ROS production is essential for the apoptotic function of E2F1 in pheochromocytoma and neuroblastoma cell lines

In this study we demonstrate that accumulation of reactive oxygen species (ROS) is essential for E2F1 mediated apoptosis in ER-E2F1 PC12 pheochromocytoma, and SH-SY5Y and SK-N-JD neuroblastoma stable cell lines. In these cells, the ER-E2F1 fusion protein is expressed in the cytosol; the addition of 4-hydroxytamoxifen (OHT) induces its translocation to the nucleus and activation of E2F1target genes. Previously we demonstrated that, in ER-E2F1 PC12 cells, OHT treatment induced apoptosis through activation of caspase-3. Here we show that caspase-8 activity did not change upon treatment with OHT. Moreover, over-expression of Bcl-xL arrested OHT-induced apoptosis; by contrast, over-expression of c-FLIP, did not have any effect on OHT-induced apoptosis. OHT addition induces BimL expression, its translocation to mitochondria and activation of Bax, which is paralleled by diminished mitochondrial enrichment of Bcl-xL. Treatment with a Bax-inhibitory peptide reduced OHT-induced apoptosis. These results point out the essential role of mitochondria on the apoptotic process driven by E2F1. ROS accumulation followed E2F1 induction and treatment with the antioxidant N-acetylcysteine, inhibited E2F1-induced Bax translocation to mitochondria and subsequent apoptosis. The role of ROS in mediating OHT-induced apoptosis was also studied in two neuroblastoma cell lines, SH-SY5Y and SK-N-JD. In SH-SY5Y cells, activation of E2F1 by the addition of OHT induced ROS production and apoptosis, whereas over-expression of E2F1 in SK-N-JD cells failed to induce either response. Transcriptional profiling revealed that many of the genes responsible for scavenging ROS were down-regulated following E2F1-induction in SH-SY5Y, but not in SK-N-JD cells. Finally, inhibition of GSK3β blocked ROS production, Bax activation and the down regulation of ROS scavenging genes. These findings provide an explanation for the apparent contradictory role of E2F1 as an apoptotic agent versus a cell cycle activator

V-ATPase, a master effector of E2F1-mediated lysosomal trafficking, mTORC1 activation and autophagy

In addition to being a master regulator of cell cycle progression, E2F1 regulates other associated biological processes, including growth and malignancy. Here, we uncover a regulatory network linking E2F1 to lysosomal trafficking and mTORC1 signaling that involves v-ATPase regulation. By immunofluorescence and time-lapse microscopy we found that E2F1 induces the movement of lysosomes to the cell periphery, and that this process is essential for E2F1-induced mTORC1 activation and repression of autophagy. Gain- and loss-of-function experiments reveal that E2F1 regulates v-ATPase activity and inhibition of v-ATPase activity repressed E2F1-induced lysosomal trafficking and mTORC1 activation. Immunoprecipitation experiments demonstrate that E2F1 induces the recruitment of v-ATPase to lysosomal RagB GTPase, suggesting that E2F1 regulates v-ATPase activity by enhancing the association of V0 and V1 v-ATPase complex. Analysis of v-ATPase subunit expression identified B subunit of V0 complex, ATP6V0B, as a transcriptional target of E2F1. Importantly, ATP6V0B ectopic-expression increased v-ATPase and mTORC1 activity, consistent with ATP6V0B being responsible for mediating the effects of E2F1 on both responses. Our findings on lysosomal trafficking, mTORC1 activation and autophagy suppression suggest that pharmacological intervention at the level of v-ATPase may be an efficacious avenue for the treatment of metastatic processes in tumors overexpressing E2F1

