Nuclear accumulation of β‐catenin is associated with endosomal sequestration of the destruction complex and increased activation of Rab5 in oral dysplasia

Abstract

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.

Wnt/β-Catenin Signaling in Oral Carcinogenesis

“…Intriguingly, whether these GTPases are altered at the early stages of oral mucosal carcinogenesis remains largely unknown. In this context, our recent studies indicate that a subset of Rab-GTPases are upregulated in oral dysplasia [ 14 ]. Specifically, the activity of Rab5, but not Rab7 or Rab11, is increased in dysplastic oral keratinocytes when compared with nondysplastic oral keratinocytes.…”

“…Despite that the nuclear accumulation of β-catenin is recognized as a recurrent event in oral dysplasia, mechanisms accounting for such phenomenon have remained elusive [ 11 , 15 , 16 , 27 ]. Recently, a link was established between this phenomenon and components of the endosomal machinery in a model that proposes that augmented Rab5 activity accounts for early endosome enlargement in dysplastic oral keratinocytes [ 14 ]. This event is followed by the increased sequestration of components of the β-catenin destruction complex, including APC, axin and GSK3β, within EEA1- and Rab5-positive compartments ( Figure 4 ).…”

“…This event is followed by the increased sequestration of components of the β-catenin destruction complex, including APC, axin and GSK3β, within EEA1- and Rab5-positive compartments ( Figure 4 ). Although this study did not explore the fate of the β-catenin destruction complex, data suggested that a subset of proteins forming this complex are targeted en route to the endo-lysosomal degradative pathway in dysplastic oral keratinocytes, since both proteins GSK3β and axin appeared enriched in fractions that were resistant against the proteinase K treatment, suggesting their accumulation in multivesicular bodies [ 14 ] (see proposed model, Figure 5 ). Confirmation of these possibilities will require further assessments by alternative approaches, including colocalization analyses of components of the destruction complex and late endosome/lysosome markers, as well as electron microscopy for ultrastructural assessments, as previously described [ 134 ].…”

“…Although further research is still required, and that more mechanistic insights should be drawn by in vitro approaches, it must be emphasized that current evidence showing the endosomal sequestration of components of the β-catenin destruction complex in patient biopsies represents a landmark finding to understand the “deregulation” of this pathway during oral carcinogenesis [ 11 , 14 ]. Intriguingly, whether this mechanism remains upregulated at late stages of carcinogenesis, namely carcinoma in situ and frank invasive OSCC, is an issue that needs to be explored in order to identify potential markers for predicting oral cancer progression.…”

“…However, unlike the extensive information available for other epithelial cancers, little is known about the molecular mechanisms accounting for the progression of early lesions and oral cancer. Specifically, studies have reported upregulation of the Wnt/β-catenin signaling pathway in oral cancer and in potentially malignant oral lesions, although the activation extent of this pathway varies according to the stage of oral carcinogenesis [ 11 , 14 , 15 , 16 ]. Here, we will review the literature that demonstrate the aberrant activation of Wnt/β-catenin in oral carcinogenesis, by first describing the components of the canonical Wnt signaling pathway and its transcriptional role over genes involved in proliferation and cell survival.…”