Sonia Mulero-Navarro

The antiproliferative activity of resveratrol results in apoptosis in MCF-7 but not in MDA-MB-231 human breast cancer cells: cell-specific alteration of the cell cycle ☆

Resveratrol, a natural phytoalexin, has gained much interest on the basis of its potential chemopreventive activity against human cancer. In this work, using the human breast cancer cell lines MCF-7 and MDA-MB-231, we have analyzed a possible mechanism by which resveratrol could interfere with cell cycle control and induce cell death. Our results show that although resveratrol inhibited cell proliferation and viability in both cell lines, apoptosis was induced in a concentration- and cell-specific manner. In MDA-MB-231, resveratrol (up to 200 microM) lowered the expression and kinase activities of positive G1/S and G2/M cell cycle regulators and inhibited ribonucleotide reductase activity in a concentration dependent manner, without a significant effect on the low expression of tumor suppressors p21, p27, and p53. These cells died by a non-apoptotic process in the absence of a significant change in cell cycle distribution. In MCF-7, resveratrol produced a significant and transient (<50 microM) increase in the expression and kinase activities of positive G1/S and G2/M regulators. Simultaneously, p21 expression was markedly induced in presence of high levels of p27 and p53. These opposing effects resulted in cell cycle blockade at the S-phase and apoptosis induction in MCF-7 cells. Thus, the antiproliferative activity of resveratrol could take place through the differential regulation of the cell cycle leading to apoptosis or necrosis. This could be influenced, among other factors, by the concentration of this molecule and by the characteristics of the target cell.

Resveratrol‐induced apoptosis in MCF‐7 human breast cancer cells involves a caspase‐independent mechanism with downregulation of Bcl‐2 and NF‐κB

Resveratrol (RES), a chemopreventive molecule, inhibits the proliferation of tumor cells of different etiologies. We previously showed that RES alters the cell cycle and induces apoptosis in MCF‐7 breast tumor cells by interfering with the estrogen receptor (ERaα)–dependent phosphoinositide 3‐kinase (PI3K) pathway. Here, we analyzed signaling downstream of PI3K, to understand the mechanisms of RES‐induced apoptosis. Apoptotic death by RES in MCF‐7 was mediated by Bcl‐2 downregulation since overexpression of this protein abolished apoptosis. Decreased Bcl‐2 levels were not related to cytochrome c release, activation of caspases 3/8 or poly(ADP‐ribose) polymerase proteolysis. However, RES decreased mitochondrial membrane potential and increased reactive oxygen species and nitric oxide production. NF‐κB, a regulator of Bcl‐2 expression, and calpain protease activity, a regulator of NF‐κB, were both inhibited by RES. The patterns for NF‐κB and calpain activities followed that of PI3K and were inhibited by LY294002. NF‐κB inhibition coincided with diminished MMP‐9 activity and cell migration. These data suggest that RES‐induced apoptosis in MCF‐7 could involve an oxidative, caspase‐independent mechanism, whereby inhibition of PI3K signaling converges to Bcl‐2 through NF‐κB and calpain protease activity. Therefore, Bcl‐2 and NF‐κB could be considered potential targets for the chemopreventive activity of RES in estrogen‐responsive tumor cells.

Fitting a xenobiotic receptor into cell homeostasis: How the dioxin receptor interacts with TGFβ signaling

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 (TGFbeta) 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 TGFbeta 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 TGFbeta-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 TGFbeta signaling. We will focus on regulatory and functional aspects by analyzing how AhR status determines TGFbeta 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-TGFbeta axis and suggest a model that could help explain some in vivo phenotypes associated to AhR deficiency.

Proteasome Inhibition Induces Nuclear Translocation and Transcriptional Activation of the Dioxin Receptor in Mouse Embryo Primary Fibroblasts in the Absence of Xenobiotics