E2F1 Regulates Cellular Growth by mTORC1 Signaling

During cell proliferation, growth must occur to maintain homeostatic cell size. Here we show that E2F1 is capable of inducing growth by regulating mTORC1 activity. The activation of cell growth and mTORC1 by E2F1 is dependent on both E2F1's ability to bind DNA and to regulate gene transcription, demonstrating that a gene induction expression program is required in this process. Unlike E2F1, E2F3 is unable to activate mTORC1, suggesting that growth activity could be restricted to individual E2F members. The effect of E2F1 on the activation of mTORC1 does not depend on Akt. Furthermore, over-expression of TSC2 does not interfere with the effect of E2F1, indicating that the E2F1-induced signal pathway can compensate for the inhibitory effect of TSC2 on Rheb. Immunolocalization studies demonstrate that E2F1 induces the translocation of mTORC1 to the late endosome vesicles, in a mechanism dependent of leucine. E2F1 and leucine, or insulin, together affect the activation of S6K stronger than alone suggesting that they are complementary in activating the signal pathway. From these studies, E2F1 emerges as a key protein that integrates cell division and growth, both of which are essential for cell proliferation

Study of the molecular mechanisms responsible for E2F1-induced Mtorc1 activation

[spa] Además de controlar la proliferación celular, E2F1 regula otros procesos biológicos asociados con la progresión del cáncer. En particular, resultados previos de nuestro grupo han demostrado que E2F1 induce el crecimiento celular mediante la activación de la vía de transducción de mTORC1. Teniendo en cuenta el papel central de esta vía en el cáncer, el objetivo de la Tesis ha sido estudiar los mecanismos moleculares que regulan la activación de mTORC1 inducida por E2F1. Este estudio nos ha permitido demostrar que E2F1 es un regulador del tráfico endosomal, de la actividad de la V-ATPase, el principal regulador del pH lisosomal, y de la autofagia. Mediante la activación de la V-ATPase, E2F1 induce la activación de mTORC1, el movimiento de los lisosomas hacia la periferia de la célula y la represión de la autofagia. Nuestros resultados en concreto han demostrado que la sobre-expresión de E2F1 induce la translocación de mTORC1 a los lisosomas y promueve su asociación con la proteína lisosomal RagB. El movimiento periférico de los lisosomas regulado por E2F1 no depende de la actividad de mTORC1, pero requiere la presencia de Raptor. La capacidad de E2F1 de reprimir la autofagia está mediada probablemente por la activación de mTORC1 y el movimiento de los lisosomas hacia la periferia celular. También hemos identificado la kinesina KIF2A cómo una nueva diana transcripcional de E2F1. Aunque esta kinesina es necesaria para la activación de mTORC1, el aumento de los niveles de KIF2A no es responsable de la activación de mTORC1 inducida por E2F1. En paralelo, E2F1 aumenta la actividad de V-ATPasa, y esta modulación es necesaria para la activación de mTORC1 y el tráfico lisosomal inducido por E2F1. E2F1 induce la asociación de RagB con la subunidad C1 V1 de la V-ATPasa (ATP6V1C1). Esta unión podría ser el mecanismo a través del cual E2F1 activa la V-ATPasa y mTORC1. Por último, E2F1 regula el ensamblaje del citoesqueleto y es necesario para la migración celular. Nuestros datos sobre el papel de E2F1 en la migración celular concuerdan con la propiedad ya descrita de E2F1 como un promotor de invasividad. Teniendo en cuenta el rol de la V-ATPase en la invasividad, nuestras observaciones relativas a la capacidad de E2F1 de regular el movimiento periférico de lisosomas y de activar la V-ATPasa nos ayudan a entender el papel de E2F1 en la invasión y metástasis.