[Cancer Research 60, 3916-3920, July 15, 2000]
© 2000 American Association for Cancer Research
Akt/Protein Kinase B Promotes Survival and Hormone-independent Proliferation of Thyroid Cells in the Absence of Dedifferentiating and Transforming Effects1
Gabriella De Vita,
Maria Teresa Berlingieri,
Roberta Visconti,
Maria Domenica Castellone,
Giuseppe Viglietto,
Gustavo Baldassarre,
Mariastella Zannini,
Alfonso Bellacosa,
Philip N. Tsichlis,
Alfredo Fusco and
Massimo Santoro2
Centro di Endocrinologia ed Oncologia Sperimentale del Consiglio Nazionale delle Ricerche, c/o Dipartimento di Biologia e Patologia Cellulare e Molecolare, 80131 Naples, Italy [G. D. V., M. T. B., R. V., M. D. C., M. Z., M. S.]; Istituto Nazionale dei Tumori "Fondazione Senatore Pascale," 80131 Naples, Italy [G. V., G. B.]; Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111 [A. B.]; Kimmel Cancer Center, Jefferson Medical College, Philadelphia, Pennsylvania 19107 [P. N. T]; and Dipartimento di Medicina Sperimentale e Clinica, 88100 Catanzaro, Italy [A. F.].
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ABSTRACT
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The Akt/protein kinase B serine/threonine kinase is a downstream
effector of phosphoinositide 3-kinase (PI3K). Akt is an important
component of mitogenic and antiapoptotic signaling pathways and is
implicated in neoplastic transformation. Thyroid cells in culture
retain a differentiated phenotype consisting of epithelial cell
morphology and the expression of several tissue-specific genes. The
survival and proliferation of these cells depend on thyrotropin and a
mixture of five additional hormones that includes insulin. The
regulation of proliferation and the expression of the thyroid
differentiation program are intimately connected processes. As a
result, oncogenes that induce hormone-independent proliferation
invariably impair the expression of the thyroid-specific
differentiation markers. Given that thyrotropin and insulin stimulate
Akt activation in thyroid cells, we set out to determine the effects of
Akt on thyroid cell proliferation, survival, and differentiation. To
this end, we expressed constitutively active myristylated Akt (myrAkt)
in PC Cl 3 thyroid cells. The myrAkt-expressing cells continued to
proliferate, even in the absence of hormones, and they were resistant
to programmed cell death induced by starvation. These effects were
paralleled by the induction of the G1 cyclins D3 and E and
by the inhibition of induction of the proapoptotic Fas, Fas ligand, and
BAD genes in starved cells. However, in marked
contrast with several other oncogenes, myrAkt did not interfere with
the expression of thyroid differentiation functions. These results
unveil the existence of an Akt-triggered thyroid cell pathway that
modulates proliferation and survival without affecting the expression
of the thyroid cell differentiated phenotype.
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INTRODUCTION
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Akt, also referred to as protein kinase B, was originally
identified as the oncogene transduced by the acute transforming
retrovirus Akt-8 (1)
. Akt lies at a nodal point of
multiple cellular signal pathways. It is activated by
PI3K3
which, in turn, is activated by tyrosine kinases via its regulatory p85
subunit or by Ras via its catalytic p110 subunit. Binding of PI3K
products to the pleckstrin homology domain results in
translocation of Akt to the plasma membrane, where it is activated by
phosphorylation of Ser-473 and Thr-308 (2)
. Akt transduces
signals that regulate multiple processes, such as endocytosis,
vesicular trafficking, glucose utilization, apoptosis, and cellular
proliferation. The PI3K/Akt pathway inhibits apoptosis through multiple
mechanisms (3)
. Akt modulates BCL-2 family members by
inducing the expression of the antiapoptotic BCL-2 protein
(4)
and by phosphorylating the proapoptotic BAD
protein (5)
. Moreover, phosphorylation by Akt prevents
nuclear localization of the forkhead transcription factors FKHRL1,
FKHR, and AFX, thus reducing the expression of FasL, a proapoptotic
cytokine (6, 7)
. Finally, Akt rescues
G1 arrest induced by growth factor withdrawal;
this effect is mediated, at least in part, by the enhanced expression
of G1 cyclins (8, 9) .
