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KRAS
G12D
-mediated oncogenic transformation of
thyroid follicular cells requires long-term TSH stimulation
and is regulated by SPRY1
Minjing Zou
1
, Essa Y Baitei
1
, Roua A Al-Rijjal
1
, Ranjit S Parhar
2
, Futwan A Al-Mohanna
3
, Shioko Kimura
4
,
Catrin Pritchard
5
, Huda BinEssa
1
, Azizah A Alanazi
3
, Ali S Alzahrani
3
, Mohammed Akhtar
6
, Abdullah M Assiri
7
,
Brian F Meyer
1
and Yufei Shi
1
KRAS
G12D
can cause lung cancer rapidly, but is not sufficient to induce thyroid cancer. It is not clear whether long-term
serum thyroid stimulating hormone (TSH) stimulation can promote KRAS
G12D
-mediated thyroid follicular cell
transformation. In the present study, we investigated the effect of long-term TSH stimulation in KRAS
G12D
knock-in mice
and the role of Sprouty1 (SPRY1)inKRAS
G12D
-mediated signaling. We used TPO-KRAS
G12D
mice for thyroid-specific
expression of KRAS
G12D
under the endogenous KRAS promoter. Twenty TPO-KRAS
G12D
mice were given anti-thyroid drug
propylthiouracil (PTU, 0.1% w/v) in drinking water to induce serum TSH and 20 mice were without PTU treatment. Equal
number of wild-type littermates (TPO-KRAS
WT
) was given the same treatment. The expression of SPRY1, a negative
regulator of receptor tyrosine kinase (RTK) signaling, was analyzed in both KRAS
G12D
-and BRAF
V600E
-induced thyroid
cancers. Without PTU treatment, only mild thyroid enlargement and hyperplasia were observed in TPO-KRAS
G12D
mice.
With PTU treatment, significant thyroid enlargement and hyperplasia occurred in both TPO-KRAS
G12D
and TPO-KRAS
WT
littermates. Thyroids from TPO-KRAS
G12D
mice were six times larger than TPO-KRAS
WT
littermates. Distinct thyroid histology
was found between TPO-KRAS
G12D
and TPO-KRAS
WT
mice: thyroid from TPO-KRAS
G12D
mice showed hyperplasia with well-
maintained follicular architecture whereas in TPO-KRAS
WT
mice this structure was replaced by papillary hyperplasia.
Among 10 TPO-KRAS
G12D
mice monitored for 14 months, two developed follicular thyroid cancer (FTC), one with
pulmonary metastasis. Differential SPRY1 expression was demonstrated: increased in FTC and reduced in papillary thyroid
cancer (PTC). The increased SPRY1 expression in FTC promoted TSH-RAS signaling through PI3K/AKT pathway whereas
downregulation of SPRY1 by BRAF
V600E
in PTC resulted in both MAPK and PI3K/AKT activation. We conclude that chronic
TSH stimulation can enhance KRAS
G12D
-mediated oncogenesis, leading to FTC. SPRY1 may function as a molecular switch
to control MAPK signaling and its downregulation by BRAF
V600E
favors PTC development.
Laboratory Investigation (2015) 95, 1269–1277; doi:10.1038/labinvest.2015.90; published online 6 July 2015
Thyroid cancer is the most common type of endocrine
malignancies and its incidence is rising rapidly in recently
years, especially among women.1Histologically, it can be
classified into papillary thyroid cancer (PTC), follicular
thyroid cancer (FTC), and anaplastic thyroid cancer (ATC).
PTC is the most common type of differentiated thyroid
carcinomas, accounting for more than 80% of thyroid cancer,
and FTC accounts for 15%.2The BRAF
V600E
is the most com-
mon genetic alterations in PTC with overall rate of 44%,3–5
and RAS (HRAS,KRAS,orNRAS) mutations are found in
about 50% of FTC and can also been found in benign
adenomas.6,7
In the earlier studies, several transgenic mouse models were
created to study the molecular mechanisms of RAS-mediated
transformation of thyroid follicular cells in vivo.8–10 Both PTC
and FTC were developed from overexpression of a mutant
RAS gene under TG promoter, which may not reflect the
activity of endogenous mutant RAS gene expressed at the
1
Department of Genetics, King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia;
2
Department of Cell Biology, King Faisal Specialist Hospital and Research
Centre, Riyadh, Saudi Arabia;
3
Department of Medicine, King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia;
4
Laboratory of Metabolism, Center
for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA;
5
Department of Biochemistry, University of Leicester, Leicester, UK;
6
Department of Pathology, King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia and
7
Department of Comparative Medicine, King Faisal Specialist
Hospital and Research Centre, Riyadh, Saudi Arabia
Correspondence: Dr Y Shi, MD, MBC 3, Department of Genetics, King Faisal Specialist Hospital and Research Centre, PO Box 3354, Riyadh 11211, Saudi Arabia.
