[CANCER RESEARCH 62, 1489 –1495, March 1, 2002]
A New Player in Oncogenesis: AUF1/hnRNPD Overexpression Leads to
Tumorigenesis in Transgenic Mice1
Agnès Gouble, Solène Grazide, Fabienne Meggetto, Pascale Mercier, Georges Delsol, and Dominique Morello2
Centre de Biologie du Développement, CNRS-UMR5547, Université Paul Sabatier, Bâtiment 4R3, 31062 Toulouse, Cedex 4 [A. G., D. M.]; Institut Claudius Régaud, INSERM
E9910, 20 rue du Pont Saint Pierre, 31052 Toulouse [S. G.]; UPR CNRS 2163 and Service d’Anatomie et de Cytologie Pathologiques, Hôpital Purpan, 31059 Toulouse, Cedex
[F. M., G. D.]; Institut de Pharmacologie et de Biologie Structurale, UMR 5089, 31062 Toulouse, Cedex [P. M.], France
ABSTRACT
AUF1/heterogeneous nuclear ribonucleoprotein D (hnRNPD) binds
to adenylate uridylate-rich elements contained in the 3ⴕ untranslated
region of many short-lived mRNAs. This binding has been shown
in vitro to control the stability of adenylate uridylate-rich elementcontaining mRNAs, including mRNAs encoding proto-oncogenes,
cytokines, or other signaling molecules. However, no studies have yet
been undertaken to identify the mRNAs subject to AUF1-mediated
regulation in vivo. The purpose of our study was to investigate the
biological functions of AUF1. Thus, we derived transgenic (Tg) mice,
which overexpress one isoform of AUF1, the p37AUF1. Mice of the three
Tg lines analyzed exhibit altered levels of expression of several target
mRNAs, such as c-myc, c-jun, c-fos, granulocyte macrophage colonystimulating factor, and tumor necrosis factor ␣. The Tg line with the
highest amount of Tg p37AUF1 protein developed sarcomas. The tumors
strongly expressed AUF1 Tg protein and Cyclin D1. Taken together,
our data show that: (a) AUF1 is a key regulatory factor of gene
expression in vivo; and (b) the deregulation of this heterogeneous
nuclear ribonucleoprotein leads to tumorigenesis.
INTRODUCTION
The expression of many genes involved in growth regulation,
including proto-oncogenes (such as c-fos, c-myc, and c-jun),
growth factors, and their receptors (GM-CSF3 and VEGF), cytokines (TNF␣), and cell cycle regulatory genes (cyclin A, B1, and
D1, p21), is mainly controlled by modulation of their mRNA
stability (1–7). This regulation is largely exerted through the
interaction of RNA-binding proteins with the ARE contained in
their 3⬘ UTR. The list of mRNAs containing the ARE has considerably increased with genome sequencing programs, as attested by
the recent construction of an ARE-containing mRNA database (8).
AUF1/hnRNPD was the first purified protein shown to mediate
ARE-directed mRNA degradation in vitro (9 –11). Subsequently,
other AUBPs were characterized, such as HuR, an ubiquitously
expressed member of the ELAV family of ribonucleoproteins (12,
13), which enhances the stability of ARE-containing mRNA (5, 6,
14 –16), and tristetraprolin, a member of a class of Cys-Cys-CysHis zinc finger proteins, which promotes deadenylation and degradation of TNF␣ and GM-CSF mRNAs (17, 18).
The mechanism(s) underlying AUBPs activities is the subject of
Received 8/15/01; accepted 1/3/02.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
1
Supported by ARC (Association pour la Recherche sur le Cancer) contract n° 9842,
ARECA network “Pôle protéomique et Cancer,” Conseil Régional, and CNRS. S. Grazide
and A. Gouble are doctoral students supported by the ARC.
2
To whom requests for reprints should be addressed, at Centre de Biologie du
Développement, CNRS-UMR5547, Université Paul Sabatier, Bâtiment 4R3, 118 Route de
Narbonne, 31062 Toulouse, Cedex 4, France. Phone: 33 5 61 55 64 73; Fax:
33 5 61 55 65 07; E-mail: [email protected].