Thyroid cells depend on TSH for proliferation (10)
. TSH
stimulates thyroid cell proliferation through PKA-dependent and
-independent pathways; PKA is, indeed, necessary but not sufficient to
stimulate thyroid cell proliferation (11)
. Recently, Cass
et al. (12)
reported that TSH stimulates
PKA-independent and PI3K-dependent Akt activation and that PI3K, in
turn, confers TSH-independent DNA synthesis to thyroid cells.
Furthermore, insulin, a well-known stimulator of the PI3K/Akt pathway,
is an important cofactor for thyroid cell proliferation
(10)
. These observations prompted us to examine the
effects of the expression of a constitutively active Akt in cultured
thyroid cells. PC Cl 3, a continuous line of Fischer rat thyroid cells,
constitutes a model to study differentiation and growth regulation in
an epithelial thyroid cell setting. PC Cl 3 cells express
differentiation markers and depend on a mixture of six hormones,
including TSH and insulin, for proliferation (13)
. The
maintenance of the thyroid differentiated phenotype and the dependence
on hormones for proliferation are strictly connected. Indeed, the
expression of different oncogenes causes the impairment of the
expression of differentiation markers and the hormone-independent
proliferation of PC Cl 3 cells (13–18)
. Here we show that
Akt delivers both mitogenic and survival signals in PC Cl 3 cells in
the absence of dedifferentiative effects. Thus, in thyroid cells
hormone-dependent proliferation can be dissociated from the expression
of differentiation markers.
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MATERIALS AND METHODS
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Plasmids and Antibodies.
The Tg, TPO, NIS, TTF-1,
and TTF-2 probes have been described previously
(19–22)
. The myrAkt construct is described elsewhere
(23)
; it contains the Akt coding sequence modified by the
addition of the Src myristylation signal and the HA epitope. The insert
was excised to be used as a probe for Northern blotting. Fas
and FasL cDNA probes were generated by reverse
transcription-PCR amplification and sequenced. The sequences were
obtained from the GenBank [accession numbers: D26112 (rat Fas); U03470
(rat FasL)]. The primers were as follows: Fas forward
(nucleotides 181–201), 5'-CAACTGCTCAGAAGGGTTAT-3' and Fasreverse (nucleotides 511–531), 5'-TTGCTGGTTCGTGTGCAAGGC-3';
FasL forward (nucleotides 731–751),
5'-GTGCTAATGGAGGAGAAGAA-3', and Fas reverse (nucleotides
1071–1091), 5'-TGATGCAGGCATTAAGGACCA-3'.
Anti-Akt and anti-phosphoAkt (Ser-473) polyclonal antibodies were from
New England Biolabs (Lake Placid, NY). Mouse monoclonal anti-HA epitope
antibodies (clone 12CA5) were from Boehringer Mannheim (Mannheim,
Germany). Antibodies for Sp1, BAD, BCL-2, cyclin D3 and E, and
secondary antibodies coupled to horseradish peroxidase were from Santa
Cruz Biotechnology (Santa Cruz, CA).
Cell Culture and Molecular Biology Techniques.
PC Cl 3 cells were grown in Coon’s modified F12 medium (Life
Technologies, Inc., Paisley, PA) supplemented with 5% calf serum (Life
Technologies, Inc.) and 6H (TSH, insulin, hydrocortisone, somatostatin,
transferrin, and glycyl-histidyl-lysine; (Sigma Chemical Co.) and
transfected as described elsewhere (13)
. For flow
cytometry, cells were harvested 48 h after reaching the confluence
or when subconfluent either in complete medium or in medium deprived of
the 6H for 96 h. Cells were fixed in methanol for 1 h at
-20°C, rehydrated in PBS for an additional hour at 4°C, and then
treated with RNase A (50 µg/ml) for 30 min. Propidium iodide (25
µg/ml) was added to the cells, and samples were analyzed with a
FACScan flow cytometer (Becton Dickinson, San Jose, CA) interfaced with
Hewlett Packard computer (Palo Alto, CA). The percentages of cells in
the G0-G1, S, and
G2-M compartments in three independent
experiments were averaged. RNA was prepared and blotted according to
standard procedures. Protein extractions and Western blot were
performed according to standard procedures. Immune complexes were
detected with the enhanced chemiluminescence kit (Amersham Corp).