E-mail: yufei@kfshrc.edu.sa
Received 18 February 2015; accepted 5 May 2015
www.laboratoryinvestigation.org | Laboratory Investigation | Volume 95 November 2015 1269
Laboratory Investigation (2015) 95, 1269–1277
©
2015 USCAP, Inc All rights reserved 0023-6837/15
physiological level. Overexpression of KRAS and HRAS could
also downregulate expression in a dose-dependent manner of
NKX2-1,TG, and TPO genes required for thyroid hormone
synthesis.11,12 Recent knock-in mouse studies have shown that
endogenous expression of either HRAS
G12V
or KRAS
G12D
is
not sufficient to induce thyroid dysfunction and cell trans-
formation under their native promoters.13,14
It is known that TSH stimulates the growth or development
of thyroid cancer and higher serum TSH is associated with
both thyroid cancer incidence and recurrence.15,16 It is not
clear whether long-term TSH stimulation can induce
KRAS
G12D
-mediated thyroid follicular cell transformation. In
the present study, we investigated the potential of long-term
TSH stimulation on thyroid cancer development in KRAS
G12D
knock-in mice targeted to express its oncoprotein in thyroid at
the physiological level. We also studied the role of SPRY1 in
the regulation of KRAS
G12D
- vs BRAF
V600E
-mediated signaling.
MATERIALS AND METHODS
Experimental Animals
LSL-KRAS
G12D
(obtained from The Jackson Laboratory, ME,
USA), LSL-BRAF
V600E
, and TPO-Cre strains have been
described previously.17–19 LSL-KRAS
G12D
and LSL-BRAF
V600E
carry a latent mutant allele of KRAS and BRAF, respectively.
Both LSL-KRAS
G12D
and LSL-BRAF
V600E
mice were kept as
heterozygotes. LSL-KRAS
G12D
or LSL-BRAF
V600E
was crossed
with TPO-Cre to generate TPO-KRAS
G12D
or TPO-BRAF
V600E
strain where KRAS
G12D
or BRAF
V600E
is conditionally expressed
in thyroid follicular cells through Cre-mediated deletion
of a floxed transcriptional stop sequence. The resulting TPO-
KRAS
G12D
or TPO-BRAF
V600E
strain expressed mutant
KRAS
G12D
or BRAF
V600E
transcripts at the physiological level
under its endogenous promoter. The study was approved by
the Animal Care and Usage Committee of the institution and
conducted in compliance with the Public Health Service
Guidelines for the Care and Use of Animals in Research.
Genotyping of TPO-KRAS
G12D
or TPO-BRAF
V600E
Strain
Genotyping of TPO-Cre mediated recombination of LSL-
KRAS
G12D
or LSL-BRAF
V600E
targeted allele has been
described previously.17,19 Briefly, the following primers were
used to detect LSL-KRAS
G12D
recombination in mouse tissue:
primer 1, 5′-GTCTTTCCCCAGCACAGTGC-3′, primer 2,
CTCTTGCCTACGCCACCAGCTC-3′, and primer 3, 5′-AG
CTAGCCACCATGGCTTGAGTAAGTCTGCA-3′. Primer
1+2 detects wild-type allele yielding a product of 622 bp.
