Non-small cell lung adenocarcinoma (NSCLC) bearing K-RasG12D mutations is one of the most prevalent types of lung cancer worldwide. Aryl hydrocarbon receptor (AHR) expression varies in human lung tumors and has been associated with either increased or reduced lung metastasis. In the mouse, Ahr also adjusts lung regeneration upon injury by limiting the expansion of resident stem cells. Here, we show that the loss of Ahr enhances K-RasG12D-driven NSCLC in mice through the amplification of stem cell subpopulations. Consistent with this, we show that K-RasG12D;Ahr−/− lungs contain larger numbers of cells expressing markers for both progenitor Clara (SCGB1A1 and CC10) and alveolar type-II (SFTPC) cells when compared to K-RasG12D;Ahr+/+-driven tumors. They also have elevated numbers of cells positive for pluripotent stem cells markers such as SOX2, ALDH1, EPCAM, LGR5 and PORCN. Typical pluripotency genes Nanog, Sox2 and c-Myc were also upregulated in K-RasG12D;Ahr−/− lung tumors as found by RNAseq analysis. In line with this, purified K-RasG12D/+;Ahr−/− lung cells generate larger numbers of organoids in culture that can subsequently differentiate into bronchioalveolar structures enriched in both pluripotency and stemness genes. Collectively, these data indicate that Ahr antagonizes K-RasG12D-driven NSCLC by restricting the number of cancer-initiating stem cells. They also suggest that Ahr expression might represent a good prognostic marker to determine the progression of K-RasG12D-positive NSCLC patients.

Mammalian embryogenesis is a complex process controlled by transcription factors that regulate the balance between pluripotency and differentiation. Transcription factor aryl hydrocarbon receptor (AhR) regulates OCT4/POU5F1 and NANOG, both essential controllers of pluripotency, stemness and early embryo development. Molecular mechanisms controlling OCT4/POU5F1 and NANOG during embryogenesis remain unidentified. We show that AhR regulates pluripotency factors and maintains the metabolic activity required for proper embryo differentiation. AhR-lacking embryos (AhR À/À ) showed a pluripotent phenotype characterized by a delayed expression of trophectoderm differentiation markers. Accordingly, central pluripotency factors OCT4/POU5F1 and NANOG were overexpressed in AhR À/À embryos at initial developmental stages. An altered intracellular localization of these factors was observed in the absence of AhR and, importantly, Oct4 had an opposite expression pattern with respect to AhR from the two-cell stage to blastocyst, suggesting a negative regulation of OCT4/POU5F by AhR. We propose that AhR is a regulator of pluripotency and differentiation in early mouse embryogenesis.

Aging impairs organismal homeostasis leading to multiple pathologies. Yet, the mechanisms and molecular intermediates involved are largely unknown. Here, we report that aged aryl hydrocarbon receptor-null mice ( AhR−/− ) had exacerbated cellular senescence and more liver progenitor cells. Senescence-associated markers β-galactosidase (SA-β-Gal), p16 Ink4a and p21 Cip1 and genes encoding senescence-associated secretory phenotype (SASP) factors TNF and IL1 were overexpressed in aged AhR−/− livers. Chromatin immunoprecipitation showed that AhR binding to those gene promoters repressed their expression, thus adjusting physiological levels in AhR+/+ livers. MCP-2, MMP12 and FGF secreted by senescent cells were overproduced in aged AhR-null livers. Supporting the relationship between senescence and stemness, liver progenitor cells were overrepresented in AhR−/− mice, probably contributing to increased hepatocarcinoma burden. These AhR roles are not liver-specific since adult and embryonic AhR-null fibroblasts underwent senescence in culture, overexpressing SA-β-Gal, p16 Ink4a and p21 Cip1 . Notably, depletion of senescent cells with the senolytic agent navitoclax restored expression of senescent markers in AhR−/− fibroblasts, whereas senescence induction by palbociclib induced an AhR-null-like phenotype in AhR+/+ fibroblasts. AhR levels were downregulated by senescence in mouse lungs but restored upon depletion of p16 Ink4a -expressing senescent cells. Thus, AhR restricts age-induced senescence associated to a differentiated phenotype eventually inducing resistance to liver tumorigenesis.