Ezrin promotes pancreatic cancer cell proliferation and invasion through activating the Akt/mTOR pathway and inducing YAP translocation

Figures & data

Figure 1 Ezrin induces pancreatic cancer cell proliferation.

Notes: (A) Endogenous expression of Ezrin and YAP was detected in different pancreatic cancer cell lines by Western blot. (B) Efficiency of Ezrin knockdown in Capan-1 cells by siRNA was verified by Western blot. (C) Efficiency of Ezrin overexpression in BxPC-3 cells by plasmid transfection was verified by Western blot. (D) Microscopic images of the cells 48 hours post-transfection. (E–G) Influence of Ezrin knockdown or overexpression on viability of Capan-1 and BxPC-3 cells as measured by colony formation, MTT assay, and Edu staining. **P<0.01.

Abbreviation: Con, control.

Figure 2 Ezrin promotes pancreatic cancer cell invasion and migration.

Notes: (A) A scratch wound-healing assay was used to determine the effects of Ezrin knockdown or overexpression on Capan-1 and BxPC-3 cell motilities. (B) For spheroid invasion assay, Capan-1 and BxPC-3 cells were embedded in Matrigel. Representative pictures of spheroids taken after 3 days and 7 days (40× magnification) were shown. Relative invasion was quantified as spheroid area difference. (C) Influence of Ezrin knockdown or overexpression on migration and invasion of Capan-1 and BxPC-3 cells was determined by transwell assay. (D) The expression of EMT-related markers, E-cadherin, ZO-1, Vimentin, snail, slug, MMP-2, and MMP-9, in Ezrin knockdown or overexpression cells was detected by Western blot. β-Actin was set as the loading control. (E) The expression of E-cadherin, Vimentin, ZO-1, and MMP-2 in Ezrin knockdown or overexpression cells was detected by immunofluorescence. Blue: DAPI; green: E-cadherin, Vimentin, and ZO-1; red: MMP-2.

Abbreviations: Con, control; EMT, epithelial–mesenchymal transition.

Figure 3 Ezrin promotes angiogenesis of pancreatic cancer.

Notes: (A) The formation of new blood vessels in CAM was quantified. (B) The expression of VEGF-A in Ezrin knockdown or overexpression cells was detected by Western blot. β-actin was set as the loading control. (C) The expression status of VEGF-A in Ezrin knockdown or overexpression cells was detected by immunofluorescence. Blue: DAPI; red: VEGF. **P<0.01.

Abbreviations: CAM, chick chorioallantoic membrane; Con, control; VEGF-A, vascular endothelial growth factor A.

Figure 4 Ezrin promotes proliferation and invasion of pancreatic cancer cells through activating Akt/mTOR pathway.

Notes: (A) The expression of Akt, p-Akt, mTOR, and p-mTOR was detected in Ezrin knockdown and overexpression cells by Western blot. β-Actin was set as the loading control. (B) The expression of Akt, mTOR, E-cadherin, and Vimentin was detected in Ezrin-overexpressed BxPC-3 cells after treatment with rapamycin or LY294002 by Western blot. β-Actin was set as the loading control. (C) Colony formation capacity was detected in Ezrin-overexpressed BxPC-3 cells after treatment with rapamycin or LY294002. (D) Invasion capacity was detected in Ezrin-overexpressed BxPC-3 cells after treatment with rapamycin or LY294002. *P<0.05, **P<0.01.

Figure 5 Ezrin regulats YAP translocation through activating PI3K/Akt pathway.

Notes: (A) The expression status of YAP and p-YAP was detected in Ezrin knockdown and overexpression cells by Western blot. (B) The cytosol and nucleus expression level of YAP and p-YAP was detected in Ezrin knockdown and overexpression cells by Western blot. (C) The cytosol and nucleus expression level of YAP and p-YAP was detected in Ezrin-overexpressed BxPC-3 cells after treatment with Rapamycin or LY294002. β-actin was set as the loading control. *P<0.05, **P<0.01.

Abbreviation: Con, control.