Primer 1+2 also detects Cre-recombined KRAS
G12D
allele
(Lox-KRAS
G12D
) yielding a product of 650 bp. This product is
larger than the wild-type allele due to the presence of LoxP
site that remains after Cre-mediated recombination. Primer 2
+3 detects the LSL-KRAS
G12D
allele yielding a product of
500 bp. Multiplex PCR containing three primers was used:
95 °C for 10 min followed by 35 cycles of amplification (95 °C
for 1 min, 60 °C for 1 min, 72 °C for 1 min with final
extension at 72 °C for 10 min). The following primers were
used to detect LSL-BRAF
V600E
recombination: primer A, 5′-
AGTCAATCATCCACAGAGACCT-3′, primer B: 5′-GCTTG
GCTGGACGTAAACTC-3′, and primer C, 5′-GCCCAGG
CTCTTTATGAGAA-3′. Primer A+C detects the wild-type
allele yielding a product of 466 bp. Primer A+C also detects
Cre-recombined allele (Lox-BRAF
V600E
) yielding a product
of 518 bp. Primer B+C detects the LSL-BRAF
V600E
allele
yielding a product of 140 bp. The PCR conditions are at
95 °C for 5 min followed by 35 cycles of amplification
(95 °C for 1 min, 60 °C for 1 min, 72 °C for 1 min with a
final extension at 72 °C for 10 min).
Anti-Thyroid Drug Treatment
TPO-KRAS
G12D
mice (4–10 weeks of age) were divided into
two groups and given 0.1% (w/v) anti-thyroid drug
propylthiouracil (PTU, Sigma-Aldrich, MO) in drinking
water ad libitum, changed once weekly, to induce serum
TSH. One group (n=10) was observed for 8 months and the
other group (n=10) was observed for 14 months. Twenty
TPO-KRAS
G12D
mice were also divided into two groups (10
in each group) without PTU treatment, and observed for 8
and 14 months, respectively. Forty wild-type mice (TPO-
KRAS
WT
, 10 in each group) were given the same treatment as
TPO-KRAS
G12D
mice.
Thyroid Hormone Measurements
Blood was collected by cardiac puncture. Serum TSH was
measured using MILLIPLEX MAP Mouse Pituitary Magnetic
Bead Panel following the manufacturer’s instruction (EMD
Millipore Corporation, Billerica, MA, USA). Serum total T4
was measured using MILLIPLEX MAP Steroid/Thyroid
Hormone Magnetic Bead Panel (EMD Millipore Corporation).
Tumor Cell Culture
A PTC tumor from a 4-month-old TPO-BRAF
V600E
mouse
was collected aseptically using blunt dissection and mechani-
cally dissociated by mincing and passaged through a 40-μM
mesh sterile screen, and suspended in DMEM/F12 growth
medium (10% fetal bovine serum, 100 units/ml penicillin,
100 μg/ml streptomycin). Cells were further dissociated by
incubation in the growth medium containing 100 U/ml type I
collagenase (Sigma-Aldrich) and 1.0 U/ml dispase I (Roche
Diagnostics, Indianapolis, IN, USA) at 37 °C rocking water
bath for 60 min. The cell suspension was washed twice and
resuspended in 10 mm culture dish with DMEM/F12 growth
medium containing 2 mU/ml bovine TSH (Sigma-Aldrich) to
establish a BVE cell line (BRAF
V600E
-induced tumor cell line).
The genetic background was confirmed by genotyping.
Quantitative Real-Time Reverse Transcriptase-PCR
Analysis for SPRY1 Expression
Total RNA was isolated from thyroid of TPO-KRAS
WT
mice
treated with PTU, PTC of TPO-BRAF
V600E
, or FTC of TPO-
KRAS
G12D
mice by the quanidinium thiocyanate-phenol-
chloroform method as described previously.20 The integrity of
TSH mediated RAS transformation of thyroid follicular cells
M Zou et al
1270 Laboratory Investigation | Volume 95 November 2015 | www.laboratoryinvestigation.org
RNA was verified by denaturing gel electrophoresis. In all, 2
μg of each total RNA was reverse-transcribed to cDNA using
the Promega RT system (Promega, Madison, WI, USA).
LightCycler DNA Master SYBR Green 1 kit was used for
quantitative real-time PCR analysis.21 The cDNA mix was
diluted 10-fold, and 2 μl of the dilution was used for real-time
PCR. PCR primers for the 114-bp SPRY1 cDNA fragment are
5′-GCGGAGGCCGAGGATTT-3′(forward) and 5′-ATCAC
CACTAGCGAAGTGTGGC-3′(reverse). The forward primer
spans the junction of exon 1 and exon 2 over 1.7 kb intron 1
so that contaminated genomic DNA will not be amplified.
The SPRY1 cDNA fragment was verified by DNA sequencing.