3
The abbreviations used are: GM-CSF, granulocyte macrophage colony-stimulating
factor; RT-PCR, reverse transcription-PCR; TCR, T-cell receptor; VEGF, vascular endothelial growth factor; TNF, tumor necrosis factor; ARE, adenylate uridylate-rich element;
RTQ, real-time quantitative; WT, wild-type; UTR, untranslated region; Endo, endogeneous; AUBP, adenylate uridylate-rich element-RNA-binding proteins; Tg, transgene;
HE, hematoxylin-eosin; hnRNPD, heterogeneous nuclear ribonucleoprotein D.
intensive study. On the one hand, they rely on the nature of the ARE
with which they interact. Indeed, AREs of different mRNAs vary in
several aspects, such as their length, the proportion of uridylate
residues, the number, and the distance between the AUUUA pentameric repeats. These characteristics, as well as the mechanisms through
which they confer mRNA instability, have been the basis of their
classification into three categories (2). Thus, overlapping AUUUA
repeats are found in the class II AREs, included in the 3⬘ UTR of
mRNAs encoding cytokines/lymphokines or inflammatory mediators,
whereas these repeats are dispersed in class I AREs and absent in class
III AREs (2). On the other hand, the assembly of a protein complex on
a given ARE is controlled not only by the relative abundance and
affinity of the different AUBPs toward the ARE but also by their
ability to elicit interactions with auxiliary proteins and form various
complexes that might play different roles in mRNA fate, including
localization, (de)stabilization, or translation. These characteristics
vary depending on the type and functional state of the cell under
analysis. This might be explained, at least in part, by the contribution
of different signaling pathways, which have been shown to play an
important role in stabilization of certain ARE-containing mRNAs
(19 –24) or in the control of mRNA translation (25).
Despite important information yielded by in vitro or ex vivo studies,
these systems are limited in predicting AUBP biological functions. To
reach this goal, we constructed Tg mice overexpressing AUF1. We
chose the p37AUF1 isoform, one of the four AUF1 isoforms resulting
from alternative pre-mRNA splicing (26, 27), because it has the
strongest affinity for AREs in vitro (27) and the highest destabilizing
effect on c-fos mRNA in K562 cells (10). We also noticed previously
that p37AUF1 is less abundant than the three other isoforms in different
tissues throughout development (28, 29). Widespread Tg expression
was ensured by the ubiquitously expressed ␤-actin regulatory
sequences. We show that in the three different Tg lines analyzed,
p37AUF1 overexpression correlates with decreased or increased abundance of several ARE-containing mRNAs, depending on the class of
ARE they contain. Concomitantly, an important lethality was observed in two of the three Tg lines, one of which developed sarcomas.
MATERIALS AND METHODS
Construction of p37 AUF1 Tg Mice. The 1-kb-long NcoI/AvaII fragment of the AUF1–3 plasmid, which encodes for the murine p37AUF1
protein (a gift from G. Brewer, Piscataway, NJ), was subcloned into pKS
(Stratagene). The pCS2 ⫹ MT plasmid (250 bp; a gift from H. Weintraub’s
laboratory) encoding five human c-Myc epitopes was cloned into pKSAUF1 in a phase with the open reading frame of p37 AUF1 to give the Myc
tag p37AUF1. The ClaI/AvaII-blunted fragment containing Myc-tagged-p37
AUF1 was inserted into HindIII-blunted pBAP plasmid between human
␤actin regulatory sequences and SV40 3⬘ UTR (30). A 5.4-kb-long ClaI/
ClaI fragment containing ␤actin/Myc-tagged-p37AUF1 sequences was microinjected into fertilized (CBA ⫻ C57Bl/6) ⫻ (CBA ⫻ C57Bl/6) oocytes to
obtain Tg founders (31), which were crossed with (CBA ⫻ C57Bl/6) WT
mice and then intercrossed or back-crossed to WT mice to derive Tg lines.
Proteins Extracts and Quantitative Western Blot Analysis. Protein extracts and quantitative Western blot analysis were performed as described
previously (29). The following antibodies were used: polyclonal rabbit anti-
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SARCOMAS IN ␤-ACTIN/AUF1 TRANSGENIC MICE
AUF1 antibody (1871; gift of G. Brewer) diluted 1/6000, monoclonal antiAUF1 antibody (5B9; gift of G. Dreyfuss, Philadelphia, PA) diluted 1/1000,
and monoclonal antihuman c-Myc antibody (9E10) diluted 1/600. The uniformity of loading was checked with the gelcode blue reagent (Pierce) and by
performing parallel immunoblots using anti-␣ actin antibodies (MAB1501;
Chemicon).
