Dioxin Receptor Expression Inhibits Basal and Transforming Growth Factor β-induced Epithelial-to-mesenchymal Transition

Recent studies have emphasized the role of the dioxin receptor (AhR) in maintaining cell morphology, adhesion, and migration. These novel AhR functions depend on the cell phenotype, and although AhR expression maintains mesenchymal fibroblasts migration, it inhibits keratinocytes motility. These observations prompted us to investigate whether AhR modulates the epithelial-to-mesenchymal transition (EMT). For this, we have used primary AhR^+/+ and AhR^−/− keratinocytes and NMuMG cells engineered to knock down AhR levels (sh-AhR) or to express a constitutively active receptor (CA-AhR). Both AhR^−/− keratinocytes and sh-AhR NMuMG cells had increased migration, reduced levels of epithelial markers E-cadherin and β-catenin, and increased expression of mesenchymal markers Snail, Slug/Snai2, vimentin, fibronectin, and α-smooth muscle actin. Consistently, AhR^+/+ and CA-AhR NMuMG cells had reduced migration and enhanced expression of epithelial markers. AhR activation by the agonist FICZ (6-formylindolo[3,2-b]carbazole) inhibited NMuMG migration, whereas the antagonist α-naphthoflavone induced migration as did AhR knockdown. Exogenous TGFβ exacerbated the promigratory mesenchymal phenotype in both AhR-expressing and AhR-depleted cells, although the effects on the latter were more pronounced. Rescuing AhR expression in sh-AhR cells reduced Snail and Slug/Snai2 levels and cell migration and restored E-cadherin levels. Interference of AhR in human HaCaT cells further supported its role in EMT. Interestingly, co-immunoprecipitation and immunofluorescence assays showed that AhR associates in common protein complexes with E-cadherin and β-catenin, suggesting the implication of AhR in cell-cell adhesion. Thus, basal or TGFβ-induced AhR down-modulation could be relevant in the acquisition of a motile EMT phenotype in both normal and transformed epithelial cells.     

Immortalized mouse mammary fibroblasts lacking dioxin receptor have impaired tumorigenicity in a subcutaneous mouse xenograft model

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.     

The aryl hydrocarbon receptor, more than a xenobiotic-interacting protein

The aryl hydrocarbon (dioxin) receptor (AhR) has been studied for several decades largely because of its critical role in xenobiotic-induced toxicity and carcinogenesis. Albeit this is a major issue in basic and clinical research, an increasing number of investigators are turning their efforts to try to understand the physiology of the AhR under normal cellular conditions. This is an exciting area that covers cell proliferation and differentiation, endogenous mechanisms of activation, gene regulation, tumor development and cell motility and migration, among others. In this review, we will attempt to summarize the studies supporting the implication of the AhR in those endogenous cellular processes.     

Paradoxical roles of FAK in tumor cell migration and metastasis

Focal adhesion kinase (FAK), as a key mediator of signaling induced by integrins, plays an instrumental role in many cellular functions, including cell survival and proliferation. Many studies have reported that FAK is a positive regulator of normal cell migration and cancer cell metastasis. However, emerging evidence shows that FAK—under certain oncogenic signaling, such as that initiated by activated Ras and some growth factor receptor kinases—negatively regulates cancer cell migration. Activated Ras may promote tumor cell migration by dephosphorylation of FAK at Y397 and facilitation of focal adhesion turnover at the leading edge of cells.     

Cyclase‐associated protein 1 is a key negative regulator of milk synthesis and proliferation of bovine mammary epithelial cells

Adenylyl cyclase‐associated protein (CAP) is a highly conserved protein. Previous reports have suggested that CAP1 may be a negative regulator of cellular proliferation, migration, and adhesion and the development of cell carcinomas. The molecular mechanism of CAP1 regulation of downstream pathways, as well as how CAP1 is regulated by environmental stimuli and upstream signalling, is not well understood. In this present study, we assessed the role of CAP1 in milk synthesis and proliferation of bovine mammary epithelial cells. Using gene overexpression and silencing methods, CAP1 was found to negatively regulate milk synthesis and proliferation of cells via the PI3K‐mTOR/SREBP‐1c/Cyclin D1 signalling pathway. Hormones, such as prolactin and oestrogen, and amino acids, such as methionine and leucine, stimulate MMP9 expression and trigger CAP1 degradation, and thus, abrogate its inhibition of synthesis of milk protein, fat, and lactose by and proliferation of bovine mammary epithelial cells. The results of our study help deepen our understanding of the regulatory mechanisms underlying milk synthesis and aid in characterizing the molecular mechanisms of CAP1. Previous reports have suggested that CAP1 is a negative regulator of cellular proliferation and anabolism, but the molecular mechanisms are largely unknown. In this present study, we identified CAP1 as a negative regulator of milk synthesis and proliferation of bovine mammary epithelial cells. Our results will deepen our understanding of the regulatory mechanisms underlying milk synthesis and aid in exploring the molecular mechanisms of CAP1.     