[eng] The oncogenic properties of E2F1 have been traditionally associated with the role of the oncogene in proliferation, but during the last few years many other functions associated with this family of transcription factors have been emerging. We focused on the study of the oncogenic properties of E2F1 not related to the cell cycle progression. In particular, previous results of our research group demonstrated that the overexpression of mammal E2F1 induces cellular growth by activating the mTORC1 pathway. Taking into account the well known implication of the mTOR pathway in cancer, the general aim of the Thesis is to elucidate the molecular mechanism by which E2F1 regulates mTORC1. This led us to novel observations concerning the ability of E2F1 in regulating V-ATPase activity, intracellular trafficking and autophagy repression. Specifically, we demonstrated that E2F1 enhances the activity of V-ATPase, the major regulator of lysosomal pH. By modulating this activity, E2F1 is capable of regulating lysosomal biology. This leads to the activation of mTORC1, to the relocalization of lysosomes to the cell periphery and to the repression of the autophagy flux. We showed that, similarly to the amino acids signaling, E2F1 activation pro- motes the translocation of mTOR to lysosomes and also induces an increase in the binding of mTORC1 to the lysosomal protein RagB. Our immunofluorescence analysis revealed that mTORC1 activity is not necessary for the peripheral movement of lysosomes regulated by E2F1. However, the depletion of Raptor totally abrogates this response. The ability of E2F1 to act as an autophagy repressor is due to its capacity in driving lysosomes to cell periphery and also in activating mTORC1. Moreover, we identified kinesin KIF2A as a novel transcriptional target of E2F1. Although KIF2A basal levels are required for E2F1-induced mTORC1 activation, the increase in KIF2A levels triggered by E2F1 does not contribute to mTORC1 activation. E2F1 enhances V-ATPase activity, and this modulation is required for E2F1-induced lysosomal trafficking and mTORC1 activation. E2F1 promotes an increase in the binding of the C1 subunit of the V1 domain, ATP6V1C1, to the V-ATPase/RagB lysosomal complex, suggesting that such binding could be the mechanism through which E2F1 enhances V-ATPase activity. Finally, we showed that E2F1 activation leads to a general re-arrangement of the cytoskeleton and that, moreover, is required to promote cell migration. Several studies indicated a high correlation between E2F1 overexpression and metastasis. Our data showing that E2F1 is required for cell migration are in agreement with the theory of E2F1 being a promotor of invasiveness. Taking into account the implication of V-ATPase in cancer, our novel observations concerning the ability of E2F1 to regulate the peripheral movement of lysosomes and to enhance V-ATPase activity help us to better understand the role of E2F1 in invasion and metastasis