Immunohistochemical Analysis. Deparaffined sections (5 ␮m) of normal and pathological mouse tissue samples fixed in formol were stained
with H&E or subjected to immunostaining. Tg expression in adult tissues
and tumors was analyzed using the 9E10 antibody which reacts with myc
Fig. 1. Schematic representation of the myc-tagged p37AUF1 Tg. It contains 3 kb of 5⬘
tagged AUF1 protein. All tumors were also analyzed with a number of human ␤-actin noncoding sequences, the first exon, the first intron, and the beginning of
other antibodies, specific to or cross-reacting with mouse antigens, to the second exon of the human ␤-actin gene, the murine p37 cDNA, as well as the SV40
site. Five copies of the Myc tag have been inserted
determine the nature of undifferentiated tumors occurring in these animals. 3⬘ UTR, including a polyadenylation
in the NH2-terminal part of the p37AUF1 protein, which each contains an ATG translation
The panel for immunostaining included antibodies against high molecular initiation codon.
weight cytokeratins (KL1; Immunotech, Marseille; dilution 1/50), muscle
actin HHF35 (DAKO A/S; dilution 1/100), ␣ smooth muscle actin 1A5
(Immunotech; dilution 1/2), desmine (DAKO A/S; dilution 1/50), S100 RESULTS
protein (DAKO A/S; dilution 1/800), Neuron-Specific Enolase (DAKO
Design of ␤-actin/AUF1 Tg. The design of the ␤-actin/AUF1 Tg
A/S; dilution 1/150), lymphocyte-associated antigens, such as B (CD79a;
DAKO A/S; dilution 1/10) and T (CD3; DAKO A/S; dilution 1/2), and (Fig. 1) was based on three main considerations: (a) the p37 AUF1
mouse leukocyte common antigens (CD45R/B220; PharMingen; undilut- cDNA was placed under the control of human ␤-actin regulatory
ed). Cyclin D1 was detected using DCS-6 (dilution 1/25) antibody from sequences (including the promoter, the first noncoding exon and the
Novocastra. Immunostaining was revealed by the streptavidin-biotin- first intron) to drive Tg expression in a wide variety of tissues, as
peroxidase complex method using DAKO StrepABComplex/HRP Duet shown for other reporters (36, 37); (b) a tag corresponding to five
(Mouse/Rabbit) Kit (code no. K0492; DAKO A/S), without prior successive human c-Myc epitopes was fused in frame to the NH
2
trypsinization, as described elsewhere (32, 33). When necessary, immunoterminus of the p37AUF1 protein to discriminate between Tg and
staining on paraffin sections was performed using the method described by
endogenous AUF1 expression and to follow the Tg cellular localizaShi et al. (34), with some modifications (32).
tion; and (c) the p37AUF1 cDNA was linked to the late SV40 3⬘ UTR,
DNA Analysis. The hypothesis that the tumors could have a T or B
including
a polyadenylation site to avoid any possible post-transcriplymphoid origin was tested by a Southern blot analysis performed on DNA
tional
control
of Tg expression via AUF1 3⬘ UTR (38). Three different
extracted from different tumors and tail of the same animal. The DNAs were
Tg
lines,
AUF1-1,
-2, and -3, were derived (see “Materials and
EcoRI or PvuII restricted and probed with a DJ4 or TCR␣ probe, respectively,
Methods”), which carried between 5 and 10 copies of the Tg in their
which allows one to observe immunoglobulin heavy chain or TCR locus (35).
RNA Extraction and S1 Mapping Assay. Total RNAs were extracted genome, as revealed by Southern blot analysis (data not shown).
Widespread Overexpression of Tagged-AUF1 in Tg Lines. Exfrom different tissues of WT and Tg mice with the Trizol procedure following
manufacturer’s instructions (Life Technologies, Inc.). S1 mapping analysis pression of Tg was detected by Western blot analysis of proteins
was performed as described previously (35). The AUF1 probe used to quantify extracted from different adult tissues of each Tg line using the
Tg mRNAs corresponds to a 427-nt-long BamHI/BglII fragment, including the anti-Myc tag 9E10 antibody. A variable level of expression was
Myc tag and the first 183 nt of p37 AUF1 sequence. It hybridizes simultaobserved between tissues and individual lines. AUF1–1 mice exneously to Tg 427-nt long and Endo 183-nt long p37 and p42 mRNAs. The two pressed the highest amount of Tg in all of the 10 tissues analyzed,
bands observed for Tg mRNAs (Fig. 3) correspond to two alternative 3⬘ splice including the liver (Fig. 2 and data not shown). These results were
sites, one located at the junction between ␤actin intron 1 and exon 2 and the
supported by immunohistochemical analysis (data not shown).
