Institute for Research in Dental Sciences, Faculty of Dentistry, Universidad de Chile, Santiago, Chile.
Advanced Center for Chronic Diseases (ACCDiS), Universidad de Chile, Santiago, Chile.
FASEB J. 2020 Mar;34(3):4009-4025. doi: 10.1096/fj.201902345RR. Epub 2020 Jan 28.
Potentially malignant lesions, commonly referred to as dysplasia, are associated with malignant transformation by mechanisms that remain unclear. We recently reported that increased Wnt secretion promotes the nuclear accumulation of β-catenin and expression of target genes in oral dysplasia. However, the mechanisms accounting for nuclear re-localization of β-catenin in oral dysplasia remain unclear. In this study, we show that endosomal sequestration of the β-catenin destruction complex allows nuclear accumulation of β-catenin in oral dysplasia, and that these events depended on the endocytic protein Rab5. Tissue immunofluorescence analysis showed aberrant accumulation of enlarged early endosomes in oral dysplasia biopsies, when compared with healthy oral mucosa. These observations were confirmed in cell culture models, by comparing dysplastic oral keratinocytes (DOK) and non-dysplastic oral keratinocytes (OKF6). Intriguingly, DOK depicted higher levels of active Rab5, a critical regulator of early endosomes, when compared with OKF6. Increased Rab5 activity in DOK was necessary for nuclear localization of β-catenin and Tcf/Lef-dependent transcription, as shown by expression of dominant negative and constitutively active mutants of Rab5, along with immunofluorescence, subcellular fractionation, transcription, and protease protection assays. Mechanistically, elevated Rab5 activity in DOK accounted for endosomal sequestration of components of the destruction complex, including GSK3β, Axin, and adenomatous polyposis coli (APC), as observed in Rab5 dominant negative experiments. In agreement with these in vitro observations, tissue immunofluorescence analysis showed increased co-localization of GSK3β, APC, and Axin, with early endosome antigen 1- and Rab5-positive early endosomes in clinical samples of oral dysplasia. Collectively, these data indicate that increased Rab5 activity and endosomal sequestration of the β-catenin destruction complex leads to stabilization and nuclear accumulation of β-catenin in oral dysplasia.
潜在恶性病变，通常称为异型增生，与恶性转化有关，但机制尚不清楚。我们最近报道，Wnt 分泌增加促进了口腔异型增生中β-连环蛋白的核积累和靶基因的表达。然而，口腔异型增生中β-连环蛋白核再定位的机制仍不清楚。在这项研究中，我们表明β-连环蛋白破坏复合物的内体隔离允许β-连环蛋白在口腔异型增生中核积累，并且这些事件依赖于内吞蛋白 Rab5。组织免疫荧光分析显示，与健康口腔黏膜相比，口腔异型增生活检中异常积累了增大的早期内体。在细胞培养模型中，通过比较异型增生口腔角质形成细胞（DOK）和非异型增生口腔角质形成细胞（OKF6），证实了这一观察结果。有趣的是，与 OKF6 相比，DOK 显示出更高水平的活性 Rab5，这是早期内体的关键调节因子。正如通过表达显性负和组成性激活的 Rab5 突变体以及免疫荧光、亚细胞分级、转录和蛋白酶保护测定所显示的，DOK 中 Rab5 活性的增加对于β-连环蛋白和 Tcf/Lef 依赖性转录的核定位是必要的。在机制上，DOK 中 Rab5 活性的升高导致破坏复合物的成分（包括 GSK3β、Axin 和腺瘤性结肠息肉病（APC））被内体隔离，这在 Rab5 显性负实验中观察到。与这些体外观察结果一致，组织免疫荧光分析显示，在口腔异型增生的临床样本中，GSK3β、APC 和 Axin 与早期内体抗原 1 和 Rab5 阳性的早期内体的共定位增加。总之，这些数据表明，Rab5 活性的增加和β-连环蛋白破坏复合物的内体隔离导致β-连环蛋白在口腔异型增生中的稳定和核积累。
