ER-α36-mediated gastric cancer cell proliferation via the c-Src pathway

Abstract

Previously, a novel variant of estrogen receptor (ER)-α, ER-α36, was identified and cloned and reported to mainly mediate non-genomic estrogen signaling. More recently, we identified that ER-α36 is important for the invasion and lymph node metastasis of human gastric cancer. In the present study, the c-Src signaling pathway was demonstrated to be involved in the non-genomic estrogen signaling mediated by ER-α36 in SGC7901 gastric cancer cells. SGC7901 cells were subjected to the siRNA-mediated knockdown of ER-α36 (PLKO.1-PURO-SP6-ER-α36-L) or transfected with an ER-α36 upregulated expression plasmid (PLJM1-ER-α36-H) and treated with 17β-estradiol (E2β) and PP2, a c-Src protein inhibitor. The expression of ER-α36 and c-src/p-c-Src and cyclin D1 was examined by western blot analysis, and tumor cell growth was analyzed by cell proliferation and nude mouse xenograft assays. The ER variant, ER-α36, was shown to enhance gastric cancer cell proliferation through activation of the membrane-initiated c-Src signaling pathways, indicating that ER-α36 is important for the regulation of proliferation in gastric cancer. In addition, ER-α36 was shown to directly interact with c-Src by immunoprecipitation. The results of the present study indicate that the use of ER-α36 may be a targeted therapeutic approach in gastric cancer.

Keywords: ER-α36; c-src; cyclin D1; gastric cancer; proliferation.

Figure 1.

PP2 inhibits the activation of c-Src induced by E2β. (A) PP2 blocks c-Src activation in SGC7901, High36 and Low36 cell lines from days 5–11 and PP2 reduced proliferation in ∼68.91 and 91.56% of High36 and Low36 cells, respectively, at day 11. (B) E2β and/or PP2-stimulated SGC7901, High36 and Low36 cells at 0, 5 and 20 min. Cell lysates were analyzed with anti-p416-c-Src and anti-p527-c-Src antibodies. Anti-c-Src antibody was used to ensure equal loading. Western blot analysis of p-416-c-Src and p-527-c-Src expression in SGC7901, High36 and Low36 cells. E2β, 17β-estradiol.

Figure 2.

Estrogen induces cyclin D1 expression through activation of the ER-α36 pathway. (A) Western blot analysis of cyclin D1 expression in SGC7901, High36 and Low36 cell lines. Cells were treated with E2β alone. (B) Western blot analysis of cyclin D1 expression in SGC7901, High36 and Low36 cells. Cells were treated with the c-Src inhibitor, PP2 and E2β. (C) Western blot analysis of cyclin D1 expression in SGC7901, High36 and Low36 cells. Cells were treated with the PP2 c-Src inhibitor alone. (D) Estrogen induces cyclin D1 expression through activation of the ER-α36 pathway and the PP2 c-Src inhibitor downregulates cyclin D1 expression in gastric cancer cells (^*P<0.05, vs. E2β alone). ER, estrogen receptor; E2β, 17β-estradiol.

Figure 3.

ER-α36 promotes malignant growth of gastric cancer cells in nude mice. (A) The size of the xenografted tumors was examined every 4 days for 24 days. (B) Tumor weight of xenografts at day 24 (^*P<0.05, vs. SGC7901 control). (C) Western blot analysis of cyclin D1 and ER-α36 expression in nude mice. (D) Western blot analysis of cyclin D1 expression in nude mice (^*P<0.05, vs. SGC7901 control). (E) Immunohistochemistry (IHC) staining of ER-α36, c-Src and cyclin D1 in the xenografted tumors. ER-α36 and c-Src were observed at the plasma membrane. Cyclin D1 was observed in the nucleus (magnification, ×400). ER, estrogen receptor.

Figure 4.

ER-α36-c-Src interaction analysis. Extracts from SGC7901 cells were incubated with E2β and/or with PP2. Formed complexes were pulled down and analyzed using antibodies against (A) c-Src and ER-α36, (B) p416-c-Src and ER-α36 and (C) p527-c-Src and ER-α36. ER, estrogen receptor; E2β, 17β-estradiol.