other one in the Myc Tag sequence. Because of the presence of a translation
The Myc tag AUF1 protein was found to be expressed in endotheinitiation site in each one of the five Myc epitopes, the myc-tagged AUF1
lial cells of virtually all organs (heart, kidney, brain, intestine, stomproteins synthesized from both mature mRNA species will differ only in the
ach, lung, spleen, and liver). In addition, smooth muscle cells of the
number of myc epitopes in their NH2-terminal part.
RTQ RT-PCR. Each target mRNA amount was determined by RTQ intestine, stomach, and bronchus, as well as skeletal muscle cells and
RT-PCR using the appropriate primers and normalized on the basis of P0 heart muscle cells, were also positive with a predominant staining in
(ribosomal protein) mRNA level of expression. Data are indicated by 2-⌬⌬Ct the nucleus. Numerous cells in the brain and scattered lymphoid cells
in the spleen were also found to be stained. In the testis, seminiferous
values:
tubules showed Tg expression mainly in primary and secondary
2⫺[(Ct sample gene X ⫺ Ct sample P0) ⫺ (Ct calibrator gene X ⫺ Ct calibrator P0)]
where Ct indicates the number of cycles when DNA amplification is 3-fold spermatogonia.
the baseline (in linear amplification phase), calibrator corresponds to one of the
None of the available AUF1 antibodies recognized the myc-tagged
WT tissue sample.
AUF1 protein in Western blot analysis, a situation sometimes encounPrimers were chosen with the assistance of Primer Express (PE Applied tered with myc-tagged proteins [see e.g., ␤-Catenin (39) and mycBiosystems); their sequences are available on request.
tagged AUF14]; thus, we estimated the level of Tg overexpression
cDNA synthesis: reverse transcription of total RNA was performed using using an S1 nuclease protection assay with a probe hybridizing with
Superscript II RNase H⫺ reverse transcriptase (Life Technologies, Inc.) and both the Tg and the endogenous AUF1 mRNAs (see Fig. 3A and
random hexamer (250 ng of random hexamer were used for 2 ␮g of total
“Materials and Methods”). Depending on the tissue and line considRNA).
ered, the level of accumulation of Tg mRNA was 2–20 times that of
PCR amplification: PCR reactions were performed using a PE 5700 appathe sum of the endogenous p37 and p42 mRNAs, as illustrated for
ratus (PE Applied Biosystems) and the Syber Green master mix 2X (PE
Applied Biosystems) with 300 nM each primer in 25 ␮l of final reaction muscle cells in Fig. 3B. This experiment also verified that Tg expresvolume. Each appropriate diluted reverse transcript sample (5 ␮l) was used per sion did not alter endogenous AUF1 expression (Fig. 3B and 4A). This
result was confirmed by Western blot analysis showing that the
reaction. The thermal cycling conditions included an initial denaturation step
(95°C for 10 min) and 40 cycles (95°C for 15 s; 60°C for 1 min). Experiments
4
were performed in duplicates.
A-B. Shyu, personal communication.
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SARCOMAS IN ␤-ACTIN/AUF1 TRANSGENIC MICE
Fig. 2. AUF1–1 line expresses the highest level of p37AUF1 protein. Comparison of Tg
expression in the different AUF1 Tg lines by Western blot analysis. myc-tagged p37
AUF1 expression was analyzed in various tissues of WT and AUF1 Tg mice from each
line (AUF1–1, AUF1–2, and AUF1–3) using anti-Myc tag antibody (9E10). Total protein
extracts (20 ␮g) were used for each sample, except for liver, for which 100 ␮g were used.
Immunoblots were performed in parallel using anti-␣ actin antibodies to check for
uniformity of protein loading, as shown in an example for the lung at the top of the figure.
Fig. 3. Comparison of Tg and Endo AUF1 mRNA expression. An S1 nuclease
protection assay was performed using RNA extracted from the skeletal muscle from
WT and AUF1–1, AUF1–2, and AUF1–3 Tg mice and a Myc tag AUF1 uniformly
radiolabeled probe (Endo) (diagram in A), which hybridizes simultaneously to the Tg
and endogenous p37 and p42 mRNAs (Endo). The two bands observed for Tg mRNAs
(427- and 350-nt long) correspond to two alternative 3⬘ splice sites (3⬘SS; see
“Materials and Methods”).
quantity of the different p37, p40, p42, and p45 endogenous isoforms
and their relative abundance was identical between Tg and WT
protein extracts from various tissues (data not shown).