Table 1 Expression of Ezrin and YAP in pancreatic cancer and adjacent normal tissues

Diagnosis	No. of cases	Ezrin expression		YAP expression
		Positive rate (%)	Strongly positive rate (%)	Positive rate (%)	Strongly positive rate (%)
Pancreatic cancer	108	86.1** (93/108)	65.7** (71/108)	85.2** (92/108)	62.0** (67/108)
Adjacent normal tissues	79	35.4 (28/79)	3.8 (3/79)	31.6 (25/79)	2.5 (2/79)

Note: **P<0.01, compared with normal tissue.

Figure 6 Ezrin and YAP are significantly overexpressed in pancreatic cancer tissues, and related to poor prognosis.

Notes: (A) mRNA expression levels of Ezrin and YAP in pancreatic cancers and normal pancreas were obtained from the Oncomine database. (B) Survival analysis of Ezrin and YAP in pancreatic cancer patients obtained from the UALCAN dataset. (C, D) Relationships between Ezrin, YAP, and the clinicopathologically significant aspects of pancreatic cancer. (E) Images of Ezrin and YAP immunohistochemical staining in pancreatic cancer and adjacent normal pancreas tissues. (F) Kaplan–Meier analysis of Ezrin and YAP in pancreatic cancer patients.

Abbreviation: LN, lymph-node.

Table 2 Survival analyses (Cox regression model) of various factors in patients with pancreatic cancer

Factor	B	SE	Wald	HR	95% CI		P
					Lower	Upper
Univariate
Gender	0.081	0.250	0.105	1.084	0.664	1.770	0.746
Age	0.107	0.250	0.185	0.898	0.550	1.466	0.667
Location	−0.131	0.247	0.279	0.878	0.540	1.425	0.598
Tumor size	0.087	0.299	0.085	1.091	0.607	1.962	0.770
Histological grade	−0.930	0.258	12.951	0. 395	0.238	0.655	0.0003*
LN metastasis	0.659	0.244	7.286	0.517	0.321	0.835	0.007*
Clinical stage	−0.614	0.255	5.800	0.541	0.328	0.892	0.016*
N/V invasion	−0.328	0.243	1.834	0.720	0.448	1.158	0.176
Ezrin	−1.644	0.322	26.075	0.193	0.103	0.363	0.000*
YAP	−1.451	0.290	25.042	0.234	0.133	0.414	0.000*
Multivariate
YAP	−0.568	0.376	2.286	0.566	0.271	1.183	0.131
Ezrin	−1.093	0.436	6.297	0.335	0.143	0.787	0.012*
LN metastasis	0.089	0.414	0.047	0.915	0.407	2.057	0.829
Histological grade	−0.706	−0.302	5.465	0.494	0.273	0.892	0.019*
Clinical stage	−0.194	0.392	0.245	0.824	0.382	1.777	0.621

Note: * P<0.05.

Abbreviations: LN, lymph-node; N/V, nerve/vessel.

Figure 7 Expression of Ezrin and expression of YAP are statistically related in pancreatic cancer tissues.

Note: (A) Correlation between mRNA expression of Ezrin and YAP in 178 samples of pancreatic cancer patients (obtained from the Starbase dataset). (B) Microscopic images of immunohistochemical staining of Ezrin and YAP in well-differentiated and poorly differentiated pancreatic cancer tissues.

Table 3 Relationships between Ezrin and YAP expression in pancreatic cancer tissues

Ezrin expression	YAP expression				Total	r	P
	-	+	++	+++
-	14	1	0	0	15
+	1	17	4	0	22
++	1	7	31	8	47	0.821	0.000
+++	0	0	7	17	24
Total	16	25	42	25	108

Figure S1 Knockdown of Ezrin inhibits cell proliferation, invasion, and migration of Panc-1 cells. (A) Efficiency of Ezrin knockdown in Panc-1 cells by siRNA was verified by Western blot. (B) Effect of Ezrin knockdown on viability of Panc-1 cells was measured by MTT assay. (C) Influence of Ezrin knockdown on migration and invasion of Panc-1 cells was determined by transwell assay.

Abbreviation: Con, control.

Figure S2 Scheme of proposed molecular mechanism of Ezrin-induced pancreatic cancer cell proliferation, invasion, and EMT progression.

Abbreviation: EMT, epithelial–mesenchymal transition.