E2F1 regulates cellular growth by mTOR signaling

During cell proliferation, growth must occur to maintain homeostatic cell size. Here we show that E2F1 is capable of inducing growth by regulating mTORC1 activity. The activation of cell growth and mTORC1 by E2F1 is dependent on both E2F1's ability to bind DNA and to regulate gene transcription, demonstrating that a gene induction expression program is required in this process. Unlike E2F1, E2F3 is unable to activate mTORC1, suggesting that growth activity could be restricted to individual E2F members. The effect of E2F1 on the activation of mTORC1 does not depend on Akt. Furthermore, over-expression of TSC2 does not interfere with the effect of E2F1, indicating that the E2F1-induced signal pathway can compensate for the inhibitory effect of TSC2 on Rheb. Immunolocalization studies demonstrate that E2F1 induces the translocation of mTORC1 to the late endosome vesicles, in a mechanism dependent of leucine. E2F1 and leucine, or insulin, together affect the activation of S6K stronger than alone suggesting that they are complementary in activating the signal pathway. From these studies, E2F1 emerges as a key protein that integrates cell division and growth, both of which are essential for cell proliferation

ROS production is essential for the apoptotic function of E2F1 in pheochromocytoma and neuroblastoma cell lines

In this study we demonstrate that accumulation of reactive oxygen species (ROS) is essential for E2F1 mediated apoptosis in ER-E2F1 PC12 pheochromocytoma, and SH-SY5Y and SK-N-JD neuroblastoma stable cell lines. In these cells, the ER-E2F1 fusion protein is expressed in the cytosol; the addition of 4-hydroxytamoxifen (OHT) induces its translocation to the nucleus and activation of E2F1target genes. Previously we demonstrated that, in ER-E2F1 PC12 cells, OHT treatment induced apoptosis through activation of caspase-3. Here we show that caspase-8 activity did not change upon treatment with OHT. Moreover, over-expression of Bcl-xL arrested OHT-induced apoptosis; by contrast, over-expression of c-FLIP, did not have any effect on OHT-induced apoptosis. OHT addition induces BimL expression, its translocation to mitochondria and activation of Bax, which is paralleled by diminished mitochondrial enrichment of Bcl-xL. Treatment with a Bax-inhibitory peptide reduced OHT-induced apoptosis. These results point out the essential role of mitochondria on the apoptotic process driven by E2F1. ROS accumulation followed E2F1 induction and treatment with the antioxidant N-acetylcysteine, inhibited E2F1-induced Bax translocation to mitochondria and subsequent apoptosis. The role of ROS in mediating OHT-induced apoptosis was also studied in two neuroblastoma cell lines, SH-SY5Y and SK-N-JD. In SH-SY5Y cells, activation of E2F1 by the addition of OHT induced ROS production and apoptosis, whereas over-expression of E2F1 in SK-N-JD cells failed to induce either response. Transcriptional profiling revealed that many of the genes responsible for scavenging ROS were down-regulated following E2F1-induction in SH-SY5Y, but not in SK-N-JD cells. Finally, inhibition of GSK3β blocked ROS production, Bax activation and the down regulation of ROS scavenging genes. These findings provide an explanation for the apparent contradictory role of E2F1 as an apoptotic agent versus a cell cycle activator

mRNA levels changes after OHT addition in SH-SY5Y cells.

<p>RT²Profiler human oxidative stress and antioxidant defense PCR Arrays (Bioscience) were performed according to the manufacture’s protocols. Expression levels were compared between with and without OHT addition. Hypoxanthine phosphoribosyltransferase 1 gene was used as control for each gene expression calculation, and the extent of change in the expression of each gene was calculated by the ΔC_t method. Only genes whose expression was downregulated at least 2 fold are shown in the Table. We have also removed all genes whose expression change significantly between duplicates. When ΔC_t was over 12 and therefore expression was thought to be extremely low, the gene was omitted from analysis.</p

E2F1 enhanced ROS production is essential for the apoptotic function.

<p>(A) PC12 ER-E2F1 cells were serum deprived and treated with OHT for the indicated hours. ROS levels were analysed by using the oxidation-sensitive fluorescent probe H2DCFDA as described in Material and Methods. Results are presented as Mean ± SEM, for n = 6 (B) Stable ER-E2F1 PC12 cells were treated with (+) or without OHT in the presence (+) or in the absence (−) of 1 mM NAC, or in the presence (+) or in the absence of 40 mM LiCl or serum for 4 hours. After cell collection and lysis, caspase-3 activity of cell extracts was analysed by using the <i>p</i>NA colorimetric assay as indicated in Material and Methods. Results are presented as Mean ± SEM, for n = 3. (C) ER-E2F1 cells were transiently transfected with YFP-Bax, treated for 3 hours with (+) or without (−) OHT in the presence (+) or absence (−) of 1 mM NAC, and the punctuated mitochondrial clusters of YFP-Bax were quantified by immunostaining analysis. Results are presented as Mean ± SEM, for n = 3. In all of the figures, data are compared as indicated individually. Student’s <i>t</i>-test values of *p<0,05, **p<0,01 and ***p<0,0001 were considered statistically significant.</p

LiCl inhibits ROS production and Bax activation induced by E2F1.

<p>(A) PC12 ER-E2F1 cells were serum deprived and treated with OHT (+) or not (−) with OHT, in the presence (+) or in the absence (−) of 40 mM LiCl for 4 hours. ROS levels were analysed by using the oxidation-sensitive fluorescent probe H2DCFDA as described in Material and Methods. Results are presented as Mean ± SEM, for n = 4. (B) PC12 ER-E2F1 cells were transiently transfected with an YFP-Bax plasmid, serum deprived and treated with OHT (+) or not (−) with OHT, in the presence or absence of 40 mM LiCl for 3 hours. Quantification of the punctuated mitochondrial clusters of YFP-Bax achieved in the immunofluorescence assay is described in Material and Methods. Results are presented as Mean ± SEM, for n = 3. In all of the figures, data are compared as indicated individually. Student’s <i>t</i>-test values of *p<0,05 and **p<0,01were considered statistically significant.</p