Increased Accumulation of Specific ARE-mRNAs in p37AUF1
Healthy Tg Mice. AUF1 was shown previously to bind to AREs
contained in the 3⬘ UTR of RNAs encoded by a variety of early
response genes (40). To determine whether this binding results in a
change in accumulation of target mRNAs in vivo, we examined
representative mRNAs of each different class of ARE, c-myc, c-fos,
and cyclin D1 for class I; GM-CSF and TNF␣ for class II; and c-jun
for class III. These experiments were performed by RTQ RT-PCR
using RNAs from WT and Tg tissues expressing high (brain, testis, or
spleen) or low (muscle and liver) levels of endogenous AUF1 proteins
(29). Except for a 2.8-fold increase in c-fos expression in the brain of
AUF1–1 mice, we did not observe modification in the expression of
the various representative ARE-mRNAs in tissues with high-endogenous AUF1 protein expression, in any AUF1 strain (data not shown).
By contrast, in tissues with low levels of endogenous AUF1 protein
and strong ␤-actin promoter activity, such as the skeletal muscle, we
observed, in all three Tg lines, a reproducible 2.5–7-fold increase of
c-myc, c-fos, and c-jun mRNA (Fig. 5, A and B), except c-fos in line
AUF1–2. These values are certainly underestimated because their
measure was realized from crude tissue extracts in which not 100% of
the cells expressed the Tg, as mentioned above. By contrast, no
significant modification of class II ARE-containing mRNAs was
found (data not shown). We also noticed a 2–3-fold increase in the
level of c-jun and c-myc mRNAs in the liver of AUF1–1 Tg mice, the
only strain which expressed the Tg in this organ (Figs. 2 and 5D).
Overall, these results showed that AUF1 overexpression was able to
increase the expression of class I and class III ARE-containing mRNA
in vivo.
AUF1–1 Tg Mice Developed Sarcomas. To see whether p37AUF1
Tg expression could lead to abnormalities, mice of the three Tg lines
were kept under observation for 1.5 years. Numerous mice died in the
AUF1–1 (27 of 90) and AUF1–3 (12 of 35) strains, most of them
between 2 and 3 months of age. Macroscopic examination of these
mice revealed atrophy of the spleen and thymus (10% of cases), a
phenotype that we have not yet analyzed in detail. Moreover, tumors
were observed in 50% of sick or dead AUF1–1 mice. All tumors but
one were diagnosed at a late stage of their development. Although
they originated from various tissues, such as esophagus (n ⫽ 1),
gastrointestinal tract (n ⫽ 4), pancreas (n ⫽ 1), bladder (n ⫽ 1), testis
(n ⫽ 2), lung (n ⫽ 1), ureter (n ⫽ 1), and soft tissue of the head and
neck (n ⫽ 3), all tumors shared similar morphological features in that
they showed high cellularity and rich vascularization. Large necrotic
areas were present in all cases. Overall, these tumors consisted of
medium-sized cells with round to oval nuclei, sometimes slightly
irregular, and with scanty cytoplasm. Mitosis were commonly observed. Despite the large size of the tumors, metastases were only
found once. Despite variable morphological features, areas consisting
of immature spindle cells, highly suggestive of a sarcoma (sarcomalike pattern), were observed in every case (Fig. 6, A and B).
In some areas, neoplastic cells seemed to be cohesive, suggesting a
tumor from epithelial cell origin (carcinoma-like pattern), whereas in
other areas, poorly cohesive malignant cells showed a patternless
arrangement, mimicking a large cell lymphoma (lymphoma-like pattern). However, Southern blot analysis using different TCR and immunoglobulin probes excluded a lymphoid origin (see “Materials and
Methods” and data not shown). This conclusion was confirmed by the
Fig. 4. Enhanced Tg expression leads to accumulation of AUF1 target mRNAs.