Aryl hydrocarbon receptor signaling involved in the invasiveness of LNCaP cells

There is now mounting evidence that the aryl hydrocarbon receptor (AhR) plays an important role in physiologic responses such as development, cell cycle regulation, immune function and also malignant transformation in various tissues. The strong nuclear AhR expression is observed in the invasive phenotype, and an elevated nuclear AhR expression is associated with a poor prognosis of human prostate cancer. On the other hand, there are conflicting results that the AhR deficiency results in increased susceptibility to prostate tumors in mouse model. In the present study, we investigated AhR expression and its role in the growth and invasiveness of human prostate cancer cells. The AhR protein expression was detected in prostate cancer cell lines and human prostate cancer tissues. A small interfering RNA targeting AhR, constitutive active AhR expression vector, and AhR agonist and antagonist were used to moderate its expression and signaling. The induction of AhR signaling attenuated invasiveness of prostate cancer cells without affecting the cellular growth rate. These results suggest that AhR signaling in prostate cancer cells facilitates invasion of these cells, and modulation with this signaling can be a potential therapeutic target of invasive tumors.     

The dioxin receptor controls β1 integrin activation in fibroblasts through a Cbp–Csk–Src pathway

Recent studies have suggested a regulatory role for the dioxin receptor (AhR) in cell adhesion and migration. Following our previous work, we report here that the C-terminal Src kinase-binding protein (Cbp) signaling pathway controls β1 integrin activation and that this mechanism is AhR dependent. T-FGM AhR ?/? fibroblasts displayed higher integrin β1 activation, revealed by the increased binding of the activation reporter 9EG7 anti-β1 mAb and of a soluble fibronectin fragment, as well as by enhanced talin-β1 association. AhR ?/? fibroblasts also showed increased fibronectin secretion and impaired directional migration. Notably, interfering Cbp expression in AhR ?/? fibroblasts reduced β1 integrin activation, improved cell migration and rescued wild-type cell morphology. Cbp over-expression in T-FGM AhR ?/? cells enhanced the formation of inhibitory Csk–Cbp complexes which in turn reduced c-Src p-Tyr⁴¹⁶ activation and focal adhesion kinase (FAK) phosphorylation at the c-Src-responsive residues p-Tyr⁵⁷⁶ and p-Tyr⁵⁷⁷. The c-Src target and migration-related protein Cav1 was also hypophosphorylated at p-Tyr¹⁴ in AhR ?/? cells, and such effect was rescued by down-modulating Cbp levels. Thus, AhR regulates fibroblast migration by modulating β1 integrin activation via Cbp-dependent, Src-mediated signaling.     

Role of mitogen-activated protein kinases in aryl hydrocarbon receptor signaling

Human populations are increasingly exposed to a number of environmental pollutants such as polycyclic aromatic hydrocarbons, polychlorinated biphenyls and dioxins. These compounds are activators of the aryl hydrocarbon receptor (AhR) that controls the expression of many genes including those for detoxification enzymes. The regulatory mechanisms of AhR are multi-factorial and include phosphorylation by various protein kinases. Significant progress in the research of mitogen-activated protein kinases (MAPKs) has been achieved in the last decade. Isolated reports have been published on the role of MAPKs in AhR functions and vice versa, with activation of MAPKs by AhR ligands. This mini-review summarizes current knowledge on the mutual interactions between MAPKs and AhR. The majority of studies has been done on cancer-derived cell lines that have impaired cell cycle regulation and lacks the complete detoxification apparatus. We emphasize the importance of the future studies that should be done on non-transformed cells to distinguish the role of MAPKs in cancer and normal cells. Primary cultures of human or rodent hepatocytes that are equipped with a fully functional biotransformation battery or xenobiotics-metabolizing extra-hepatic tissues should be the models of choice, as the results in our experiments confirm.     

