Immortalized Mouse Mammary Fibroblasts Lacking Dioxin Receptor Have Impaired Tumorigenicity in a Subcutaneous Mouse Xenograft Model

Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias, Universidad de Extremadura, Avenida de Elvas s/n, 06071 Badajoz, Spain.
Journal of Biological Chemistry (Impact Factor: 4.57). 09/2005; 280(31):28731-41. DOI: 10.1074/jbc.M504538200
Source: PubMed


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.

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Available from: Alberto Alvarez, Oct 09, 2015
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    • "Our findings differed from studies claiming that the pathway of Ras-cRaf-Erk is activated by AhR agonists [28], [29]. Our results showed that in MCVECs treated with 3MC, Ras/c-Raf was inactivated through an AhR/RhoA-dependent mechanism (Figure 3) and that PTEN/PI3K was involved in the dephosphorylation of c-Raf/pRb2 in MCVECs (Figure 4A). "
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    ABSTRACT: We previously showed that treating vascular endothelial cells with 3-methylcholanthrene (3MC) caused cell-cycle arrest in the Go/G1 phase; this resulted from the induction of p21 and p27 and a decreased level and activity of the cyclin-dependent kinase, Cdk2. We further investigated the molecular mechanisms that modulate cell-cycle regulatory proteins through the aryl-hydrocarbon receptor (AhR)/Ras homolog gene family, member A (RhoA) dependent epigenetic modification of histone. AhR/RhoA activation mediated by 3MC was essential for the upregulation of retinoblastoma 2 (pRb2) and histone deacetylase 1 (HDAC1), whereas their nuclear translocation was primarily modulated by RhoA activation. The combination of increased phosphatase and tensin homolog (PTEN) activity and decreased phosphatidylinositide 3-kinase (PI3K) activation by 3MC led to the inactivation of the Ras-cRaf pathway, which contributed to pRb2 hypophosphorylation. Increased HDAC1/pRb2 recruitment to the E2F1 complex decreased E2F1-transactivational activity and H3/H4 deacetylation, resulting in the downregulation of cell-cycle regulatory proteins (Cdk2/4 and Cyclin D3/E). Co-immunoprecipitation and electrophoretic mobility shift assay (EMSA) results showed that simvastatin prevented the 3MC-increased binding activities of E2F1 proteins in their promoter regions. Additionally, RhoA inhibitors (statins) reversed the effect of 3MC in inhibiting DNA synthesis by decreasing the nuclear translocation of pRb2/HDAC1, leading to a recovery of the levels of cell-cycle regulatory proteins. In summary, 3MC decreased cell proliferation by the epigenetic modification of histone through an AhR/RhoA-dependent mechanism that can be rescued by statins.
    PLoS ONE 03/2014; 9(3):e92793. DOI:10.1371/journal.pone.0092793 · 3.23 Impact Factor
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    • "In contrast, knockdown of the AHR in MDA-MB-468 cells did not affect cell proliferation (Zhang et al., 2009). Immortalized mammary tumor fibroblasts derived from Ahr +/+ and Ahr −/− mice were used as models to show that the AHR was required for tumor growth in a sc mouse xenograft model, and AHR loss was associated with decreased migration and angiogenesis (VEGFR1) (Mulero-Navarro et al., 2005). Thus, the role of AHR expression in breast cancer is variable and cell context dependent, and results of in vivo studies with Ahr −/− mice crossed with a transgenic mammary tumor model have not been reported. "
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    ABSTRACT: The aryl hydrocarbon receptor (AHR) is highly expressed in multiple organs and tissues, and there is increasing evidence that the AHR plays an important role in cellular homeostasis and disease. The AHR is expressed in multiple tumor types, in cancer cell lines, and in tumors from animal models, and the function of the AHR has been determined by RNA interference, overexpression, and inhibition studies. With few exceptions, knockdown of the AHR resulted in decreased proliferation and/or invasion and migration of cancer cell lines, and in vivo studies in mice overexpressing the constitutively active AHR exhibited enhanced stomach and liver cancers, suggesting a pro-oncogenic role for the AHR. In contrast, loss of the AHR in transgenic mice that spontaneously develop colonic tumors and in carcinogen-induced liver tumors resulted in increased carcinogenesis, suggesting that the receptor may exhibit antitumorigenic activity prior to tumor formation. AHR ligands also either enhanced or inhibited tumorigenesis, and these effects were highly tumor specific, demonstrating that selective AHR modulators that exhibit agonist or antagonist activities represent an important new class of anticancer agents that can be directed against multiple tumors.
    Toxicological Sciences 06/2013; 135(1). DOI:10.1093/toxsci/kft128 · 3.85 Impact Factor
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    • "Associations between SNPs in AHRR and TGCC AHR signaling pathway has been shown to interface with estrogen and androgen receptor signaling (Jana et al., 1999; Kizu et al., 2003; Safe and Wormke, 2003; Boverhof et al., 2006; Beischlag et al., 2008; Kollara and Brown, 2010). Additionally, AHR plays a significant role in tumor promotion and progression by deregulation of cell– cell contact and inhibition of apoptosis (Mulero-Navarro et al., 2005; Diry et al., 2006; Carvajal-Gonzalez et al., 2009; Chopra et al., 2009; Dietrich and Kaina, 2010). "
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    ABSTRACT: In the Western world, testicular germ cell cancer (TGCC) is the most common malignancy of young men. The malignant transformation of germ cells is thought to be caused by developmental and hormonal disturbances, probably related to environmental and lifestyle factors because of rapidly increasing incidence of TGCC in some countries. Additionally, there is a strong genetic component that affects susceptibility. However, genetic polymorphisms that have been identified so far only partially explain the risk of TGCC. Many of the persistent environmental pollutants act through the aryl hydrocarbon receptor (AHR). AHR signaling pathway is known to interfere with reproductive hormone signaling, which is supposed to play a role in the pathogenesis and invasive progression of TGCC. The aim of the present study was to identify whether AHR-related polymorphisms were associated with risk as well as histological and clinical features of TGCC in 367 patients and 537 controls. Haplotype-tagging single-nucleotide polymorphisms (SNPs) were genotyped in genes encoding AHR and AHR repressor (AHRR). Binary logistic regression was used to calculate the risk of TGCC, non-seminoma versus seminoma, and metastasis versus localized disease. Four SNPs in AHRR demonstrated a significant allele association with risk to develop metastases (rs2466287: OR = 0.43, 95% CI 0.21-0.90; rs2672725: OR = 0.49, 95% CI: 0.25-0.94; rs6879758: OR = 0.27, 95% CI: 0.08-0.92; rs6896163: OR = 0.34, 95% CI: 0.12-0.98). This finding supports the hypothesis that compounds acting through AHR may play a role in the invasive progression of TGCC, either directly or through modification of reproductive hormone action.
    Frontiers in Endocrinology 02/2013; 4:4. DOI:10.3389/fendo.2013.00004
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