A, comparative analysis of AUF1 Tg expression. An S1 nuclease protection assay was
performed by using 20 ␮g of RNA extracted from the liver of WT (WT1 and WT2),
healthy (735 h and 737 h), or sick (F0 1 s and 563 s) AUF1–1 Tg mice and the Myc
tag AUF1 uniformly radiolabeled probe described in Fig. 3 and “Materials and
Methods.” In B, quantitative analysis of AUF1 target mRNAs. c-myc, c-fos, and c-jun
mRNA level of expression in the same RNA samples was quantified by RTQ RT-PCR.
The values are expressed relative to the values found in the liver of WT mouse,
arbitrarily expressed as 1.
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SARCOMAS IN ␤-ACTIN/AUF1 TRANSGENIC MICE
Fig. 5. Modulation of AUF1 target mRNA expression in tissues and tumors of AUF1–1 mice. Quantification by RTQ RT-PCR of expression of c-myc, c-fos, and cyclin D1 mRNAs
(class I ARE); TNF␣ and GM-CSF (class II ARE); and c-jun mRNA (class III ARE) in tissues from healthy or sick AUF1 Tg mice. A and B, values found in healthy muscle from
WT, AUF1–1, AUF1–2, and AUF1–3 mice and in AUF1–1 tumor; C, WT muscle, AUF1–1 muscle, and tumor; D, values found in the liver from WT and healthy or sick AUF1–1
mice and in the liver and tumor of L-PK-c-myc mice; E, values found in the spleen of healthy WT, AUF1–1, AUF1–2, and AUF1–3 mice and of sick AUF1–1 mice. The histograms
represent the 2 -⌬⌬Ct values found in the various Tg tissues expressed relative to 2 -⌬⌬Ct values found in the WT tissues, arbitrarily expressed as 1. In AUF1 tumors, the values are
expressed relative to the ones found in WT muscle. L-PK-c-myc tumors correspond to RNA extracted from hepatocarcinomas obtained in c-myc-overexpressing, Tg mice (46); the values
are expressed relative to that found in WT liver. n, the number of mice under analysis. The values corresponding to the liver of the sick F0 n° 1 mouse have not been included in these
histograms but are shown in Fig. 4B. Note that the scale is different in each histogram.
absence of reactivity of the tumor cells with a panel of antibodies
directed against lymphocyte-associated antigens (CD3, CD45, and
CD79a). Immunostaining with 9E10 antibody showed that all these
tumors were strongly and uniformly positive for expression of the
AUF1 Tg product (Fig. 6, C and D). This suggests a clonal origin of
the tumor and a direct correlation between Tg expression and neoplasia. The staining was mostly in the nucleus of neoplastic cells although a weak cytoplasmic staining was also noted (Fig. 6, C and D
and data not shown). Except for S100 protein, which was found to be
positive in a proportion of neoplastic cells, all of the other antibodies
directed against muscle actin (HHF-35), vimentin, neurone-specific
enolase, and cytokeratin were unreactive with neoplastic cells. A
weak but significant staining of some cells was noted with anti-␣
smooth muscle actin 1A5 antibody. Together with the lack of morphological features characteristic of an established category of malignant tumors, negative staining with a panel of specific antibodies led
us to make the diagnosis of “undifferentiated sarcoma, possibly of
smooth muscle cell origin.”
Modulation of AUF1 Target mRNA Expression in Different
Tissues of Mice Developing Tumors and in Tumors. We analyzed
by quantitative RT-PCR the level of expression of representative
ARE-containing mRNAs in apparently normal tissues (heart, spleen,
and liver) of sick or dead animals, as well as in their tumors. Because
we observed previously a synchronous expression of AUF1 and HuR,
we checked whether HuR level of expression could have been modified on p37AUF1 overexpression. However, we observed no change
neither in the muscle of healthy Tg mice nor in the five tumors
analyzed (from testis, neck, or bladder). Because most tumors were
highly vascularized, we also studied VEGF expression, a class III
ARE mRNA (8). However, we did not observe significant difference
in VEGF mRNA level of expression between tumors and muscle (data
not shown). By contrast, we noticed a 4- and 2-fold decrease in the
level of expression of TNF␣ and GM-CSF mRNAs, respectively, and
a 2-fold increase in c-fos mRNA expression in the spleen of sick mice
compared with the spleen of WT or healthy mice (Fig. 5E). In
addition, c-myc expression was increased 2–3-fold in the heart (data
not shown), and c-fos and c-jun mRNA expression was also considerably increased in the liver of tumor-bearing animals (mean of 12 and
7 times, respectively; Figs. 4B and 5D). This increase was directly
correlated with a high AUF1 Tg mRNA expression. Indeed, as revealed by S1 nuclease analysis, the liver of the two animals analyzed
in this experiment that developed tumors (F0 n° 1 and F2 n° 563), and
contained high amounts of myc-tagged AUF1 mRNA, also abundantly
expressed c-fos and c-jun mRNAs (Fig. 4, A and B).