ABSTRACT The aryl hydrocarbon receptor (AHR) is a transcription factor that is highly conserved during evolution and shares important structural features with the Drosophila developmental regulatorsSim and Per. Although much is known about the mechanism of AHR activation by xenobiotics, little information is available regarding its activation by endogenous stimuli in the absence of exogenous ligand. In this study, using embryonic primary fibroblasts, we have analyzed the role of proteasome inhibition on AHR transcriptional activation in the absence of xenobiotics. Proteasome inhibition markedly reduced cytosolic AHR without affecting its total cellular content. Cytosolic AHR depletion was the result of receptor translocation into the nuclear compartment, as shown by transient transfection of a green fluorescent protein-tagged AHR and by immunoblot analysis of nuclear extracts. Gel retardation experiments showed that proteasome inhibition induced transcriptionally active AHR-ARNT heterodimers able to bind to a consensus xenobiotic-responsive element. Furthermore, nuclear AHR was transcriptionally active in vivo, as shown by the induction of the endogenous target gene CYP1A2. Synchronized to AHR activation, proteasome inhibition also induced a transient increase in AHR nuclear translocator (ARNT) at the protein and mRNA levels. Since nuclear levels of AHR and ARNT are relevant for AHR transcriptional activation, our data suggest that proteasome inhibition, through a transient increase in ARNT expression, could promote AHR stabilization and accumulation into the nuclear compartment. An elevated content of nuclear AHR could favor AHR-ARNT heterodimers able to bind to xenobiotic-responsive elements and to induce gene transcription in the absence of xenobiotics. Thus, depending on the cellular context, physiologically regulated proteasome activity could participate in the control of endogenous AHR functions.

The Dioxin Receptor Regulates the Constitutive Expression of the Vav3 Proto-Oncogene and Modulates Cell Shape and Adhesion

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.

Overexpression of latent transforming growth factor-β binding protein 1 (LTBP-1) in dioxin receptor-null mouse embryo fibroblasts

The aryl hydrocarbon receptor (AhR) is a transcriptional regulator of genes involved in xenobiotic metabolism. Increasingly clear is also the role of the AhR in the control of cell growth and proliferation. By analyzing differential patterns of gene expression between wild-type (AhR+/+) and null (AhR–/–) mouse embryo fibroblasts (MEF), we have identified latent transforming growth factor-β binding protein 1 (LTBP-1) as a negatively AhR-regulated gene in the absence of xenobiotics. Ltbp-1 mRNA and protein expression were markedly increased in AhR–/– MEF. Furthermore, secreted LTBP-1 was elevated in the culture medium and the extracellular matrix of AhR-null MEF. Actinomycin D inhibited Ltbp-1 mRNA overexpression, suggesting regulation at the transcriptional level. AhR activation by dioxin (TCDD) downregulated Ltbp-1, again suggesting an AhR-regulated mechanism. Treatment of AhR+/+ MEF with transforming growth factor-β(TGF-β) downregulated AhR and, simultaneously, increased Ltbp-1, further supporting the role of this receptor in LTBP-1 expression. AhR–/– conditioned medium had higher levels of active and total TGF-β activity, suggesting a role for LTBP-1 in maintaining extracellular TGF-β concentrations. TGF-β did not appear to directly regulate Ltbp-1 given that addition of TGFβ neutralizing antibody or TGFβ protein to AhR–/– MEF had no effect on Ltbp-1 expression. AhR–/– MEF had lower levels of matrix metalloproteinase 2 (MMP-2) activity, which could not be attributable to MMP-2 mRNA downregulation or MMP-inhibitors Timp-1 and Timp-2 overexpression. These data identify LTBP-1 as one of the few AhR-regulated genes not involved in xenobiotic metabolism and also support the implication of the AhR in controlling TGFβ activity and cell proliferation.

New Trends in Aryl Hydrocarbon Receptor Biology

Traditionally considered as a critical intermediate in the toxic and carcinogenic response to dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin, TCDD), the Aryl hydrocarbon/Dioxin receptor (AhR) has proven to be also an important regulator of cell physiology and organ homeostasis. AhR has become an interesting and actual area of research mainly boosted by a significant number of recent studies analyzing its contribution to the proper functioning of the immune, hepatic, cardiovascular, vascular and reproductive systems. At the cellular level, AhR establishes functional interactions with signaling pathways governing cell proliferation and cell cycle, cell morphology, cell adhesion and cell migration. Two exciting new aspects in AhR biology deal with its implication in the control of cell differentiation and its more than likely involvement in cell pluripotency and stemness. In fact, it is possible that AhR could help modulate the balance between differentiation and pluripotency in normal and transformed tumor cells. At the molecular level, AhR regulates an increasingly large array of physiologically relevant genes either by traditional transcription-dependent mechanisms or by unforeseen processes involving genomic insulators, chromatin dynamics and the transcription of mobile genetic elements. AhR is also closely related to epigenetics, not only from the point of view of target gene expression but also with respect to its own regulation by promoter methylation. It is reasonable to consider that deregulation of these many functions could have a causative role, or at least contribute to, human disease. Consequently, several laboratories have proposed that AhR could be a valuable tool as diagnostic marker and/or therapeutic target in human pathologies. An additional point of interest is the possibility of regulating AhR activity by endogenous non-toxic low weight molecules agonist or antagonist molecules that could be present or included in the diet. In this review, we will address these molecular and functional features of AhR biology within physiological and pathological contexts.

