MAPK/ERK-Dependent Translation Factor Hyperactivation and Dysregulated Laminin γ2 Expression in Oral Dysplasia and Squamous Cell Carcinoma

Abstract

Lesions displaying a variety of dysplastic changes precede invasive oral and epidermal squamous cell carcinoma (SCC); however, there are no histopathological criteria for either confirming or staging premalignancy. SCCs and dysplasias frequently contain cells that abnormally express the γ2 subunit of laminin-332. We developed cell culture models to investigate γ2 dysregulation. Normal human keratinocytes displayed density-dependent repression of γ2, whereas premalignant keratinocytes and SCC cells overexpressed γ2 and secreted laminin assembly intermediates. Neoplastic cells had hyperactive EGFR/MAPK(ERK) signaling coordinate with overexpressed γ2, and EGFR and MEK inhibitors normalized γ2 expression. Keratinocytes engineered to express HPV16 E6 or activated mutant HRAS, cRAF1, or MEK1 lost density repression of γ2 and shared with neoplastic cells signaling abnormalities downstream of ERK, including increased phosphorylation of S6 and eIF4 translation factors. Notably, qPCR results revealed that γ2 overexpression was not accompanied by increased γ2 mRNA levels, consistent with ERK-dependent, eIF4B-mediated translation initiation of the stem-looped, 5'-untranslated region of γ2 mRNA in neoplastic cells. Inhibitors of MEK, but not of TORC1/2, blocked S6 and eIF4B phosphorylation and γ2 overexpression. Immunostaining of oral dysplasias identified γ2 overexpression occurring within fields of basal cells that had elevated p-S6 levels. These results reveal a causal relationship between ERK-dependent translation factor activation and laminin γ2 dysregulation and identify new markers of preinvasive neoplastic change during progression to SCC.

Phosphorylated S6 as an immunohistochemical biomarker of vulvar intraepithelial neoplasia

“…P-S6 is present in the upper layers and absent in the basal and parabasal layers of normal oral epithelium, but frequently is present in cells of the basal layer in oral dysplasias and squamous cell carcinomas. 29 Laminin g 2 is undetectable in normal basal cells but frequently is present in the cytoplasm of basal cells in oral and epidermal dysplasias. 24,29 In the present study, p-S6 and laminin g 2 were scored as positive when present in any basal cells in the section, regardless of the number or proportion of positive cells.…”

“…Immunostaining in Normal Vulvar Epithelium, Vulvar Carcinoma, and Classic and Differentiated VIN Our recent identification of p-S6(S235/236) as a readout for EGFR/ERK signal pathway hyperactivation in neoplastic oral epithelium and the presence of p-S6-positive basal cells in oral dysplastic lesions and squamous cell carcinomas 29 provided the rationale to examine VINs and vulvar carcinomas for this biomarker. We immunostained seven differentiated VINs, six of which were associated with carcinoma, and nine classic VINs, six of which were associated with carcinoma, for p-S6.…”

“…29 Increased S6(S235/236) phosphorylation (p-S6) proved to be detectable immunohistochemically in the basal cell layer of oral dysplasias and squamous cell carcinomas, preceding and accompanying laminin g 2 overexpression. 29 We therefore sought in the present study to determine whether p-S6 in the basal cell layer is a typical feature of VIN and other potentially premalignant vulvar lesions.…”

“…[26][27][28] Our recent study identified EGFR/ERK signal pathway hyperactivation as necessary and sufficient to drive laminin g 2 overexpression in cultured human premalignant epithelial and squamous cell carcinoma cell lines and also found that the HPV16 E6 oncoprotein also induces laminin g 2 overexpression. 29 Laminin g 2 overexpression proved to require the ERK-activated RSK kinase and to correlate with increased phosphorylation of the translation factor eIF4B at S442 and of ribosomal protein S6 at S235/236. 29 Increased S6(S235/236) phosphorylation (p-S6) proved to be detectable immunohistochemically in the basal cell layer of oral dysplasias and squamous cell carcinomas, preceding and accompanying laminin g 2 overexpression.…”

“…29 Laminin g 2 overexpression proved to require the ERK-activated RSK kinase and to correlate with increased phosphorylation of the translation factor eIF4B at S442 and of ribosomal protein S6 at S235/236. 29 Increased S6(S235/236) phosphorylation (p-S6) proved to be detectable immunohistochemically in the basal cell layer of oral dysplasias and squamous cell carcinomas, preceding and accompanying laminin g 2 overexpression. 29 We therefore sought in the present study to determine whether p-S6 in the basal cell layer is a typical feature of VIN and other potentially premalignant vulvar lesions.…”