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.

Human embryonic stem (hES) cells can generate cells expressing p63, K14, and involucrin, which have been proposed to be keratinocytes. Although these hES-derived, keratinocyte-like (hESderK) cells form epithelioid colonies when cultured in a fibroblast feeder system optimal for normal tissue-derived keratinocytes, they have a very short replicative lifespan unless engineered to express HPV16 E6E7. We report here that hESderK cells undergo senescence associated with p16INK4A expression, unrelated to telomere status. Transduction to express bmi1, a repressor of the p16INK4A/p14ARF locus, conferred upon hESderK cells and keratinocytes a substantially extended lifespan. When exposed to transforming growth factor beta or to an incompletely processed form of Laminin-332, three lifespan-extended or immortalized hESderK lines that we studied became directionally hypermotile, a wound healing and invasion response previously characterized in keratinocytes. In organotypic culture, hESderK cells stratified and expressed involucrin and K10, as do epidermal keratinocytes in vivo. However, their growth requirements were less stringent than keratinocytes. We then extended the comparison to endoderm-derived, p63+/K14+ urothelial and tracheobronchial epithelial cells. Primary and immortalized lines of these cell types had growth requirements and hypermotility responses similar to keratinocytes and bmi1 expression facilitated their immortalization by engineering to express the catalytic subunit of telomerase (TERT). In organotypic culture, they stratified and exhibited squamous metaplasia, expressing involucrin and K10. Thus, hESderK cells proved to be distinct from all three normal p63+ cell types tested. These results indicate that hESderK cells cannot be identified conclusively as keratinocytes or even as ectodermal cells, but may represent an incomplete form of, or deviation from, normal p63+ lineage development.

Overexpression of the basement membrane protein Laminin γ2 (Lamγ2) is a feature of many epidermal and oral dysplasias and all invasive squamous cell carcinomas (SCCs). This abnormality has potential value as an immunohistochemical biomarker of premalignancy but its mechanism has remained unknown. We recently reported that Lamγ2 overexpression in culture is the result of deregulated translation controls and depends on the MAPK-RSK signaling cascade. Here we identify eIF4B as the RSK downstream effector responsible for elevated Lamγ2 as well as MYC protein in neoplastic epithelial cells. Premalignant dysplastic keratinocytes, SCC cells, and keratinocytes expressing the E6 oncoprotein of human papillomavirus (HPV) type 16 displayed MAPK-RSK and mTOR-S6K1 activation and overexpressed Lamγ2 and MYC in culture. Immunohistochemical staining of oral dysplasias and SCCs for distinct, RSK- and S6K1-specific S6 phosphorylation events revealed that their respective upstream pathways become hyperactive at the same time during neoplastic progression. However, pharmacologic kinase inhibitor studies in culture revealed that Lamγ2 and MYC overexpression depends on MAPK-RSK activity, independent of PI3K-mTOR-S6K1. eIF4B knockdown reduced Lamγ2 and MYC protein expression, consistent with the known requirement for eIF4B to translate mRNAs with long, complex 5′ untranslated regions (5′-UTRs). Accordingly, expression of a luciferase reporter construct preceded by the Lamγ2 5′-UTR proved to be RSK-dependent and mTOR-independent. These results demonstrate that RSK activation of eIF4B is causally linked to elevated Lamγ2 and MYC protein levels during neoplastic progression to invasive SCC. These findings have potential clinical significance for identifying premalignant lesions and for developing targeted drugs to treat SCC.