Lung mucoepidermoid carcinoma (MEC) is a very poorly characterized rare subtype of non-small-cell lung cancer (NSCLC) associated with more favorable prognoses than other forms of intrathoracic malignancies. We have previously identified that heme oxygenase-1 (HO-1, encoded by HMOX1) inhibits MEC tumor growth and modulates the transcriptome of microRNAs. Here we investigate the role of a major upstream regulator of HO-1 and a master regulator of cellular antioxidant responses, transcription factor Nrf2, in MEC biology. Nrf2 overexpression in the NCI-H292 MEC cell line mimicked the phenotype of HO-1 overexpressing cells, leading to inhibition of cell proliferation and migration and down-regulation of oncogenic miR-378. HMOX1 silencing identified HO-1 as a major mediator of Nrf2 action. Nrf2- and HO-1 overexpressing cells exhibited strongly diminished expression of multiple matrix metalloproteinases and inflammatory cytokine interleukin-1β, which was confirmed in an NCI-HO-1 xenograft model. Overexpression of HO-1 altered not only human MMP levels in tumor cells but also murine MMP levels within tumor microenvironment and metastatic niche. This could possibly contribute to decreased metastasis to the lungs and inhibitory effects of HO-1 on MEC tumor growth. Our profound transcriptome analysis and molecular characterization of the mucoepidermoid lung carcinoma helps to understand the specific clinical presentations of these tumors, emphasizing a unique antitumoral role of the Nrf2-HO-1 axis.

Mammalian DNA methylation is a critical epigenetic mechanism orchestrating gene expression networks in many biological processes. However, investigation of the functions of specific methylation events remains challenging. Here, we demonstrate that fusion of Tet1 or Dnmt3a with a catalytically inactive Cas9 (dCas9) enables targeted DNA methylation editing. Targeting of the dCas9-Tet1 or -Dnmt3a fusion protein to methylated or unmethylated promoter sequences caused activation or silencing, respectively, of an endogenous reporter. Targeted demethylation of the BDNF promoter IV or the MyoD distal enhancer by dCas9-Tet1 induced BDNF expression in post-mitotic neurons or activated MyoD facilitating reprogramming of fibroblasts into myoblasts, respectively. Targeted de novo methylation of a CTCF loop anchor site by dCas9-Dnmt3a blocked CTCF binding and interfered with DNA looping, causing altered gene expression in the neighboring loop. Finally, we show that these tools can edit DNA methylation in mice, demonstrating their wide utility for functional studies of epigenetic regulation.

Recently we have shown that hypoxia as well as overexpression of the stable form of hypoxia-inducible factor-1α (HIF-1α) diminished the expression of interleukin-8 (IL-8) by inhibition of the Nrf2 transcription factor in HMEC-1 cells. Because HIF isoforms may exert different effects, we aimed to examine the influence of HIF-2α on IL-8 expression in endothelial cells. In contrast to HIF-1α, overexpression of HIF-2α obtained by adenoviral transduction resulted in increased expression of IL-8 in an Nrf2-independent way. Importantly, HIF-2α augmented the activity of SP-1, a transcription factor involved in IL-8 regulation and known coactivator of c-Myc. Additionally, HIF-1 decreased, whereas HIF-2 increased, c-Myc expression, and silencing of Mxi-1, a c-Myc antagonist, restored IL-8 expression downregulated by HIF-1α or hypoxia. Accordingly, binding of c-Myc to the IL-8 promoter was abolished in hypoxia. Importantly, both severe (0.5% O(2)) and mild (5% O(2)) hypoxia diminished IL-8 expression despite the stabilization of both HIF-1 and HIF-2. This study reveals the opposite roles of HIF-1α and HIF-2α in the regulation of IL-8 expression in endothelial cells. However, despite stabilization of both isoforms in hypoxia the effect of HIF-1 is predominant, and downregulation of IL-8 expression in hypoxia is caused by attenuation of Nrf2 and c-Myc.

While intrinsic changes in aging hematopoietic stem cells (HSCs) are well characterized, it remains unclear how extrinsic factors affect HSC aging. Here, we demonstrate that cells in the niche-endothelial cells (ECs) and CXCL12-abundant reticular cells (CARs)-highly express the heme-degrading enzyme, heme oxygenase 1 (HO-1), but then decrease its expression with age. HO-1-deficient animals (HO-1-/- ) have altered numbers of ECs and CARs that produce less hematopoietic factors. HSCs co-cultured in vitro with HO-1-/- mesenchymal stromal cells expand, but have altered kinetic of growth and differentiation of derived colonies. HSCs from young HO-1-/- animals have reduced quiescence and regenerative potential. Young HO-1-/- HSCs exhibit features of premature exhaustion on the transcriptional and functional level. HO-1+/+ HSCs transplanted into HO-1-/- recipients exhaust their regenerative potential early and do not reconstitute secondary recipients. In turn, transplantation of HO-1-/- HSCs to the HO-1+/+ recipients recovers the regenerative potential of HO-1-/- HSCs and reverses their transcriptional alterations. Thus, HSC-extrinsic activity of HO-1 prevents HSCs from premature exhaustion and may restore the function of aged HSCs.