Head and neck squamous cell carcinoma (HNSCC) is the sixth most common type of cancer affecting humans worldwide. To determine the potential mechanisms by which chronic tobacco and alcohol abuse lead to HNSCC of the oral cavity, we have used both the 4-nitroquinoline-1-oxide (4-NQO) murine oral carcinogenesis and the Meadows-Cook alcohol models. In this study, we treated mice with 4-NQO in drinking water for 10 weeks and then administered 20% (w:v) ethanol (EtOH) for another 10 weeks. We observed increased levels and/or activation of signaling proteins [p38 mitogen-activated protein kinase (MAPK), β-catenin and Erk 1/2] that are typically altered during HNSCC initiation in humans. We found that EtOH administration alone increased the expression of p38 MAPK but not Erk 1/2 MAPK. Total β-catenin levels in the tongues increased by 2- to 3-fold after 4-NQO treatment, with or without EtOH. However, EtOH combined with 4-NQO reduced phosphorylated β-catenin levels, whereas 4-NQO treatment alone did not. These data implicate EtOH as a regulator of β-catenin signaling in this HNSCC model. We also utilized K14-CreER(TAM); ROSA26 mice to mark permanently stem/progenitor cells in the tongue epithelia. We found that 4-NQO alone and 4-NQO plus EtOH treatment resulted in massive, horizontal expansion of stem/progenitor cell populations arising from single stem cells in the basal layer of the epithelia. This expansion is consistent with carcinogen-associated, symmetric division of stem/progenitor cells. Our data suggest that specific therapeutic targets for prevention of HNSCC of the oral cavity associated with both alcohol and tobacco use are p38 MAPK and β-catenin.

Embryonic stem cells (ESCs) have both the ability to self-renew and to differentiate into various cell lineages. Retinoic acid (RA), a metabolite of Vitamin A, has a critical function in initiating lineage differentiation of ESCs through binding to the retinoic acid receptors (RARs). Additionally, the Wnt signaling pathway plays a role in pluripotency and differentiation, depending on the activation status of the canonical and noncanonical pathways. The activation of the canonical Wnt signaling pathway, which requires the nuclear accumulation of β-catenin and its interaction with Tcf1/Lef at Wnt response elements, is involved in ESC stemness maintenance. The noncanonical Wnt signaling pathway, through actions of Tcf3, can antagonize the canonical pathway. We show that RA activates the noncanonical Wnt signaling pathway, while concomitantly inhibiting the canonical pathway. RA increases the expression of ligands and receptors of the noncanonical Wnt pathway (Wnt 5a, 7a, Fzd2 and Fzd6), downstream signaling, and Tcf3 expression. RA reduces the phosphorylated β-catenin level by 4-fold, though total β-catenin levels don't change. We show that RA signaling increases the dissociation of Tcf1 and the association of Tcf3 at promoters of genes that regulate stemness (e.g. NR5A2,Lrh-1) or differentiation (eg. Cyr61, Zic5). Knockdown of Tcf3 increases Lrh-1 transcript levels in mESCs and prevents the RA-associated, ∼4-fold increase in Zic5, indicating that RA requires Tcf3 to effect changes in Zic5 levels. We demonstrate a novel role for RA in altering the activation of these two Wnt signaling pathways and show that Tcf3 mediates some actions of RA during differentiation.

We compared the changes in global gene expression between an early stage (the termination of the carcinogen treatment and prior to the appearance of frank tumors) and a late stage (frank squamous cell carcinoma (SCC)) of tongue carcinogenesis induced by the carcinogen 4-nitroquinoline 1-oxide (4-NQO) in a mouse model of human oral cavity and esophageal squamous cell carcinoma. Gene ontology and pathway analyses show that increases in “cell cycle progression” and “degradation of basement membrane and ECM pathways” are early events during SCC carcinogenesis and that changes in these pathways are even greater in the actual tumors. Myc, NFκB complex (NFKB1/RELA), and FOS transcription networks are the major transcriptional networks induced in early stage tongue carcinogenesis. Decreases in metabolism pathways, such as in “tricarboxylic acid cycle” and “oxidative phosphorylation”, occurred only in the squamous cell carcinomas and not in the early stages of carcinogenesis. We detected increases in ALDH1A3, PTGS2, and KRT1 transcripts in both the early and late stages of carcinogenesis. The identification of the transcripts and pathways that change at an early stage of carcinogenesis provides potentially useful information for early diagnosis and for prevention strategies for human tongue squamous cell carcinomas.

