Induction of the Ras activator Son of Sevenless 1 by environmental pollutants mediates their effects on cellular proliferation.

Abstract

TCDD (2,3,7,8-tetrachlorodibenzodioxin), a highly persistent environmental pollutant and a human carcinogen, is the ligand with the highest affinity for the Aryl Hydrocarbon Receptor (AhR) that induces via the AhR, xenobiotic metabolizing enzyme genes as well as several other genes. This pollutant elicits a variety of systemic toxic effects, which include cancer promotion and diverse cellular alterations that modify cell cycle progression and cell proliferation. Large-scale studies have shown that the expression of Son of Sevenless 1 (SOS1), the main mediator of Ras activation, is one of the targets of dioxin in human cultured cells. In this study, we investigated the regulation of the previously uncharacterized SOS1 gene promoter by the AhR and its ligands in the human hepatocarcinoma cell line, HepG2. We found that several environmental pollutants (AhR ligands) induce SOS1 gene expression by increasing its transcription. Chromatin immunoprecipitation experiments demonstrated that the AhR binds directly and activates the SOS1 gene promoter. We also showed that dioxin treatment leads to an activated Ras-GTP state, to ERK activation and to accelerated cellular proliferation. All these effects were mediated by SOS1 induction as shown by knock down experiments. Our data indicate that dioxin-induced cellular proliferation is mediated, at least partially, by SOS1 induction. Remarkably, our studies also suggest that SOS1 induction leads to functional effects similar to those elicited by the well-characterized oncogenic Ras mutations.

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Accepted Manuscript
Title: Induction Of The Ras Activator Son Of Sevenless 1 By
Environmental Pollutants Mediates Their Effects On Cellular
Proliferation
Authors: St´ ephane Pierre, Anne-Sophie Bats, Aline Chevallier,
Linh-Chi Bui, Ariane Ambolet-Camoit, Mich` ele Garlatti,
Martine Aggerbeck, Robert Barouki, Xavier Coumoul
PII:
DOI:
Reference:
S0006-2952(10)00759-8
doi:10.1016/j.bcp.2010.10.003
BCP 10735
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3-10-2010
5-10-2010
Please cite this article as: Pierre S, Bats A-S, Chevallier A, Bui L-C, Ambolet-Camoit
A, Garlatti M, Aggerbeck M, Barouki R, Coumoul X, Induction Of The Ras Activator
Son Of Sevenless 1 By Environmental Pollutants Mediates Their Effects On Cellular
Proliferation, Biochemical Pharmacology (2010), doi:10.1016/j.bcp.2010.10.003
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peer-00649887, version 1 - 9 Dec 2011
Author manuscript, published in "Biochemical Pharmacology 81, 2 (2010) 304"
DOI : 10.1016/j.bcp.2010.10.003

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Title page

INDUCTION OF THE RAS ACTIVATOR SON OF SEVENLESS 1 BY
ENVIRONMENTAL POLLUTANTS MEDIATES THEIR EFFECTS ON CELLULAR
PROLIFERATION
Stéphane Pierre 1,2,‡, Anne-Sophie Bats 1,2, 3,‡, Aline Chevallier 1,2, Linh-Chi Bui 1,2,
Ariane Ambolet-Camoit 1,2, Michèle Garlatti1,2, Martine Aggerbeck 1,2, Robert Barouki
1,2, 4, Xavier Coumoul 1,2,†
1INSERM UMR-S 747, Toxicologie Pharmacologie et Signalisation Cellulaire, 45 rue des
Saints Pères, 75006 Paris France
2Université Paris Descartes, Centre universitaire des Saints-Pères, 45 rue des Saints Pères,
75006 Paris France
3AP-HP, Hôpital Européen Georges Pompidou, Service de Chirurgie Gynécologique
Cancérologique, 75015 Paris France
4AP-HP, Hôpital Necker-Enfants Malades, service de biochimie métabolique, Paris, France
‡ These authors contributed equally to this work.

Running title : Regulation of Ras activator SOS1 by dioxin.

†Address correspondance to : Xavier Coumoul, INSERM UMR-S 747 45 rue des Saints
Pères 75006 Paris France Phone: +33 1 42 86 33 59 Fax : +33 1 42 86 38 68 E-mail:
xavier.coumoul@parisdescartes.fr
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Abstract

TCDD (2,3,7,8-TetraChloroDibenzoDioxin), a highly persistent environmental pollutant
and a human carcinogen, is the ligand with the highest affinity for the Aryl
Hydrocarbon Receptor (AhR) that induces via the AhR, xenobiotic metabolizing enzyme
genes as well as several other genes. This pollutant elicits a variety of systemic toxic
effects, which include cancer promotion and diverse cellular alterations that modify cell
cycle progression and cell proliferation. Large-scale studies have shown that the
expression of Son of Sevenless 1 (SOS1), the main mediator of Ras activation, is one of
the targets of dioxin in human cultured cells. In this study, we investigated the
regulation of the previously uncharacterized SOS1 gene promoter by the AhR and its
ligands in the human hepatocarcinoma cell line, HepG2. We found that several
environmental pollutants (AhR ligands) induce SOS1 gene expression by increasing its
transcription. Chromatin Immunoprecipitation experiments demonstrated that the AhR
binds directly and activates the SOS1 gene promoter. We also showed that dioxin
treatment leads to an activated Ras-GTP state, to ERK activation and to accelerated
cellular proliferation. All these effects were mediated by SOS1 induction as shown by
knock down experiments. Our data indicate that dioxin-induced cellular proliferation is
mediated, at least partially, by SOS1 induction. Remarkably, our studies also suggest
that SOS1 induction leads to functional effects similar to those elicited by the well-
characterized oncogenic Ras mutations.






