Mechanisms of breast cancer progression induced by environment-polluting aryl hydrocarbon receptor agonists.

Breast cancer is the most common invasive malignancy among women worldwide and constitutes a complex and heterogeneous disease. Interest has recently grown in the role of the aryl hydrocarbon receptor (AhR) in breast cancer and the contribution of environment-polluting AhR agonists. Here, we present a literature review addressing AhR ligands, including pesticides hexachlorobenzene and chlorpyrifos, polycyclic aromatic hydrocarbons, polychlorinated dibenzo-p-dioxins and dibenzofurans, polychlorinated biphenyls, parabens, and phthalates. The objectives of this review are a) to summarize recent original experimental, preclinical, and clinical studies on the biological mechanisms of AhR agonists which interfere with the regulation of breast endocrine functions, and b) to examine the biological effects of AhR ligands and their impact on breast cancer development and progression. We discuss biological mechanisms of action in cell viability, cell cycle, proliferation, epigenetic changes, epithelial to mesenchymal transition, and cell migration and invasion. In addition, we examine the effects of AhR ligands on angiogenic processes, metastasis, chemoresistance, and stem cell renewal. We conclude that exposure to AhR agonists stimulates pathways that promote breast cancer development and may contribute to tumor progression. Given the massive use of industrial and agricultural chemicals, ongoing evaluation of their effects in laboratory assays and preclinical studies in breast cancer at environmentally relevant doses is deemed essential. Likewise, awareness should be raised in the population regarding the most harmful toxicants to eradicate or minimize their use.

Breast cancer progression and kynurenine pathway enzymes are induced by hexachlorobenzene exposure in a Her2-positive model.

Breast cancer is one of the leading cancers among women worldwide. Given the evidence that pesticides play an important role in breast cancer, interest has grown in pesticide impact on disease progression. Hexachlorobenzene (HCB), an aryl hydrocarbon receptor (AhR) ligand, promotes triple-negative breast cancer cell migration and invasion. Estrogen receptor ß (ERß) inhibits cancer motility, while G protein-coupled ER (GPER) modulates the neoplastic transformation. Tryptophan is metabolized through the kynurenine pathway by indoleamine-2,3-dioxygenase (IDO) and tryptophan-2,3-dioxygenase (TDO), with kynurenine signaling activation often predicting worse prognosis in cancer. In this context, we examined the HCB (0.005; 0.05; 0.5 and 5 µM) effect on LM3 cells, a human epidermal growth factor receptor 2 (HER2)-positive breast cancer model. Results show that HCB increases IDO and TDO mRNA levels and promotes cell viability, proliferation and migration through the AhR pathway. Moreover, HCB boosts mammosphere formation, vascular endothelial growth factor and cyclooxygenase-2 expression and reduces IL-10 levels. For some parameters, U-shaped or inverted U-shaped dose-response curves are shown. HCB alters ER levels, reducing ERß while increasing GPER. These results demonstrate that exposure to environmentally relevant concentrations of HCB up-regulates the kynurenine pathway and dysregulates ERß and GPER levels, collaborating in HER2-positive breast cancer progression.

Extracellular acidosis stimulates breast cancer cell motility through aryl hydrocarbon receptor and c-src kinase activation.

A reduction in extracellular pH (pHe) is a characteristic of most malignant tumors. The aryl hydrocarbon receptor (AhR) is a transcription factor localized in a cytosolic complex with c-Src, which allows it to trigger nongenomic effects through c-Src. Considering that the slightly acidic tumor microenvironment promotes breast cancer progression in a similar way to the AhR/c-Src axis, our aim was to evaluate whether this pathway could be activated by low pHe. We examined the effect of pHe 6.5 on AhR/c-Src axis using two breast cancer cell lines (MDA-MB-231 and LM3) and mammary epithelial cells (NMuMG) and found that acidosis increased c-Src phosphorylation only in tumor cells. Moreover, the presence of AhR inhibitors prevented c-Src activation. Low pHe reduced intracellular pH (pHi), while amiloride treatment, which is known to reduce pHi, induced c-Src phosphorylation through AhR. Analyses were conducted on cell migration and metalloproteases (MMP)-2 and -9 activities, with results showing an acidosis-induced increase in MDA-MB-231 and LM3 cell migration and MMP-9 activity, but no changes in NMuMG cells. Moreover, all these effects were blocked by AhR and c-Src inhibitors. In conclusion, acidosis stimulates the AhR/c-Src axis only in breast cancer cells, increasing cell migration and MMP-9 activity. Although the AhR activation mechanism still remains elusive, a reduction in pHi may be thought to be involved. These findings suggest a critical role for the AhR/c-Src axis in breast tumor progression stimulated by an acidic microenvironment.