Immune-complex kinase assay was performed as described by using 500
µg of protein lysates and 0.1 µg/ml histone 2B as a substrate
(23)
.
DNA Fragmentation Analysis and TUNEL Assay.
Cells (2 x 106 per sample) were
lysed in 0.5% Triton X-100, 5 mM Tris buffer (pH 7.4), and
20 mM EDTA for 20 min at 4°C. After centrifugation at
14,000 rpm in a microcentrifuge, supernatants were extracted with
phenol-chloroform and precipitated in ethanol. Soluble DNA was
incubated with 50 µg/ml RNase A for 1 h and electrophoresed on a
1.2% agarose gel. For TUNEL, an equal number (5 x 103) of cells from the different lines was seeded
onto single-well Costar glass slides. Cells were fixed in 4% (w/v)
paraformaldehyde, and then, they were permeabilized by the addition of
0.1% Triton X-100/0.1% sodium citrate. Slides were rinsed twice with
PBS, air-dried, and subjected to the TUNEL reaction (Boehringer
Mannheim). Apoptotic nuclei were visualized by FAST RED staining (Dako
Co., Carpinteria, CA).
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RESULTS
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myrAkt Expression Does Not Alter the Thyroid Differentiated
Phenotype.
A myristylated, constitutively activated HA-tagged form of Akt (myrAkt)
was transfected in PC Cl 3 cells. Two pools of several clones
(PC-Akt-1a and PC-Akt-1b) were obtained by G418 selection and expanded
for further studies. The expression of the transfected construct was
demonstrated both at the RNA and protein level. PC Cl 3 cells were
found to express endogenous Akt mRNA; the messenger of the transfected
construct was detected in the two transfected populations as a band of
a lower molecular weight with respect to the endogenous transcript
(Fig. 1A
). Furthermore, the myrAkt protein was visualized in the two
populations by using anti-HA epitope antibodies (Fig. 1B
). Then, Akt was immunoprecipitated from cells maintained
in 0.1% serum and subjected to an in vitro kinase assay
using histone 2B as a substrate. An active Akt kinase was detected in
both transfected populations (Fig. 1C
, right);
endogenous Akt was inactive in parental cells, but it was promptly
activated when cells were stimulated with insulin (1
µM for 10 min; Fig. 1C
,
left). Direct blotting with phospho-Akt antibodies, which
detect Akt only when phosphorylated at Ser-473, and thus active,
confirmed these findings (Fig. 1C
, right).
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Fig. 1. myrAkt expression in transfected PC Cl 3 cells.
A, 20 µg of RNA were subjected to Northern blotting
with an Akt cDNA probe. Actin was used for normalization.
B, 50 µg of protein lysates were subjected to Western
blotting; the filter was stained with anti-HA epitope monoclonal
antibodies, and immunocomplexes were revealed by enhanced
chemiluminescence. Anti-Sp1 antibodies were used for normalization.
C, Akt was immunoprecipitated from protein lysates from
cells cultured for 3 days in the presence of 0.1% calf serum. Where
indicated, cells were treated with insulin. The immunoprecipitates were
incubated with histone 2B and labeled ATP; the reaction product was
analyzed by SDS-PAGE, followed by autoradiography. Fifty µg of the
lysates were immunoblotted with anti-Akt or anti-phosphoAkt
(pSer473)-specific antibodies.
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Expression of the activated myrAkt did not cause major changes in the
morphology of PC Cl 3 cells (Fig. 2A
). Expression of several oncogenes in PC Cl 3 cells resulted
in impaired expression of the thyroid differentiated phenotype.
