Department of Surgery, Cancer Biology, and Vanderbilt-Ingram Cancer Center, Vanderbilt University Medical Center, Nashville, Tennessee 37232, USA.
Gastroenterology. 2011 Nov;141(5):1738-48.e1-2. doi: 10.1053/j.gastro.2011.06.070. Epub 2011 Jul 7.
BACKGROUND & AIMS: Dopamine and adenosine 3',5'-cyclic monophosphate-regulated phosphoprotein, Mr 32000 (DARPP-32), is overexpressed during gastric carcinogenesis. Gastric tumors can become resistant to gefitinib, an inhibitor of the epidermal growth factor receptor (EGFR). We investigated the role of DARPP-32 in gastric tumor resistance to gefitinib.
Cell survival was determined by clonogenic survival and ATP-Glo Viability Assays. Apoptosis was assessed by Annexin-V and immunoblot analyses. The association between DARPP-32 and EGFR was evaluated by immunofluorescence and co-immunoprecipitation assays. Findings were validated in mice with gastric xenograft tumors. DARPP-32 expression was reduced using small hairpin RNAs in the human gastric cancer cell lines SNU-16 and MKN-45 cells.
Overexpression of DARPP-32 in MKN-28 cells, which do not normally express DARPP-32, blocked gefitinib-induced apoptosis and increased the drug's IC(50) 10-fold, compared to that of control cells (P < .01). Reduced expression of DARPP-32 in SNU-16 cells increased the sensitivity to gefitinib (P < .01). DARPP-32 activated phosphatidylinositol-3-kinase-AKT signaling, increased stability of the EGFR, and suppressed EGF- or gefitinib-induced degradation of the EGFR. DARPP-32 colocalized with EGFR on the cell membrane in a complex with EGFR and the EGF receptor ERBB3. DARPP-32-mediated resistance to gefitinib resulted from increased phosphorylation of and interaction between EGFR and ERBB3, which led to phosphorylation of AKT (at serine 473). Knockdown of DARPP-32 in gastric cancer cells reduced the mean size of tumors in mice and increased their response to gefitinib.
DARPP-32 promotes resistance of gastric cancer cells to gefitinib by promoting interaction between EGFR and ERBB3 and activating phosphatidylinositol-3-kinase-AKT signaling.
多巴胺和腺苷 3'，5'-环单磷酸调节磷蛋白，Mr32000（DARPP-32）在胃癌发生过程中过度表达。胃肿瘤可能对表皮生长因子受体（EGFR）抑制剂吉非替尼产生耐药性。我们研究了 DARPP-32 在胃肿瘤对吉非替尼耐药中的作用。
通过集落形成生存和 ATP-Glo 活力测定法测定细胞存活。通过 Annexin-V 和免疫印迹分析评估细胞凋亡。通过免疫荧光和共免疫沉淀测定评估 DARPP-32 与 EGFR 之间的关联。在具有胃异种移植肿瘤的小鼠中验证了这些发现。使用小发夹 RNA 降低人胃癌细胞系 SNU-16 和 MKN-45 细胞中的 DARPP-32 表达。
在不通常表达 DARPP-32 的 MKN-28 细胞中过表达 DARPP-32 可阻止吉非替尼诱导的细胞凋亡，并使药物的 IC50 增加 10 倍，与对照细胞相比（P <.01）。在 SNU-16 细胞中降低 DARPP-32 的表达可增加对吉非替尼的敏感性（P <.01）。DARPP-32 激活了磷脂酰肌醇-3-激酶-AKT 信号通路，增加了 EGFR 的稳定性，并抑制了 EGF 或吉非替尼诱导的 EGFR 降解。DARPP-32 在细胞膜上与 EGFR 共定位，形成与 EGFR 和表皮生长因子受体 ERBB3 的复合物。DARPP-32 介导的对吉非替尼的耐药性是由于 EGFR 和 ERBB3 之间的相互作用增加和磷酸化，从而导致 AKT（丝氨酸 473 位）磷酸化。在胃癌细胞中敲低 DARPP-32 可减少小鼠肿瘤的平均大小，并增加其对吉非替尼的反应。
DARPP-32 通过促进 EGFR 和 ERBB3 之间的相互作用并激活磷脂酰肌醇-3-激酶-AKT 信号通路，促进胃肿瘤细胞对吉非替尼的耐药性。