Angiogenesis signaling in breast cancer models is induced by hexachlorobenzene and chlorpyrifos, pesticide ligands of the aryl hydrocarbon receptor.

Breast cancer incidence is increasing globally and pesticides exposure may impact risk of developing this disease. Hexachlorobenzene (HCB) and chlorpyrifos (CPF) act as endocrine disruptors, inducing proliferation in breast cancer cells. Vascular endothelial growth factor-A (VEGF-A), cyclooxygenase-2 (COX-2) and nitric oxide (NO) are associated with angiogenesis. Our aim was to evaluate HCB and CPF action, both weak aryl hydrocarbon receptor (AhR) ligands, on angiogenesis in breast cancer models. We used: (1) in vivo xenograft model with MCF-7 cells, (2) in vitro breast cancer model with MCF-7, and (3) in vitro neovasculogenesis model with endothelial cells exposed to conditioned medium from MCF-7. Results show that HCB (3 mg/kg) and CPF (0.1 mg/kg) stimulated vascular density in the in vivo model. HCB and CPF low doses enhanced VEGF-A and COX-2 expression, accompanied by increased levels of nitric oxide synthases (NOS), and NO release in MCF-7. HCB and CPF high doses intensified VEGF-A and COX-2 levels but rendered different effects on NOS, however, both pesticides reduced NO production. Moreover, our data indicate that HCB and CPF-induced VEGF-A expression is mediated by estrogen receptor and NO, while the increase in COX-2 is through AhR and NO pathways in MCF-7. In conclusion, we demonstrate that HCB and CPF environmental concentrations stimulate angiogenic switch in vivo. Besides, pesticides induce VEGF-A and COX-2 expression, as well as NO production in MCF-7, promoting tubulogenesis in endothelial cells. These findings show that pesticide exposure could stimulate angiogenesis, a process that has been demonstrated to contribute to breast cancer progression.

Impact of endocrine disruptor hexachlorobenzene on the mammary gland and breast cancer: The story thus far.

Breast cancer incidence is increasing globally and exposure to endocrine disruptors has gained importance as a potential risk factor. Hexachlorobenzene (HCB) was once used as a fungicide and, despite being banned, considerable amounts are still released into the environment. HCB acts as an endocrine disruptor in thyroid, uterus and mammary gland and was classified as possibly carcinogenic to human. This review provides a thorough analysis of results obtained in the last 15 years of research and evaluates data from assays in mammary gland and breast cancer in diverse animal models. We discuss the effects of environmentally relevant HCB concentrations on the normal mammary gland and different stages of carcinogenesis, and attempt to elucidate its mechanisms of action at molecular level. HCB weakly binds to the aryl hydrocarbon receptor (AhR), activating both membrane (c-Src) and nuclear pathways. Through c-Src stimulation, AhR signaling interacts with other membrane receptors including estrogen receptor-α, insulin-like growth factor-1 receptor, epidermal growth factor receptor and transforming growth factor beta 1 receptors. In this way, several pathways involved in mammary morphogenesis and breast cancer development are modified, inducing tumor progression. HCB thus stimulates epithelial cell proliferation, preneoplastic lesions and alterations in mammary gland development as well as neoplastic cell migration and invasion, metastasis and angiogenesis in breast cancer. In conclusion, our findings support the hypothesis that the presence and bioaccumulation of HCB in high-fat tissues and during highly sensitive time windows such as pregnancy, childhood and adolescence make exposure a risk factor for breast tumor development.