Differentiated thyroid cells express a set of thyroid-specific genes,
such as Tg, TPO, and the sodium/iodide symporter
(NIS). The expression of these genes is under the control of
a set of thyroid-specific transcription factors, including TTF-1 and
TTF-2, a homeodomain-containing protein (19, 20)
, and a
forkhead factor (21)
, respectively, and PAX-8, a
paired-domain transcription factor (22)
. Oncogene-mediated
dedifferentiation of PC Cl 3 cells is characterized by a reduced
expression of some or all of the thyroid-specific genes which, in turn,
is caused, in most of the cases, by a reduced expression of the TTF.
Thus, to assess whether myrAkt expression affected thyroid
differentiation, we monitored Tg, TPO, and
NIS mRNA levels. Activated Akt did not alter their
expression (Fig. 2B
). Accordingly, TTF-1,
TTF-2 (Fig. 2B
), and PAX-8 (not shown)
expression was also unaffected by myrAkt.
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Fig. 2. myrAkt expression does not alter differentiation of PC Cl
3 cells. A, phase-contrast micrographs of parental PC Cl
3 and PC-Akt-1a and PC-Akt-1b cells. B, Northern blot
analysis (20 µg of RNA) of thyroid-specific gene expression in PC Cl
3 and PC-Akt cells grown in complete medium (calf serum and 6H). Actin
was used for normalization.
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myrAkt Promotes Hormone-independent Proliferation of PC Cl 3 Cells.
Cultured thyroid cells depend on a mixture of 6H for proliferation.
Oncogene-mediated transformation is accompanied by the loss of this
dependence (13–18)
. We investigated the effects of myrAkt
on the growth properties of thyroid cells. Cell cycle kinetics of
parental and myrAkt cells was examined by flow cytometry in different
growth conditions: (a) in the logarithmic phase of growth;
(b) upon 6H deprivation (96 h); and (c) after
48 h they had reached confluence, when parental PC Cl 3 cells are
contact inhibited. Fig. 3A
shows that parental cells were
G1-arrested by hormone deprivation and
confluence. In contrast, a significant fraction of myrAkt cells
remained in the S and G2-M compartments, even in
conditions of 6H deprivation and confluence (Fig. 3A
).
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Fig. 3. myrAkt overcomes growth arrest induced by 6H deprivation
and confluence. A, parental and PC-Akt-1a cells were
harvested in the logarithmic phase of growth (columns
1), after 96 h of 6H deprivation (columns
2), after 48 h they had reached confluence (columns
3) and analyzed by flow cytometry. The percentage of cells in
each phase of the cell cycle is depicted in a bar graph. The results
are representative of three independent experiments. B,
50 µg of protein lysates, obtained from cells treated as described
above, were subjected to Western blotting; the filter was stained with
anti-cyclin D3 and E antibodies, and immunocomplexes were revealed by
enhanced chemiluminescence. Anti-Sp1 antibodies were used for
normalization. The results were confirmed on the PC-Akt-1b cell
population (not shown).
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Akt induces the expression of D-type cyclins (8, 9)
.
Cyclin D3 is the most abundant D-type cyclin expressed in thyroid
cells, and it is essential for TSH-induced thyroid cell proliferation
(24)
. We analyzed the expression of G1 cyclins (D3 and E)
in PC Cl 3 and PC-Akt cells. Cells were harvested in the logarithmic
phase of growth (Fig. 3B
, Lane 1),
after 96 h of 6H withdrawal (Fig. 3B
, Lane
2), or at confluence (Fig. 3B
, Lane
3). In parental cells, the expression of cyclins D3 and E
was down-modulated by 6H deprivation and by contact inhibition. In
marked contrast, this down-regulation was not observed in PC-Akt cells.
Cyclin D3 and E expression was only slightly induced by myrAkt in cells
in the logarithmic phase of growth (Fig. 3B
,
Lanes 1).
myrAkt Promotes Survival of PC Cl 3 Cells.
