Department of Oral and Maxillofacial Surgery, University Hospital Regensburg, Franz-Josef-Strauss-Allee 11, 93053 Regensburg, Germany and.
Carcinogenesis. 2013 Nov;34(11):2622-8. doi: 10.1093/carcin/bgt211. Epub 2013 Jun 5.
P-cadherin belongs to a family of Ca(2+)-dependent homophilic cell-cell adhesion proteins that are important for correct cellular localization and tissue integrity in the oral epithelium. P-cadherin is only expressed in the basal and suprabasal cell layers of the oral epithelium, but in advanced oral squamous cell carcinoma (OSCC), a reduced membranous and an enhanced cytoplasmic truncated P-cadherin level is observed. In this study, we investigated the impact of presenilin (PS) 1/γ-secretase on P-cadherin processing in OSCC. Western blot analyses showed an enhanced PS1 expression in OSCC cell lines and in primary oral keratinocytes (POK) isolated from primary OSCC tissue (OSCC POK) compared with POKs isolated from normal oral mucosa. Immunocytochemical stainings and co-immunoprecipitation experiments revealed a cytoplasmic colocalization and a direct interaction of P-cadherin and PS1 in OSCC POKs. Blocking of PS1/γ-secretase activity by the PS1/γ-secretase inhibitors and N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester, another specific γ-secretase inhibitor yielded a 100 kDa P-cadherin band on western blots of OSCC cell line extracts. Small interfering RNA knockdown of PS1 equally generated a 100 kDa P-cadherin form in OSCC POKs. Mass spectrometry analyses and experiments with the glycosylation inhibitor tunicamycin characterized the appearing 100 kDa P-cadherin band as the unglycosylated full-length form of P-cadherin. On the functional level, cell attachment assays demonstrated an enhanced cell adhesion after PS1/γ-secretase inhibition only in the transiently P-cadherin expressing OSCC cell line PCI52 but not in the PCI52 control cells. In summary, our results show that PS1/γ-secretase contributes to P-cadherin processing and to reduced cell adhesion in OSCC.
P-钙黏蛋白属于钙（Ca2+）依赖性同亲性细胞-细胞黏附蛋白家族，对于口腔上皮细胞的正确细胞定位和组织完整性非常重要。P-钙黏蛋白仅在口腔上皮的基底和超基底细胞层表达，但在晚期口腔鳞状细胞癌（OSCC）中，观察到膜性降低和细胞质截断 P-钙黏蛋白水平升高。在这项研究中，我们研究了早老素（PS）1/γ-分泌酶对 OSCC 中 P-钙黏蛋白加工的影响。Western blot 分析显示，与来自正常口腔黏膜的 POK 相比，OSCC 细胞系和从原发性 OSCC 组织中分离的原发性口腔角质形成细胞（POK）（OSCC POK）中 PS1 的表达增强。免疫细胞化学染色和共免疫沉淀实验显示，在 OSCC POK 中，P-钙黏蛋白和 PS1 存在细胞质共定位和直接相互作用。PS1/γ-分泌酶抑制剂和另一种特异性 γ-分泌酶抑制剂 N-[N-（3,5-二氟苯乙酰基）-L-丙氨酰]-S-苯甘氨酸叔丁酯阻断 PS1/γ-分泌酶活性，在 OSCC 细胞系提取物的 Western blot 上产生了 100 kDa 的 P-钙黏蛋白带。PS1 的小干扰 RNA 敲低同样在 OSCC POK 中产生了 100 kDa 的 P-钙黏蛋白形式。质谱分析和使用糖基化抑制剂衣霉素的实验将出现的 100 kDa P-钙黏蛋白带鉴定为未经糖基化的全长 P-钙黏蛋白形式。在功能水平上，细胞附着实验表明，仅在瞬时表达 P-钙黏蛋白的 OSCC 细胞系 PCI52 中，PS1/γ-分泌酶抑制后细胞黏附增强，而在 PCI52 对照细胞中则没有。总之，我们的结果表明 PS1/γ-分泌酶有助于 P-钙黏蛋白加工和 OSCC 中细胞黏附的降低。