Activation of c-Src/HER1/STAT5b and HER1/ERK1/2 signaling pathways and cell migration by hexachlorobenzene in MDA-MB-231 human breast cancer cell line.

Hexachlorobenzene (HCB) is a widespread environmental pollutant. It is a dioxin-like compound and a weak ligand of the aryl hydrocarbon receptor (AhR) protein. HCB is a tumor cocarcinogen in rat mammary gland and an inducer of cell proliferation and c-Src kinase activity in MCF-7 breast cancer cells. This study was carried out to investigate HCB action on c-Src and the human epidermal growth factor receptor (HER1) activities and their downstream signaling pathways, Akt, extracellular-signal-regulated kinase (ERK1/2), and signal transducers and activators of transcription (STAT) 5b, as well as on cell migration in a human breast cancer cell line, MDA-MB-231. We also investigated whether the AhR is involved in HCB-induced effects. We have demonstrated that HCB (0.05µM) produces an early increase of Y416-c-Src, Y845-HER1, Y699-STAT5b, and ERK1/2 phosphorylation. Moreover, our results have shown that the pesticide (15 min) activates these pathways in a dose-dependent manner (0.005, 0.05, 0.5, and 5µM). In contrast, HCB does not alter T308-Akt activation. Pretreatment with a specific inhibitor for c-Src (4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo[3,4-d]pyrimidine [PP2]) prevents Y845-HER1 and Y699-STAT5b phosphorylation. AG1478, a specific HER1 inhibitor, abrogates HCB-induced STAT5b and ERK1/2 activation, whereas 4,7-orthophenanthroline and α-naphthoflavone, two AhR antagonists, prevent HCB-induced STAT5b and ERK1/2 phosphorylation. HCB enhances cell migration evaluated by scratch motility and transwell assays. Pretreatment with PP2, AG1478, and 4,7-orthophenanthroline suppresses HCB-induced cell migration. These results demonstrate that HCB stimulates c-Src/HER1/STAT5b and HER1/ERK1/2 signaling pathways in MDA-MB-231. c-Src, HER1, and AhR are involved in HCB-induced increase in cell migration. The present study makes a significant contribution to the molecular mechanism of action of HCB in mammary carcinogenesis.

Central role of extracellular signal-regulated kinase and Toll-like receptor 4 in IL-10 production in regulatory dendritic cells induced by Trypanosoma cruzi.

Several Trypanosoma cruzi molecules that stimulate macrophages activity were described as Toll-like receptor 2 (TLR2) ligands. Besides, the models of dendritic cells (DC) are poorly characterised. We have previously demonstrated that live-trypomastigotes (Tp) plus lipopolysaccharide (LPS) induce DC with tolerogenic properties that produce high levels of interleukin (IL)-10 and an impaired capacity to induce lymphoproliferation. Here, we show that the regulatory phenotype was observed with heat-killed trypomastigotes (Tphk) stimulation, ruling out DC infection. T. cruzi induced a particular DC activation state increasing LPS-activation of extracellular regulated kinase (ERK) 1/2 and signal transducer and activator of transcription (STAT) 3. Inhibition of ERK down-regulated IL-10 production and restored DC stimulatory capacity, showing the importance of this pathway in the DC modulation. A recent work shows that signalling via TLR4 and TLR2 induces a synergism in anti-inflammatory cytokine production in murine DC. Upon TLR2 and TLR4 stimulation using Pam(3)Cys or LPS and Tphk in DC from TLR2 knock out (KO) or TLR4-mutant mice, we showed that high levels of IL-10 were independent of TLR2 but associated with TLR4 and NF-kappaB signallization. Although sialic acid has been described as a molecule responsible of DC inhibition, we determine that it is not associated with T. cruzi-IL-10 modulatory response. In conclusion, all these findings demonstrate a key role of ERK and TLR4 in association with NF-kappaB in IL-10 modulation induced by T. cruzi and suggest that this regulatory effect involves parasite-DC interactions not described yet.