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.
Dendritic cells (DCs) promote tolerance or immunity depending on their maturation state, which is enhanced or accelerated upon MEK-ERK signaling pathway inhibition. We have determined the ...contribution of MEK-ERK activation to the profile of gene expression of human immature monocyte-derived dendritic cells (MDDCs) and peripheral blood myeloid DCs. ERK inhibition altered the expression of genes that mediate Chemokine (C-C motif) ligand 19 (CCL19)–directed migration (CCR7) and low-density lipoprotein (LDL) binding (CD36, SCARB1, OLR1, CXCL16) by immature DCs. In addition, ERK upregulated CCL2 expression while impairing the expression of DC maturation markers (RUNX3, ITGB7, IDO1). MEK-ERK–regulated genes exhibited an overrepresentation of cognate sequences for the aryl hydrocarbon receptor (AhR) transcription factor, whose transcriptional and DNA-binding activities increased in MDDCs upon exposure to the MEK1/2 inhibitor U0126. Therefore, the MEK-ERK signaling pathway regulates antigen capture, lymph node homing, and acquisition of maturation-associated genes, and its contribution to the maintenance of the immature state of MDDCs and myeloid DCs is partly dependent on the activity of AhR. Since pharmacologic modulation of the MEK-ERK signaling pathway has been proposed as a potential therapeutic strategy for cancer, our findings indicate that ERK inhibitors might influence antitumor responses through regulation of critical DC effector functions.
•Aryl hydrocarbon receptor (AhR) mediates the ERK-dependent maintenance of the immature state of monocyte-derived dendritic cells (MDDCs).•MEK-ERK regulates antigen capture, lymph node homing, and the acquisition of maturation-associated genes in MDDCs.
Angiogenesis has key roles in development and in the progression of human diseases such as cancer. Consequently, identifying the novel markers and regulators of angiogenesis is a critical task. The ...dioxin receptor (AhR) contributes to vascular homeostasis and to the endothelial response to toxins, although the mechanisms involved are largely uncharacterized. Here, we show that AhR-null mice (AhR−/−) have impaired angiogenesis in vivo that compromises tumor xenograft growth. Aortic rings emigration experiments and RNA interference indicated that AhR−/− endothelial cells failed to branch and to form tube-like structures. Such a phenotype was found to be vascular endothelial growth factor (VEGF)-dependent, as AhR−/− aortic endothelial cells (MAECs) secreted lower amounts of active VEGF-A and their treatment with VEGF-A rescued angiogenesis in culture and in vivo. Further, the addition of anti-VEGF antibody to AhR+/+ MAECs reduced angiogenesis. Treatment under hypoxic conditions with 2-methoxyestradiol suggested that HIF-1α modulates endothelial VEGF expression in an AhR-dependent manner. Importantly, AhR-null stromal myofibroblasts produced increased transforming growth factor-β (TGFβ) activity, which inhibited angiogenesis in human endothelial cells (HMECs) and AhR−/− mice, whereas the co-culture of HMECs with AhR−/− myofibroblasts or with their conditioned medium inhibited branching, which was restored by an anti-TGFβ antibody. Moreover, VEGF and TGFβ activities cooperated in modulating angiogenesis, as the addition of TGFβ to AhR−/− MAECs further reduced their low basal VEGF-A activity. Thus, AhR modulates angiogenesis through a mechanism requiring VEGF activation in the endothelium and TGFβ inactivation in the stroma. These data highlight the role of AhR in cardiovascular homeostasis and suggest that this receptor can be a novel regulator of angiogenesis during tumor development.
Alterations in tissue-specific gene expression greatly affect cell function. Transcription factors (TFs) interact with cis-acting binding sites in noncoding enhancer promoter regions. Transposable ...elements (TEs) are abundant and similarly represented among mammalian genomes. TEs are important in gene regulation, but their function is not well understood. We have characterized a TE containing functional TF-binding sites for the carcinogen-activated dioxin receptor xenobiotic responsive element (XRE) and the epithelial-mesenchymal transition regulator Slug (Slug site). A Mus promoter database was scanned for XREs to predict coregulation with other TFs. We identified an overrepresented (1,398 genes) B1 retrotransposon containing XRE and Slug sites within 35 bp of each other (designated as B1-X35S). This B1-X35S retrotransposon differed from classic B1s by the presence of the Slug site and by its differential nucleotide conservation outside the X35S region. Phylogenetically, B1-X35S appeared recently in evolution, close to the B1-B subfamily. Comparative gene expression in 61 mouse tissues revealed that B1-X35S-containing genes had lower median expression levels than those with canonical B1 TEs, suggesting a repressive role for X35S. Indeed, X35S was functional and able to bind aryl hydrocarbon (dioxin) receptor (AhR) and Slug and, importantly, to repress cis-reporter genes. Moreover, AhR and Slug were recruited to X35S in vivo and repressed the endogenous expression of X35S-containing genes. Our results demonstrate the existence of a widely present B1 subfamily in the mouse. Because AhR and Slug are relevant in tumor development and differentiation, X35S may represent a genome-wide regulatory mechanism and a tool to modulate gene expression.