Cyclin D1 overexpression has been described in various neoplasia
(reviewed in Ref. 41). Because cyclin D1 mRNA contains in its 3⬘
UTR an ARE which could be a target for AUF1 (7), we quantified by
RTQ RT-PCR analysis the level of cyclin D1 mRNA in the AUF1
tumors and observed a considerable increase (20 –50-fold) in its
expression compared with WT and AUF1–1 muscle (Fig. 5C). This
increase was accompanied by a strong expression of Cyclin D1
protein, as revealed by immunostaining (Fig. 6, E and F). No comparable staining was observed on adjacent healthy tissue nor on other
tissues of healthy Tg or WT mice (data not shown). This overexpression was not observed in healthy liver, spleen, and muscle of AUF1–1
Tg mice nor in hepatocarcinomas obtained in L-PK-c-myc Tg mice,
which overexpressed c-myc in the livers (Fig. 5, C and D and data not
shown). Cyclin D1 overexpression in AUF1–1 tumors was not because of cyclin D1 gene rearrangement or deletion as revealed by
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Fig. 6. Histopathological and phenotypic analysis of
AUF1–1 tumors. Examples of tumors arising in the
esophagus (A, C, and E) and the testis (B, D, and F). A
and B, histopathological features after H&E staining
(⫻500). C and D, analysis of the Tg expression in the
tumors by immunostaining with the anti-Myc tag antibody (⫻500). Note the strong nuclear and weaker cytoplasmic staining. E and F, immunostaining with antiCyclin D1 antibody (E, ⫻500; F, ⫻800). Note the strong
nuclear staining of virtually all neoplastic cells.
Southern blot analysis using 5⬘ and 3⬘ cyclin D1 probes (data not
shown).
DISCUSSION
In this study, we show that overexpression of p37AUF1, the isoform
with the highest affinity for AREs (27), modifies the accumulation
level of several ARE-containing mRNAs in vivo. Their increased or
decreased levels of expression can be correlated with a premature
death or occurrence of tumors.
AUF1/hnRNPD has been described ex vivo and in vitro as a protein
able to bind to the AREs contained in the 3⬘ UTR of a broad variety
of functionally different mRNAs (40) and participate in their destabilization (1, 4, 10, 11, 42– 44). Therefore, it was surprising to observe
that, in vivo, in various tissues of overexpressing AUF1 Tg mice, there
is an increase in the level of accumulation of mRNAs containing
either a class I ARE, such as c-fos and c-myc, or a class III ARE, like
c-jun. The only mRNAs whose expression is significantly decreased
are the GM-CSF and TNF␣, which both contain a class II ARE. The
importance of the class II ARE in the control of mRNA stability in
vivo has been demonstrated recently by the analysis of Tg mice in
which the ARE of the TNF␣ and GM-CSF was deleted (23, 45). This
deletion leads to an accumulation of TNF␣ and GM-CSF mRNAs,
which in turn results in immune disorders leading in the case of
GM-CSF to embryonic death (45). The inverse correlation observed between the level of GM-CSF mRNA accumulation and that
of AUF1 led to the postulate that AUF1 could play a major role in
the control of GM-CSF mRNA level of expression (45). Our results
indeed support this conclusion. It would now be interesting to test
whether the destabilizing activity of AUF1 is restricted to GM-CSF
and TNF␣ or if it affects also other class II ARE-containing RNAs,
such as those encoding interleukins, IFNs, or hematopoetic cell
growth factors (8). These experiments are in progress.