LTBP-1 blockade in dioxin receptor-null mouse embryo fibroblasts decreases TGF-β activity : Role of extracellular proteases plasmin and elastase

In mouse embryonic fibroblasts (MEF) lacking dioxin receptor (AhR), high levels of latent transforming growth factor‐β (TGF‐β)‐binding protein‐1 (LTBP‐1) correlated with increased TGF‐β1 activity, an observation suggesting that LTBP‐1 could contribute to maintain TGF‐β1 levels. Here, using small interfering RNAs (siRNA), we have first analyzed if LTBP‐1 expression affected TGF‐β1 activity in MEF cells. We have then determined how LTBP‐1 levels could alter the activity of extracellular proteases known to activate TGF‐β1, and finally, whether protease inhibition could reduce TGF‐β1 activation. LTBP‐1 inhibition by siRNA in AhR−/− MEF decreased the amount of active TGF‐β1 and reduced plasminogen activators (PA)/plasmin and elastase activities and thrombospondin‐1 (TSP‐1) expression, without significantly affecting their mRNA levels. On the contrary, LTBP‐1 siRNA restored matrix metalloproteinase‐2 (MMP‐2) activity in AhR−/− MEF. Interestingly, whereas a TGF‐β1 neutralizing antibody mimicked many of the LTBP‐1 siRNA effects on extracellular proteases, addition of recombinant TGF‐β1 protein increased proteases activity over basal levels in AhR−/− MEF. These proteases contributed to TGF‐β activation since their specific inhibitors reduced active TGF‐β levels in these cells. These results suggest that LTBP‐1 contributes to TGF‐β1 activation in MEF, possibly by influencing the activities of PA/plasmin, elastase, TSP‐1, and MMP‐2. TGF‐β1, on the other hand, could be also involved in maintaining the activity of these extracellular proteases. Thus, LTBP‐1 appears to play a role in TGF‐β1 activation through a process involving extracellular protease activities, which, in turn, could be affected by TGF‐β1 levels. J. Cell. Biochem.

Myeloid Dysregulation in a Human Induced Pluripotent Stem Cell Model of PTPN11-Associated Juvenile Myelomonocytic Leukemia

Somatic PTPN11 mutations cause juvenile myelomonocytic leukemia (JMML). Germline PTPN11 defects cause Noonan syndrome (NS), and specific inherited mutations cause NS/JMML. Here, we report that hematopoietic cells differentiated from human induced pluripotent stem cells (hiPSCs) harboring NS/JMML-causing PTPN11 mutations recapitulated JMML features. hiPSC-derived NS/JMML myeloid cells exhibited increased signaling through STAT5 and upregulation of miR-223 and miR-15a. Similarly, miR-223 and miR-15a were upregulated in 11/19 JMML bone marrow mononuclear cells harboring PTPN11 mutations, but not those without PTPN11 defects. Reducing miR-223s function in NS/JMML hiPSCs normalized myelogenesis. MicroRNA target gene expression levels were reduced in hiPSC-derived myeloid cells as well as in JMML cells with PTPN11 mutations. Thus, studying an inherited human cancer syndrome with hiPSCs illuminated early oncogenesis prior to the accumulation of secondary genomic alterations, enabling us to discover microRNA dysregulation, establishing a genotype-phenotype association for JMML and providing therapeutic targets.

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-Tyr(416) activation and focal adhesion kinase (FAK) phosphorylation at the c-Src-responsive residues p-Tyr(576) and p-Tyr(577). The c-Src target and migration-related protein Cav1 was also hypophosphorylated at p-Tyr(14) 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.