Although the dioxin receptor, the aryl hydrocarbon receptor (AhR), is considered a major regulator of xenobiotic-induced carcinogenesis,
its role in tumor formation in the absence of xenobiotics is ...still largely unknown. Trying to address this question, we have
produced immortalized cell lines from wild-type (T-FGM-AhR+/+) and mutant (T-FGM-AhR-/-) mouse mammary fibroblasts by stable
co-transfection with the simian virus 40 (SV-40) large T antigen and proto-oncogenic c-H-Ras. Both cell lines had a myofibroblast
phenotype and similar proliferation, doubling time, SV-40 and c-H-Ras expression and activity, and cell cycle distribution.
AhR+/+ and AhR-/- cells were also equally able to support growth factor- and anchorage-independent proliferation. However,
the ability of T-FGM-AhR-/- to induce subcutaneous tumors (leimyosarcomas) in NOD/SCID-immunodeficient mice was close to 4-fold
lower than T-FGM-AhR+/+. In culture, T-FGM-AhR-/- had diminished migration in collagen-I and decreased lamellipodia formation.
VEGFR-1/Flt-1, a VEGF receptor that regulates cell migration and blood vessel formation, was also down-regulated in AhR-/-
cells. Signaling through the ERK-FAK-PKB/AKT-Rac-1 pathway, which contributes to cell motility and invasion, was also significantly
inhibited in T-FGM-AhR-/-. Thus, the lower tumorigenic potential of T-FGM-AhR-/- could result from a compromised adaptability
of these cells to the in vivo microenvironment, possibly because of an impaired ability to migrate and to respond to angiogenesis.
As our knowledge on the mechanisms that control cell function increases, more complex signaling pathways and quite intricate cross-talks among regulatory proteins are discovered. Establishing ...accurate interactions between cellular networks is essential for a healthy cell and different alterations in signaling are known to underline human disease. Transforming growth factor beta (TGFβ) is an extracellular cytokine that regulates such critical cellular responses as proliferation, apoptosis, differentiation, angiogenesis and migration, and it is assumed that the latency-associated protein LTBP-1 plays a relevant role in TGFβ targeting and activation in the extracellular matrix (ECM). The dioxin receptor (AhR) is a unique intracellular protein long studied because of its critical role in xenobiotic-induced toxicity and carcinogenesis. Yet, a large set of studies performed in cellular systems and
in vivo animal models have suggested important xenobiotic-independent functions for AhR in cell proliferation, differentiation and migration and in tissue homeostasis. Remarkably, AhR activity converges with TGFβ-dependent signaling through LTBP-1 since cells lacking AhR expression have phenotypic alterations that can be explained, at least in part, by the coordinated regulation of both proteins. Here, we will discuss the existence of functional interactions between AhR and TGFβ signaling. We will focus on regulatory and functional aspects by analyzing how AhR status determines TGFβ activity and by proposing a mechanism through which LTBP-1, a novel AhR target gene, mediates such effects. We will integrate ECM proteases in the AhR-LTBP-1-TGFβ axis and suggest a model that could help explain some
in vivo phenotypes associated to AhR deficiency.