Our results indicate that depending on the class of ARE, the level
of p37AUF1 overexpression relative to the other isoforms and the
abundance and/or activity of the auxiliary factors with which it
interacts, AUF1 may act either as a stabilizing or a destabilizing
factor. Several lines of evidence show a direct correlation between
p37AUF1 overexpression and modulation of target mRNA expression:
(a) the modulation is only observed in tissues where the Tg expression
is high compared with endogenous AUF1; e.g., in muscle cells, where
endogenous AUF1 expression is very low (29) and the ␤-actin regulatory sequences are highly active, we found a significant and reproducible increase in the level of c-myc, c-fos, and c-jun mRNA expression; (b) this increase is independent of the Tg integration site as it is
observed in the three independent Tg lines; (c) in the liver of AUF1
tumor-bearing animals, the higher expression of the tested target
mRNAs correlates with the high level of p37AUF1 expression; and (d)
finally, none of the modifications observed in Tg normal or tumoral
tissues were found in WT tissues or in other models of tumors proved
to have normal AUF1 expression. In effect, we did not find any
modification of c-fos, c-jun, and cyclin D1 mRNA expression in the
hepatocarcinoma obtained in L-PK-c-myc Tg mice, which constitutively express very high levels of c-myc in their liver (46). Although
we have not analyzed in depth the effect of AUF1 on target mRNA
expression, we believe that it is likely post-transcriptional, because
there are many reports showing that AUF1 is an RNA-binding protein
affecting mRNA stability (1, 4, 9 –11, 42– 44, 47).
Despite various types of investigations, the precise classification
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SARCOMAS IN ␤-ACTIN/AUF1 TRANSGENIC MICE
of the AUF1-associated tumors has proven to be difficult. Although we made the diagnosis of undifferentiated sarcoma, the
weak staining for muscle-specific actin could indicate an undifferentiated leiomyosarcoma. This hypothesis is supported by the
strong activity of the ␤-actin regulatory sequences in muscle cells
from which these tumors are supposed to develop and which
exhibit a strong expression of the Tg.
In the AUF1 tumors, beside an accumulation of c-myc and c-fos
mRNA similar to that found in the muscle, there is a considerable
increase in cyclin D1 mRNA and protein expression. The correlation
between overexpression of a key regulator of the G1 phase of the cell
cycle and tumorigenesis has already been established not only in
mouse mammary tumor virus-cyclin D1 Tg mice, which develop
mammary adenocarcinomas (48), but also in various human neoplasia, including breast cancers, malignant lymphomas, and chronic
lymphocytic leukemia (reviewed in Ref. 41). In the latter malignancies, Cyclin D1 overexpression is associated with DNA rearrangement, corresponding either to a translocation, as documented in 40 –
70% of cases of mantle cell lymphomas (reviewed in Ref. 49), or a
small deletion in the 3⬘ UTR of cyclin D1 mRNA (50). Interestingly,
it was shown recently that this long 3⬘ UTR contains a 390-base
element that binds AUF1 (7). We cannot formally exclude the hypothesis of a transcriptional up-regulation of cyclin D1 expression.
However, because Cyclin D1 overexpression was detected in all tested
AUF1 tumors, a clonal regulatory mutation is unlikely, and we favor
the hypothesis of a trans-acting regulatory disturbance mediated by
AUF1 overexpression. Accumulation of this cell regulator could favor
rapid cell division, progressive loss of differentiation markers, and
tumorigenesis.
An analysis of the human ARE-containing mRNA database suggests that the proportion of mRNAs with AREs could be as high as
8% (8). Thus, there are many candidates for post-transcriptional
regulation by AUF1. Using the p37AUF1 Tg mice, we have now shown
for the first time that this heterogeneous nuclear ribonucleoprotein
acts as a trans-acting factor in vivo and that its overexpression is
associated with tumors. This Tg model thus constitutes an invaluable
tool to identify which of the potential AUF1 target mRNAs are
involved in physiological processes and/or in tumorigenesis.
ACKNOWLEDGMENTS
We thank Jacques Auriol for his help with mice and genetic screen. We also
thank Michel March for excellent technical work with tissue sections. We
thank Dr. Christine Perret for RNA samples from L-PK-c-myc Tg mice. We
also thank Dr. Martin Van Der Valk for his pertinent advice on tumor
characterization and Drs. Alain Vincent and Agamemnon J. Carpousis for
critical comments and suggestions on the manuscript. The quantitative RTPCR analysis was done in Marie-Paule Roth’s laboratory. The Tg mice were
derived in the Service Transgenèse Toulouse (CNRS). Immunohistochemical
analysis was realized using the “Plateforme d’histopathologie expérimentale.”
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A New Player in Oncogenesis: AUF1/hnRNPD Overexpression
Leads to Tumorigenesis in Transgenic Mice
Agnàs Gouble, Solàne Grazide, Fabienne Meggetto, et al.
Cancer Res 2002;62:1489-1495.
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