The mRNA level of housekeeping gene glyceraldehyde-3-
phosphate dehydrogenase (GAPDH) was used as an internal
control, and a 155-bp PCR product was amplified using the
following two primers: 5′-ATGTTCCAGTATGACTCCACT
CACG-3′(forward) and 5′-GAAGACACCAGTAGACT
CCACGACA-3′(reverse). The PCR conditions are 95 °C for
30 s followed by 30 cycles of amplification (95 °C for 10 s, 50 °
C for 5 s, and 72 °C for 10 s). The resulting concentration of
SPRY1 PCR products was normalized by comparison with
GAPDH and was used to determine the relative mRNA level
of SPRY1 in thyroid tumors.
Histology and Immunohistochemistry
Histology and immunohistochemical staining was described
previously.22 Briefly, 4-μm-thick formalin-fixed paraffin-
embedded tissue sections were prepared and stained with
hematoxylin and eosin or SPRY1 (1:50 dilution, Abcam,
Cambridge, MA, USA), p-ERK, or p-AKT antibody (1:50
dilution, Cell Signaling Technology, Danvers, MA, USA).
Histological diagnosis was performed by a thyroid pathologist
(MA) blinded to the genotype and the treatment status of the
animal. DAKO LSAB+kit, HRP was used for immunostaining
(DAKO, Carpinteria, CA, USA). The sections were counter-
stained with Mayer's haematoxylin.
Regulation of TSH Signaling by SPRY1
Mouse SPRY1 cDNA in pCMV6 expression vector was
obtained from OriGene (Rockville, MD, USA). BVE cell line
was transfected with either pCMV6 vector or mSPRY1/
pCMV6 using Lipofectamine (Invitrogen, CA). The culture
medium was changed to DMEM/F12 growth medium 16 h
after transfection, and the transfected cells were cultured for
additional 48 h in the presence or absence of 10 mU/ml
bTSH. The expression of SPRY1, phospho-ERK, and
phospho-AKT was determined by western blot analysis.
Western Blot Analysis
BVE cell line was transfected with mouse SPRY1 cDNA under
the control of CMV promoter (Origen). 40 μg of protein
was loaded onto a 12% SDS-polyacrylamide gel. Proteins
were transferred onto a PVDF membrane and subject to
western blot analysis using anti-phospho-ERK 1/2, and
phosphor-AKT antibody (1:1000, Cell Signaling Technology)
or anti-SPRY1 (1:1000, Abcam).
Bioinfomatic Anylysis of Human PTC Samples
The expression profiles of SPRY1,SPRY2,SPRY3, and SPRY4
in the TCGA data set of 572 PTC samples23 were analyzed
using UCSC cancer genomics browser (https://genome-
cancer.soe.ucsc.edu/proj/site/hgHeatmap/).
Statistical Analysis
Unpaired Student’st-test (two-tailed) was used. A P-value of
0.05 or less was considered as significant.
RESULTS
Without PTU treatment, only mild thyroid enlargement and
hyperplasia were observed in TPO-KRAS
G12D
mice (Figure 1b).
The thyroids were about two times larger as compared with
those from the TPO-KRAS
WT
mice. Thyroid cancer develop-
ment was not observed among 10 mice monitored for up to
14 months. With PTU treatment, significant thyroid enlarge-
ment with multinodular goiters and hyperplasia occurred in
both TPO-KRAS
G12D
and TPO-KRAS
WT
mice (Figure 2a).
Massive thyroid enlargement was found in TPO-KRAS
G12D
mice: thyroids of TPO-KRAS
G12D
mice were about six times
larger than TPO-KRAS
WT
mice (118.75 ±13.15 mg vs
21.11 ±6.33 mg, Po0.001, Figure 2b), indicating that TSH
can cooperate with KRAS
G12D
to promote thyroid growth and
hyperplasia. Interestingly, thyroid histology of TPO-
KRAS
G12D
mice showed nodular hyperplasia with well-
maintained follicular architecture whereas in TPO-KRAS
WT
mice such follicular architecture was replaced by papillary
hyperplasia (Figure 2c). However, PTC was not diagnosed due
to lack of characteristic nuclear features such as nuclear
grooves, intranuclear cytoplasmic inclusion, fine chromatin
texture, and nuclear envelope irregularity. As shown in
Figure 2d, serum TSH was significantly elevated in PTU-
treated mice as compared with the control (24 370.8 ±12 942.5
vs 426.4 ±9.65 pg/ml, Po0.01). Serum T4 was significantly
reduced in PTU-treated mice as compared with the control
(5.21 ±1.31 vs 22.68 ±6.23 ng/ml, Po0.05). Among 10 TPO-
KRAS
G12D
mice monitored for 14 months, two developed
follicular thyroid cancer (FTC, Figure 3a) and one with
pulmonary metastasis (Figure 3b). Since the cellular features
were indistinguishable between follicular hyperplasia and
cancer, the diagnosis of FTC was based on the invasion of
surrounding tissues such as blood vessel and neck muscle
(Figure 3a), and pulmonary metastasis (Figure 3b). Among 10
TPO-KRAS
WT
mice monitored for 14 months under PTU
treatment, no tumor development was observed.
