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. 2006 Nov;97(11):1211-6.
doi: 10.1111/j.1349-7006.2006.00313.x. Epub 2006 Sep 5.

EphA4 receptor, overexpressed in pancreatic ductal adenocarcinoma, promotes cancer cell growth

Megumi Iiizumi  1 , Masayo Hosokawa, Akio Takehara, Suyoun Chung, Toru Nakamura, Toyomasa Katagiri, Hidetoshi Eguchi, Hiroaki Ohigashi, Osamu Ishikawa, Yusuke Nakamura, Hidewaki Nakagawa
Affiliations

EphA4 receptor, overexpressed in pancreatic ductal adenocarcinoma, promotes cancer cell growth

Megumi Iiizumi et al. Cancer Sci. 2006 Nov.

Abstract

To isolate novel diagnostic markers and drug targets for pancreatic ductal adenocarcinoma (PDAC), we previously performed expression profile analysis of PDAC cells using a genome-wide cDNA microarray combined with laser microdissection. Among dozens of up-regulated genes identified in PDAC cells, we herein focused on one tyrosine kinase receptor, Eph receptor A4 (EphA4), as a molecular target for PDAC therapy. Immunohistochemical analysis validated EphA4 overexpression in approximately half of the PDAC tissues. To investigate its biological function in PDAC cells, we knocked down EphA4 expression by siRNA, which drastically attenuated PDAC cell viability. In concordance with the siRNA experiment, PDAC-derivative cells that were designed to constitutively express exogenous EphA4 showed a more rapid growth rate than cells transfected with mock vector, suggesting a growth-promoting effect of EphA4 on PDAC cells. Furthermore, the expression analysis for ephrin ligand family members indicated the coexistence of ephrinA3 ligand in PDAC cells with EphA4 receptor, and knockdown of ephrinA3 by siRNA also attenuated PDAC cell viability. These results suggest that the EphA4-ephrinA3 pathway is likely to be a promising molecular target for pancreatic cancer therapy.

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Figures

Figure 1
Figure 1
Overexpression of EphA4 in pancreatic ductal adenocarcinoma (PDAC) cells. (A) Semi‐quantitative RT‐PCR validated that EphA4 expression was up‐regulated in the microdissected PDAC cells compared with normal pancreatic duct cell (ND) that were also microdissected and several vital organs (lung, heart, liver, kidney and bone marrow). Expression of ACTB served as the quantitative control. (B) Northern blot analysis showed the strong expression of EphA4 in PDAC cell lines, MIA‐PaCa2 and PK‐59, and the normal brain, while no expression was observed in vital organs including heart, lung, liver, and kidney. (C) Immunohistochemical study using anti‐EphA4 antibody. Intense staining was observed in PDAC cells in two representative specimens (PDAC1 and PDAC2), while acinar cells and normal ductal epithelium in normal pancreatic tissue showed no staining (original magnification × 200).
Figure 2
Figure 2
Knockdown effect on EphA4 by siRNA attenuated pancreatic ductal adenocarcinoma (PDAC) cell viability. Three EphA4 siRNA expression vectors (EphA4‐198si, ‐486si, and ‐1313si) and an EGFP siRNA expression vector (EGFPsi) as a negative control were transfected into (A, B, C) MIA‐PaCa2 and (D, E, F) PK‐59 cells. (A and D) Knockdown effect on EphA4 was validated by Western blot analysis, with ACTB level as a quantitative control. EphA4‐1313si revealed a strong knockdown effect, while EphA4‐198si, EphA4‐486si and EGFPsi did not show any effect on the level of EphA4. Transfection with EphA4‐1313si vector resulted in drastic reduction of the numbers of viable cells measured by (B and E) MTT assay and (C and F) the number of colonies formed, compared with the cells transfected with other siRNA expression vectors that did not showed their knockdown effect on EphA4. ABS, absorbance at 490 nm (630 nm reference), measured with a microplate reader.
Figure 3
Figure 3
Expression of exogenous EphA4 promoted pancreatic ductal adenocarcinoma (PDAC) cell growth. (A) Western blot analysis of three Panc‐1 derivative cells (Panc1‐EphA4 #9, #13, and #16) expressing exogenous EphA4 constitutively and those transfected with mock vector (Panc1‐Mock #6 and #25). Exogenous introduction of EphA4 expression was validated with anti‐FLAG tag antibody. ACTB served as a loading control. (B) The growth measurement by MTT assay demonstrates that the three Panc1‐EphA4 clones (#9, #13 and #16, solid lines) grew significantly more rapidly than the two Panc1‐mock clones (#6, and #25, dashed lines) (P = 0.0358). X‐axis represents day point after seeding and Y‐axis represents relative growth rate that was calculated in absorbance of the diameter by comparison with the absorbance value of day 1 as a control. Each average is plotted with bars representing standard error. These experiments were performed in triplicate.
Figure 4
Figure 4
Expression pattern of ephrin ligands in pancreatic ductal adenocarcinoma (PDAC) cells. Semi‐quantitative RT‐PCR in (A) clinical PDAC cells and (B) PDAC cell lines demonstrated that, among ephrin ligands A1‐5 and B1‐3, ephrinA3 (EFNA3) expression pattern was relatively correlated with that of EphA4. ACTB served as a quantitative control. ND, normal pancreatic ductal cells; TP, total pancreas. (C) Immunohistochemical staining in PDAC tissue microarray demonstrated that ephrinA3 (EFNA3) expression (upper panel) was correlated with EphA4 expression (lower panel). Three representative specimens with both EFNA3 and EphA4 positive (Cases 1 and 2) and with both negative (Case 3) are shown (original magnification × 200).
Figure 5
Figure 5
Knockdown effect on ephrinA3 (EFNA3) by siRNA attenuated pancreatic ductal adenocarcinoma (PDAC) cell viability. Three EFNA3 siRNA expression vectors (EFNA3‐539si, ‐689si, and ‐1584si) and an EGFP siRNA expression vector (EGFPsi) as a negative control were transfected into MIA‐PaCa2 cells. (A) Knockdown effect on EFNA3 was validated by Western blot analysis with ACTB level as a quantitative control. EFNA3‐689si and EFNA3‐539si revealed a strong knockdown effect on EFNA3 expression, while EFNA3‐1584si and EGFPsi did not show any effect on EFNA3 expression level. Transfection with EFNA3‐689si and EFNA3‐539si resulted in a drastic reduction of (B) the numbers of viable cells measured by MTT assay and (C) the number of colonies formed, compared with the cells transfected with siRNA expression vectors that did not show a knockdown effect on EFNA3. ABS, absorbance at 490 nm (630 nm reference), measured with a microplate reader.
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