We evaluated whether myrAkt suppressed starvation-induced apoptosis of
PC Cl 3 cells. Subconfluent cells were kept for 72 h in the
presence of complete medium (5% calf serum and 6H) or in 0.1% calf
serum, and internucleosomal DNA fragmentation was evaluated by the
TUNEL assay. Apoptosis was measured in 10 randomly selected microscopic
fields. Apoptotic nuclei were rare (<2%) in the presence of complete
medium (data not shown). After 72 h of starvation, 30 ± 5% PC Cl 3 cell nuclei were apoptotic. On the contrary, <5%
of cells were apoptotic in the case of PC-Akt-1a and PC-Akt-1b
populations. A representative field and the average results of two
independent experiments are reported in Fig. 4
, A and B, respectively. Similar results were
obtained when a different apoptotic stimulus (2 ng/ml vincristine) was
used (data not shown). To confirm these results, DNA fragmentation was
evaluated by DNA electrophoresis. Soluble DNA extracted from starved PC
Cl 3 cells showed the characteristic ladder-like electrophoretic
pattern indicating apoptotic cell death; myrAkt expression clearly
protected thyroid cells from DNA fragmentation (Fig. 4C
).
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Fig. 4. Starvation induces apoptosis in parental but not PC-Akt
cells. An equal number of cells (5 x 103)
from the three lines were seeded onto single-well Costar glass slides.
Cells were maintained for 72 h in 0.1% calf serum and subjected
to the TUNEL reaction. A representative field is shown in
A. The average percentages of apoptotic nuclei,
calculated by counting a minimum of 200 cells in 10 randomly selected
fields of each specimen in two independent experiments, are reported in
B. Variations between single experiments were < 20% of the mean; bars, SD.
C, cells were maintained in complete medium or for
72 h in 0.1% calf serum (Starv.). Cells
(2 x 106 cells per sample) were lysed, and
soluble DNA was recovered by centrifugation, resuspended, and
electrophoresed on a 1.2% agarose gel stained with ethidium bromide.
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BAD and BCL-2 have been implicated in survival effects of Akt (4, 5)
. Thus, we evaluated BAD and BCL-2 expression in proliferating
and starved thyroid cells. Both proteins underwent significant
expression changes upon starvation of parental PC Cl 3 cells. BAD
protein was barely detectable in proliferating cells, and it was
up-regulated after starvation, whereas BCL-2 was down-regulated upon
starvation (Fig. 5A
). These changes were virtually abrogated by myrAkt
expression. Fas is a proapoptotic receptor found on the surface of many
different cells. When bound to the FasL, Fas initiates a series of
intracellular events culminating in cell death. Akt has been implicated
in controlling FasL (6)
and Fas
(25)
gene transcription. We investigated whether
Fas and FasL gene expression was altered in
PC-Akt cells. Both transcripts were virtually undetectable in
proliferating PC Cl 3 cells and markedly up-regulated upon starvation.
These changes were totally prevented in myrAkt-transfected cells (Fig. 5B
).
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Fig. 5. Expression of BAD, BCL-2, Fas, and FasL in PC-Akt cells.
A, the expression of BAD and BCL-2 was evaluated by
Western blotting (50 µg of cell lysate) on cells maintained for
72 h in complete medium or in 0.1% calf serum
(Starv.). Anti-tubulin antibodies were used for
normalization. B, a Northern blot (20 µg RNA) was
performed with Fas and FasL rat cDNA-specific probes. Actin was used
for normalization. The results were confirmed on the PC-Akt-1b cell
population (not shown).
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DISCUSSION
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Here we show that a constitutively active myrAkt renders PC Cl 3
thyroid cells insensitive to 6H deprivation- and confluence-induced
growth arrest and promotes their survival under starvation. Promotion
of survival is not the general end result of oncogene expression in
thyroid cells. Indeed, for instance, activated Ras (26)
promotes apoptosis of thyroid cells. The effects of myrAkt on
cellular proliferation and survival may be mediated via effects on gene
expression. Here we provide evidence that myrAkt prevents G1 cyclin
down-regulation by hormone deprivation and contact inhibition. D-type
cyclin expression is stimulated by Akt also in other cell systems
(8, 9) . myrAkt also affects the expression of genes that
regulate apoptosis. Thus, myrAkt prevents the up-regulation of BAD and
Fas-FasL and the down-regulation of BCL-2 induced by starvation.