Keywords : Dioxin, AhR, SOS1, Ras, Cell proliferation
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expression of SOS proteins have not been addressed and little is known about the regulation
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1. Introduction
The Ras signalling pathway is involved in cell growth and differentiation. Mutations
in the Ras proto-oncogene have been described in a large variety of human malignancies [1].
These mutations result in a constitutively active Ras protein, which is one of the most
common Ras dysregulations in cancers [1, 2]. Three different Ras genes encoding the H-Ras,
K-Ras and N-Ras 21 kDa proteins were discovered 30 years ago [1, 3]. The activated Ras
proteins physically associate with and activate Raf-1, a serine/threonine kinase which triggers
a kinase cascade that results in the activation of the extracellular signal-regulated protein
kinases (ERKs of the MAPK family). Ras acts as a functional hub by activating multiple
downstream pathways that participate in cell growth and differentiation. The activity of Ras is
regulated by two sets of proteins: guanine-nucleotide exchange factors (GEFs) and GTPase-
activating proteins (GAPs). GEFs stimulate the release of the Ras bound GDP which then
allows GTP to bind and activate Ras. Conversely, GAPs stimulate Ras GTPase activity and
lead to the basal GDP-bound state.
Several pathways, which lead to the active Ras state, have been described in
mammalian cells. The best-characterized pathway employs the activation of tyrosine kinase
receptors to elicit binding of various proteins to the phosphorylated receptor, most notably the
growth factor receptor bound 2 (Grb2) protein bound to the guanine nucleotide exchange
factor SOS1. The complex is targeted to the plasma membrane, allowing the GEF function of
SOS1 to activate Ras by GDP/GTP exchange [4-6]. SOS1 can activate other GTP-binding
proteins [4] and other effectors also control Ras activity. The most extensively characterized
non-physiological conditions under which increased SOS activitiy has been observed result
from gain of function mutations. For example, SOS1 mutations have been described in the
Noonan syndrome, a developmental disorder that is characterized by short stature, facial
dysmorphia, congenital heart defects and skeletal anomalies [7, 8]. These gain of function
mutations, which lead to the activation of Ras and were the first examples of activating GEF
mutations associated with human disease. Surprisingly, the implications of increased
of the gene promoter.
Dioxin is a member of the PolyChlorinated dibenzo Dioxins (PCDD) family which is
a class of highly persistent environmental contaminants. 2,3,7,8-TetraChloroDibenzoDioxin
(TCDD), the most extensively characterized dioxin, has been classified in 1997 as a “human
carcinogen” by the International Agency for Research on Cancer (IARC) [9]. This pollutant is
the ligand with the greatest affinity for the Aryl Hydrocarbon Receptor (AhR). TCDD via the
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AhR, induces various toxicities including chloracne, wasting syndrome, teratogenicity,
immunotoxicity, neurotoxicity, tumor promotion and carcinogenesis [10]. The AhR is a
cytosolic, ligand-activated transcription factor which, upon activation, translocates to the
nucleus where it forms a heterodimer complex with AhR Nuclear Translocator (ARNT). This
complex binds to specific Xenobiotic Responsive Elements (XRE) which are characterized by
a 5’-GCGTG-3’ consensus core and induces specific target genes which include xenobiotic
metabolizing enzymes (XME) such as the cytochrome P450 (CYP) 1 family [11]. In addition
to detoxification functions, the activation of this receptor has been shown to elicit diverse
cellular effects including cell proliferation [12, 13]. These cellular alterations might mediate
part of the long-term toxicity of the AhR ligands in animals and humans [14]. Indeed, AhR-
null mice display several defects of development and proliferation including thymus and liver
hypotrophy [15]. Numerous studies using AhR defective cells or AhR invalidating strategies
have clearly established a role for this receptor in cell cycle regulation and proliferation [16].
The dysregulation of these cellular pathways may disrupt normal fetal development and favor
cancer progression. The AhR gene targets that are involved in the proliferative effects of this
receptor are not completely identified. To this end, we have examined large-scale
toxicogenomic analyses of dioxin effects and focused on regulatory proteins that control
cellular growth, in particular, the main mediator of Ras activation, SOS1.
In the present work, we investigated, for the first time, the regulation of the previously
uncharacterized SOS1 gene promoter by the AhR and its ligands in the human
hepatocarcinoma cell line, HepG2. We show that several environmental pollutants (AhR
ligands) induce SOS1 gene expression via increased transcription. Moreover, considering the
critical role of SOS1 in controlling Ras activity, we also demonstrate that induction of SOS1
by AhR ligands leads to a predominant Ras-GTP state, to MAPK activation and to cell
growth. The results point towards SOS1 gene induction as being a critical step for the
carcinogenic effects mediated by dioxins.
peer-00649887, version 1 - 9 Dec 2011