MAPK/RSK-dependent, mTOR/S6K1-independent activation of eIF4B and Lamγ2 mRNA translation.

<p><b>A</b>) Western blot analysis of confluent cultures of SCC-68, the premalignant oral keratinocyte line POE9n, and normal primary keratinocyte strain N engineered to express the JH26 mutant of HPV16 E6 (N/E6(JH26). Cultures were treated for 24 hr with the indicated kinase inhibitors and then analyzed for levels of Lamγ2 and MYC protein and for the phosphorylated, activated forms of signaling proteins and translation factors. The Lamγ2 band shown is the 155 kD intracellular form and not the 105 kD form that predominates after secretion and proteolytic processing. The bar graphs below show densitometric analysis of Lamγ2 and p-EIF4B levels in each drug treatment condition relative to untreated control cultures of each line, as described in panel B. <b>B</b>) SCC-13 cells transfected with the reporter constructs pDL-N, pDL-N/(Lamγ2 5′-UTR), and pDL-N/(ODC 5-′UTR) with or without the RSK inhibitor BI-D1870 or the mTORC1/2 inhibitor Ku-0063794 and analyzed for Renilla and Firefly luciferase activity. Reduction caused by BI-D1780 in Lamγ2 5′UTR- and ODC 5′UTR-dependent expression had P values for significance of 0.0043 and 0.01, respectively.</p

Human oral epithelial lesions examined for increased p-S6 and Laminin γ2 by immunohistochemical staining.

<p>Formalin-fixed, paraffin-embedded specimens of oral lesions were immunostained for p-S6(S235), p-S6(S240), and Lamγ2 and the percentage of the total dysplasia positive in the basal cell layer for these markers determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078979#s2" target="_blank">Methods</a>. Basal layer p-S6(235) and p-S6(240) immunostaining always corresponded precisely. n.d.: not determined.</p

Coincidence of p-S6(S235) and p-S6(S240) detectable in human oral dysplastic lesions and SCCs <i>in vivo</i>.

<p>Sections of normal (<b>A</b>) and dysplastic (<b>B-C</b>) epithelium, and invasive SCC (<b>D</b>), stained with H&E and immunostained for Lamγ2, p-S6(S240) and p-S6(S235). Scale bar: 200 µm. Enlarged insets of some regions are shown for easier viewing of p-S6 staining patterns. Panel A shows a region of Case 2, panel B of case 8, panel C of case 7, and panel D of case 4 as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078979#pone-0078979-t001" target="_blank">Table 1</a>. Note that normal epithelium did not express Lamγ2 and neither S6 phosphorylation event was detectable in the basal cells. Cells in dysplasias and SCCs always showed coincidence of the two S6 phosphorylation events. Dysplasias varied with respect to frequency and intensity of Lamγ2 expression and S6 phosphorylation, with Lamγ2 cells representing a subset of p-S6 positive basal cells and invasive SCCs contained many Lamγ2 and basal layer p-S6 positive regions.</p