We investigated the effects of bexarotene (a retinoid X receptor agonist), CD1530 (a retinoic acid receptor γ selective agonist), and the combination of these two drugs for the prevention of oral carcinogenesis induced by the carcinogen 4-nitroquinoline 1-oxide (4-NQO) in a mouse model of human oral-cavity and esophageal squamous-cell carcinoma previously generated in our laboratory. We observed decreased numbers of neoplastic tongue lesions and reduced lesion severity in the 4-NQO plus CD1530 (4N+C) and 4-NQO plus bexarotene plus CD1530 (4N+B+C) groups compared with the 4-NQO group. RNA-Seq analyses showed increases in transcripts in cell proliferation/cell cycle progression pathways in the 4-NQO vs. the untreated group. In addition, β-catenin and matrix metallopeptidase 9 (MMP9) protein levels and reactive oxygen species (ROS), as assessed by 4-hydroxynonenal (4-HNE) staining, were elevated in tongue tissues 17 wk after the termination of the 4-NQO treatment. The 4N+B, 4N+C, and 4N+B+C groups showed dramatically lower levels of β-catenin, MMP9, and 4-HNE staining compared with the 4-NQO group. The major reduction in 4-HNE staining in the retinoid treatment groups suggests a novel mechanism of action, reduction of ROS, by which bexarotene and CD1530 inhibit carcinogenesis.cancer prevention | retinoic acid receptor gamma agonist | retinoid X receptor | tongue squamous cell carcinoma | oral cancer

Background Head and neck squamous cell carcinoma (HNSCC) is a cancer that is characterized by its high morbidity and mortality rates. While tobacco use and alcohol consumption are two major contributing factors for HNSCC carcinogenesis, how the combination of tobacco and alcohol increases HNSCC risk is not understood. Methods We combined the 4-nitroquinoline-1-oxide (4-NQO) oral carcinogenesis and Meadows-Cook alcohol mouse models to elucidate the molecular events and to identify novel biomarkers associated with oral cancer development. Results By genome-wide RNA-seq of tongue samples (three mice per group) we identified changes in transcripts that mediate alcohol metabolism and oxidative stress (Aldh2, Aldh1a3, Adh1, Adh7, and Cyp2a5) in mice treated with 4-NQO followed by ethanol (4-NQO/EtOH) as compared to the vehicle control/untreated samples (V.C./Untr.). We measured major, global increases in specific histone acetylation and methylation epigenetic marks (H3K27ac, H3K9/14ac, H3K27me3, and H3K9me3) in the oral cavities of V.C./EtOH, 4-NQO/Untr. and 4-NQO/EtOH treatment groups compared to the V.C./Untr. group. We detected changes in histone epigenetic marks near regulatory regions of genes involved in ethanol metabolism by chromatin immunoprecipitation (ChIP). For instance, the Aldh2 promoter showed increased H3K27me3 marks, and Aldh2 mRNA levels were reduced by 10-fold in 4NQO/EtOH vs. V.C./Untr. tongue samples. 4-NQO/EtOH treatment also caused increases in markers of oxidative stress, including 4-HNE, MCT4/Slc16a3, and TOM20, as measured by immunohistochemistry. Conclusions We delineate a mechanism by which 4-NQO and ethanol can regulate gene expression during the development of HNSCC, and suggest that histone epigenetic marks and oxidative stress markers could be novel biomarkers and targets for the prevention of HNSCC.