ArgBP2 and the SoHo family of adapter proteins in oncogenic diseases

ArgBP2, a member of the SoHo family of adapter proteins, is a regulator of actin-dependent processes such as cell adhesion and migration. Recent data from our lab revealed that by regulating adhesion and migration of pancreatic cancer cells, ArgBP2 is endowed with an anti-tumoral function. We could show that part of the molecular mechanism involved the interaction of ArgBP2 with the Arp2/3 activator WAVE1, the tyrosine phosphatase PTP-PEST, and the tyrosine kinase c-Abl. As ArgBP2 shares common structural organization and overlapping functions with the two other members of this protein family, CAP and Vinexin, it raises the question whether these two other proteins could also be involved in cancer diseases. The control of cell migration being an important issue in tumor treatment, these recent findings suggest that ArgBP2 family-dependent signaling pathways represents potential targets for the development of therapeutic strategies, and highlight the importance of elucidating their molecular mechanisms of cytoskeletal regulation.Key words: ArgBP2, pancreatic cancer, SoHo family of proteins, adapter protein, CAP/ponsin, vinexin, cell signaling, actin cytoskeleton, adhesion, migrationAcquisition of migration properties by cancerous cells is a crucial feature for cancer cell invasion and metastasis and, therefore, the control of cell migration is an important issue in tumor treatment.¹ As molecular mechanisms governing cell motility are common to non-neoplasic cells and cancer cells, the elucidation of these mechanisms is crucial in order to better understand how the loss of control of cell adhesion, migration and cytoskeletal organization participate to tumorigenesis.     

Carboxyl terminus of hsc70-interacting protein (CHIP) can remodel mature aryl hydrocarbon receptor (AhR) complexes and mediate ubiquitination of both the AhR and the 90 kDa heat-shock protein (hsp90) in vitro

The regulation of the aryl hydrocarbon receptor (AhR) protein levels has been an area of keen interest, given its important role in mediating the cellular adaptation and toxic response to several environmental pollutants. The carboxyl terminus of hsc70-interacting protein (CHIP) ubiquitin ligase was previously associated with the regulation of the aryl hydrocarbon receptor, although the mechanisms were not directly demonstrated. In this study, we established that CHIP could associate with the AhR at cellular levels of these two proteins, suggesting a potential role for CHIP in the regulation of the AhR complex. The analysis of the sucrose-gradient-fractionated in vitro translated AhR complexes revealed that CHIP can mediate hsp90 ubiquitination while cooperating with unidentified factors to promote the ubiquitination of mature unliganded AhR complexes. In addition, the immunophilin-like protein XAP2 was able to partially protect the AhR from CHIP-mediated ubiquitination in vitro. This protection required the direct interaction of the XAP2 with the AhR complex. Surprisingly, CHIP silencing in Hepa-1c1c7 cells by siRNA methods did not reveal the function of CHIP in the AhR complex, because it did not affect well-characterized activities of the AhR nor affect its steady-state protein levels. However, the presence of potential compensatory mechanisms may be confounding this particular observation. Our results suggest a model where the E3 ubiquitin ligase CHIP cooperates with other ubiquitination factors to remodel native AhR-hsp90 complexes and where co-chaperones such as the XAP2 may affect the ability of CHIP to target AhR complexes for ubiquitination.     

Serum osteopontin, an enhancer of tumor metastasis to bone, promotes B16 melanoma cell migration

Tumor malignancy is associated with several features such as proliferation ability and frequency of metastasis. Since tumor metastasis shortens patients' lifetime, establishment of therapy for anti-metastasis is very important. Osteopontin (OPN), which abundantly expressed in bone matrix, is involved in cell adhesion, migration, extracellular matrix (ECM) invasion and cell proliferation via interaction with its receptor, that is, alphavbeta3 integrin. OPN is believed to be a positive regulator of tumor metastasis in vivo. However, how OPN regulates metastasis is largely unknown. Here, we explore the role of OPN in cell migration. Serum from wild-type mice induced cell migration of B16 melanoma cells, while serum from OPN-deficient mouse suppressed this event. The presence of recombinant OPN significantly enhanced cell migration compared to albumin containing medium. OPN-induced cell migration was suppressed by inhibiting the ERK/MAPK pathway indicating that OPN-induced cell migration depends on this pathway. Overexpression of OPN in these cancer cells per se promoted cell proliferation and tended to increase B16 cell migration suggesting that OPN promotes bone metastasis by playing dual roles both in host microenvironment and in tumor cell itself. In conclusion, the elevated OPN expression in host tissue and tumor cell itself promotes tumor cell migration reading to tumor metastasis, suggesting that neutralization of OPN-induced signal might be effective in suppression of tumor metastasis.