyclin E reverse	GAGCCTCTGGATGGTGCAAT
p27 forward	CTGCAACCGACGATTCTTCTACT
p27 reverse	CTTCTGAGGCCAGGCTTCTT
CTGF forward	GTTACCAATGACAACGCCTCCT
CTGF reverse	TGCACTTTTTGCCCTTCTTAATGT
Actin forward	TCCACGAAACTACCTTCAACTCC
Actin reverse	TCTGCATCCTGTCGGCAAT

Figure 1 Expression pattern of WWC3 protein in gastric cancer tissues.

Notes: (A) Positive cytoplasmic WWC3 staining in normal gastric tissues. (B) Positive WWC3 overexpression in a case of adenocarcinoma. (C) Weak WWC3 expression in a case of tubular adenocarcinoma. (D) Negative WWC3 expression in gastric cancer (magnification, 400×). (E) One section containing both cancer tissue and its corresponding normal adjacent tissue was used. Lower expression of WWC3 could be observed in cancer tissue (magnification, 100×). (F) Kaplan–Meier analysis showed that patients with low WWC3 had worse overall survival than patients with high WWC3 protein.

Table 2 Distribution of WWC3 status in gastric cancer according to clinicopathological characteristics

Characteristics	Number of patients	WWC3 low expression	WWC3 high expression	P-value
Age (years)				0.8437
<60	52	23	29
≥60	59	25	34
Gender				0.8848
Male	84	36	48
Female	27	12	15
Differentiation				0.6412
Poor	55	25	30
Well-moderate	56	23	33
Tumor invasion (T)				0.0864
T1+T2	40	13	27
T3+T4	71	35	36
Lymph node metastasis				0.0076
Absent	41	11	30
Present	70	37	33
TNM stage				0.0005
I+II	51	13	38
III	60	35	25
Relapse				0.0109
Negative	40	10	30
Positive	55	28	27

Abbreviation: TNM, tumor, node, metastases.

Table 3 Univariate and multivariate analyses for predictive factors in patients with gastric carcinoma (Cox regression model)

Factors	Univariate		Multivariate
	Hazard ratio (95% CI)	P-value	Hazard ratio (95% CI)	P-value
Age	1.250 (0.729–2.144)	0.4177	1.181 (0.676–2.064)	0.5589
Gender	0.911 (0.488–1.700)	0.7685	0.789 (0.410–1.516)	0.4761
T stage	1.606 (1.180–2.185)	0.0026	1.431 (1.024–1.999)	0.0359
Nodal status	1.631 (1.297–2.052)	<0.001	1.410 (1.073–1.854)	0.0137
WWC3	0.711 (0.543–0.931)	0.0133	1.107 (0.821–1.494)	0.5042
Differentiation	0.750 (0.439–1.279)	0.2906	0.679 (0.383–1.205)	0.1861
Recurrence	25.01 (9.413–66.45)	<0.001	28.913 (9.677–86.384)	<0.001

Abbreviation: CI, confidence interval.

Figure 2 WWC3 inhibits gastric cancer cell proliferation.

Notes: (A) Protein and mRNA expression of WWC3 and YAP in gastric cancer cell lines was lower than that in normal GES-1 cell line. HGC-27 cell line has relatively low expression and SGC-7901 cell line has relatively high expression. (B) Western blot and PCR analysis showed that plasmid transfection significantly increased WWC3 level in HGC-27 cells and siRNA decreased WWC3 expression in SGC-7901 cells. (C) MTT assay showed that WWC3 overexpression inhibited the proliferation rate in HGC-27 cell line. WWC3 knockdown promoted proliferation rate in SGC-7901 cell line. (D) Colony formation assay showed that WWC3 knockdown decreased colony numbers while its overexpression increased colony formation ability. *P<0.05.
Abbreviations: siRNA, small interfering RNA; PCR, polymerase chain reaction.

Figure 3 WWC3 regulates gastric cancer cell invasion and cell cycle progression.

Notes: (A) Matrigel invasion assays showed that WWC3 overexpression inhibited HGC-27 cell invasion while WWC3 knockdown promoted SGC-7901 cell invasion. (B) WWC3 overexpression increased the percentage of G1 cells and decreased the percentage of S phase cells. Conversely, WWC3 siRNA treatment decreased G1 phase cells and increased S phase cells compared with control. *P<0.05.
Abbreviation: siRNA, small interfering RNA.

Figure 4 WWC3 regulates cell cycle-related proteins and Hippo signaling pathway.

Notes: (A) WWC3 overexpression downregulated cyclin D1, cyclin E, CTGF, total YAP, and nuclear YAP and upregulated p27 level in HGC-27 cells. Knockdown of WWC3 upregulated the protein levels of cyclin D1, cyclin E, CTGF, total YAP, and nuclear YAP and downregulated p27 expression in SGC-7901 cells. The change of p21 and p53 was not significant. (B) Real-time PCR analysis showed that WWC3 overexpression downregulated cyclin D1, cyclin E, and CTGF and upregulated p27 mRNA in HGC-27 cells. Knockdown of WWC3 upregulated the protein levels of cyclin D1, cyclin E, and CTGF mRNA and downregulated p27 mRNA in SGC-7901 cells. (C) Luciferase reporter assay showed that WWC3 overexpression downregulated while its depletion upregulated 8xGTIIC luciferase activity, which indicates the YAP/TEAD transcription activity. *P<0.05.
Abbreviation: PCR, polymerase chain reaction.

Figure S1 Validation of antibody and transfection efficiency.

Notes: (A) The specificity of the antibody was validated by using gastric cancer tissue, corresponding normal tissue, and cell lines with WWC3 overexpression and siRNA depletion. Western blot was used to examine WWC3 with this antibody. The Western blot bands of cells were strong when WWC3 was overexpressed and were weak when depleted. Gastric cancer tissues and normal tissues showed similar single bands at the same molecular weight. (B) Expression of WWC1 and WWC2 proteins in gastric cancer cell lines and GES-1 cell line. (C) The knockdown and overexpression efficiency were checked at different time points (at 2, 4, and 8 days after transfection) using Western blot. The transfection of plasmid significant upregulated WWC3 protein at day 8. The knockdown efficiency was most significant at day 2 and could still be observed at day 8.

Abbreviation: siRNA, small interfering RNA.