Transcriptional regulation of Fas-FasL and of
BCL-2 genes by Akt was proposed previously in other cell
systems (4, 6, 25)
. Our findings indicate that, in
addition, Akt delivers a signal that inhibits BAD protein accumulation
induced by starvation. Akt is known to phosphorylate BAD, leading to
the binding of BAD to 14-3-3 proteins (5)
. Whether this
process results in increased BAD protein degradation or whether BAD is
transcriptionally regulated by Akt in thyroid cells remains to be
established.
Oncogenes of different categories such as growth factors (fibroblast
growth factor; Ref. 15
), tyrosine kinases (Src and
RET/PTC; Refs. 13
and 17
),
serine/threonine kinases (Raf and Mos; Ref. 13
and
14
), and nuclear proteins (E1A and p53; Refs.
16
and 18
) cause impaired expression of
differentiation genes in PC Cl 3 cells along with the induction of cell
proliferation. Akt is unique in that it does not interfere with the
thyroid differentiated program, although it promotes
hormone-independent proliferation. This demonstrates that control of
differentiation and of TSH-dependent proliferation and survival can be
dissociated in thyroid cells.
Deregulation of survival and mitogenic signals can be a critical step
toward tumorigenesis. Because Akt induces both survival and
proliferation independent on the physiological stimuli and insensitive
to contact inhibition, a possible involvement of Akt in thyroid cancer
can be postulated. Regarding this possibility, we would like to point
out that: (a) amplification of PI3K and
Akt family genes is involved in human cancers
(2)
; (b) thyroid neoplasia can be a consequence
of the germ-line inactivation of the PTEN tumor suppressor
gene, which negatively regulates the PI3K/Akt pathway
(27)
; and (c) somatic activation of Ras and
tyrosine kinase genes is frequently found in thyroid neoplasia, and Akt
activation is one of the end results common to these genetic
alterations. However, because Akt does not alter the morphology and
differentiation status of thyroid cells, it is unlikely to induce
thyroid cancer by itself. Experiments in progress have been
designed to address potential cooperation of Akt with other
oncogenes. Furthermore, promotion of apoptosis is regarded as the
underlying mechanism for several chronic inflammatory diseases.
Hashimoto thyroiditis is an autoimmune disease resulting from
Fas-mediated thyrocyte destruction (28)
. According to our
data, the Fas-FasL system is regulated by the PI3K/Akt pathway. This
suggests the intriguing possibility that the PI3K/Akt pathway could be
involved in Hashimoto thyroiditis.
By signaling through multiple intracellular pathways, TSH stimulates
the proliferation of thyroid cells. PI3K is implicated in TSH and
insulin stimulation of thyroid cell growth. Our data indicate that Akt
is likely to mediate PI3K mitogenic and survival signals in thyroid
cells.
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ACKNOWLEDGMENTS
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We thank R. Di Lauro for the probes to study differentiated
thyroid markers. We thank C. Garbi and G. Chiappetta for the help with
the TUNEL assay and A. M. Cirafici for help with cell transfections.
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FOOTNOTES
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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 This study was supported by the Associazione
Italiana per la Ricerca sul Cancro, by European Community Grant
BMH4-CT96-0814, and by the Programma Biotecnologie legge 95/95
[Ministero dell’Università e della Ricerca Scientifica e
Tecnologica (MURST 5%)]. 
2 To whom requests for reprints should be
addressed, at Centro di Endocrinologia ed Oncologia Sperimentale del
CNR, Università di Napoli "Federico II," via S. Pansini 5,
80131 Naples, Italy. Phone: 39-081-7463056; Fax: 39-081-7463037;
E-mail: masantor{at}unina.it
3 The abbreviations used are: PI3K,
phosphoinositide 3-kinase; TSH, thyrotropin; PKA, protein kinase A; HA,
hemagglutin antigen; 6H, six hormones; TUNEL, terminal
desoxynucleotidyl transferase-mediated desoxyUTP nick end labeling;
myrAKT, myristylated AKT; Tg, thyroglobulin; TPO, thyroperoxidase; TTF,
thyroid-specific transcription factor; Fas L, Fas ligand.
Received 1/27/00.
Accepted 5/17/00.
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ABSTRACT
INTRODUCTION