The dioxin receptor (AhR) modulates cell plasticity and migration, although the signaling involved remains unknown. Here, we report a mechanism that integrates AhR into these cytoskeleton-related ...functions. Immortalized and mouse embryonic fibroblasts lacking AhR (AhR-/-) had increased cell area due to spread cytoplasms that reverted to wild-type morphology upon AhR re-expression. The AhR-null phenotype included increased F-actin stress fibers, depolarized focal adhesions, and enhanced spreading and adhesion. The cytoskeleton alterations of AhR-/- cells were due to down-regulation of constitutive Vav3 expression, a guanosine diphosphate/guanosine triphosphate exchange factor for Rho/Rac GTPases and a novel transcriptional target of AhR. AhR was recruited to the vav3 promoter and maintained constitutive mRNA expression in a ligand-independent manner. Consistently, AhR-/- fibroblasts had reduced Rac1 activity and increased activation of the RhoA/Rho kinase (Rock) pathway. Pharmacological inhibition of Rac1 shifted AhR+/+ fibroblasts to the null phenotype, whereas Rock inhibition changed AhR-null cells to the AhR+/+ morphology. Knockdown of vav3 transcripts by small interfering RNA induced cytoskeleton defects and changes in adhesion and spreading mimicking those of AhR-null cells. Moreover, vav3-/- MEFs, as AhR-/- mouse embryonic fibroblasts, had increased cell area and enhanced stress fibers. By modulating Vav3-dependent signaling, AhR could regulate cell shape, adhesion, and migration under physiological conditions and, perhaps, in certain pathological states.
The dioxin (AhR) receptor can have oncogenic or tumor suppressor activities depending on the phenotype of the target cell. We have shown that AhR knockdown promotes melanoma primary tumorigenesis and ...lung metastasis in the mouse and that human metastatic melanomas had reduced AhR levels with respect to benign nevi.
Mouse melanoma B16F10 cells were engineered by retroviral transduction to stably downregulate AhR expression, Aldh1a1 expression or both. They were characterized for Aldh1a1 activity, stem cell markers and migration and invasion in vitro. Their tumorigenicity in vivo was analyzed using xenografts and lung metastasis assays as well as in vivo imaging.
Depletion of aldehyde dehydrogenase 1a1 (Aldh1a1) impairs the pro-tumorigenic and pro-metastatic advantage of melanoma cells lacking AhR expression (sh-AhR). Thus, Aldh1a1 knockdown in sh-AhR cells (sh-AhR + sh-Aldh1a1) diminished their migration and invasion potentials and blocked tumor growth and metastasis to the lungs in immunocompetent AhR+/+ recipient mice. However, Aldh1a1 downmodulation in AhR-expressing B16F10 cells did not significantly affect tumor growth in vivo. Aldh1a1 knockdown reduced the high levels of CD133(+)/CD29(+)/CD44(+) cells, melanosphere size and the expression of the pluripotency marker Sox2 in sh-AhR cells. Interestingly, Sox2 increased Aldh1a1 expression in sh-AhR but not in sh-AhR + sh-Aldh1a1 cells, suggesting that Aldh1a1 and Sox2 may be co-regulated in melanoma cells. In vivo imaging revealed that mice inoculated with AhR + Aldh1a1 knockdown cells had reduced tumor burden and enhanced survival than those receiving Aldh1a1-expressing sh-AhR cells.
Aldh1a1 overactivation in an AhR-deficient background enhances melanoma progression. Since AhR may antagonize the protumoral effects of Aldh1a1, the AhR(low)-Aldh1a1(high) phenotype could be indicative of bad outcome in melanoma.
The contribution of environmental pollutants to liver fibrosis is an important and poorly explored issue. In vitro studies suggest that the environmental pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin ...(TCDD) and other aryl hydrocarbon receptor (AhR) ligands induce several genes that are known to be upregulated during liver fibrosis. Our aim was to determine whether exposure to such pollutants can lead to liver fibrosis and to characterize the mechanisms of action. Mice were treated for 2, 14, or 42 days, once a week with 25 µg/kg of TCDD. Gene and protein expression, in vitro and in vivo, as well as liver histology were investigated for each treatment. Treatment of mice with TCDD for 2 weeks modified the hepatic expression of markers of fibrosis such as collagen 1A1 and α-smooth muscle actin. This is not observed in AhR knockout mice. Following 6 weeks of treatment, histological features of murine hepatic fibrosis became apparent. In parallel, the levels of inflammatory cytokines (interleukin-1 beta, tumor necrosis factor α) and of markers of activated fibroblasts(fibroblast-specific protein 1) were found to be upregulated. Interestingly, we also found increased expression of genes of the TGF-β pathway and a concomitant decrease of miR-200a levels. Because the transcription factors of the Snail family were shown to be involved in liver fibrosis, we studied their regulation by TCDD. Two members of the Snail family were increased, whereas their negative targets, the epithelial marker E-cadherin and Claudin 1, were decreased. Further, the expression of mesenchymal markers was increased. Finally, we confirmed that Snai2 is a direct transcriptional target of TCDD in the human hepatocarcinoma cell line, HepG2. The AhR ligand, TCDD, induces hepatic fibrosis by directly regulating profibrotic pathways.
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