The Sprouty (SPRY) family of proteins is involved in the
negative feedback regulation of growth factor-mediated
MAPK activation.24 To understand why TSH stimulation
caused only FTC, but not PTC, we investigated SPRY1
expression in thyroid tumors from TPO-KRAS
G12D
and TPO-
BRAF
V600E
mice. SPRY1 mRNA level was significantly higher
TSH mediated RAS transformation of thyroid follicular cells
M Zou et al
www.laboratoryinvestigation.org | Laboratory Investigation | Volume 95 November 2015 1271
in KRAS
G12D
-induced FTC (9.23 ±0.56) as compared with
KRAS
WT
control (1.03 ±0.15, Po0.01, Figure 4a). SPRY1
expression was also increased in KRAS
G12D
thyroid without
PTU treatment (2.1 ±0.2, Po0.01, Figure 4a), but the
expression level was much lower than under PTU treatment,
suggesting that the increased SPRY1 expression may be a
compensatory mechanism to counter TSH-mediated growth
stimulation. In contrast, SPRY1 mRNA level was significantly
lower in BRAF
V600E
-induced PTC (0.31 ±0.17) as compared
with BRAF
WT
control (1.00 ±0.12, Po0.05, Figure 4b). The
serum TSH was greater than 50 000 pg/ml (beyond the
detection limit of the kit) and T4 was 3.87±0.68 in TPO-
BRAF
V600E
mice (n=6) as compared with 445.2 ±72.21 in
TSH and 29.03 ±3.62 in T4 from TPO-BRAF
WT
mice (n=6),
consistent with previous report that TPO-BRAF
V600E
mice
have severe hypothyroidism.13 The increased SPRY1 expres-
sion in FTC was confirmed by immunohistochemistry
analysis. As shown in Figure 4c, increased SPRY1 and
p-AKT expression was observed in FTC (b and e) with
normal p-ERK expression (h). In PTC, both p-ERK and
p-AKT expression were increased (f and i). Although down-
regulation of SPRY1 expression in PTC was not clearly shown
by immunohistochemistry (Figure 4cC), it was confirmed by
western blot analysis, showing inverse correlation between
SPRY1 expression and p-ERK activation (Figure 4d).
To further investigate the role of SPRY1 in the regulation of
TSH-mediated signaling, we used BVE cell line (derived from
BRAF
V600E
-induced PTC tumor) as a model, for it has low
SPRY1 expression. The cell line was transfected with mouse
SPRY1 cDNA and treated with or without TSH. As shown in
Figure 5, SPRY1 expression reduced p-ERK expression, and
TSH stimulation only activated p-ATK when SPRY1
expression was increased. When SPRY1 expression was
reduced, TSH stimulation increased both p-ERK and p-ATK
expression (Figure 5). These data suggest that SPRY1 may act
as a molecular switch to control thyroid follicular cells to go
through either MAP kinase (RAF-MEK-ERK) or PI3 kinase
(PI3K/AKT) signaling pathway. When SPRY1 is upregulated
by KRAS
G12D
, MAPK pathway is inhibited and only PI3K/AKT
pathway is utilized, resulting in FTC. When SPRY1 is
downregulated by BRAF
V600E
, thyroid follicular cells may go
through both MAPK and PI3K/AKT pathways to initiate PTC.
Finally, we performed bioinformatic mining of the recent
TCGA data set of 572 human PTC samples to compare SPRY
gene (SPRY1,SPRY2,SPRY3, and SPRY4) expression between
classic PTC (CPTC) and follicular variant PTC (FVPTC).
FVPTC shares many molecular and pathological features with
FTC. RAS mutation is frequently detected in FVPTC as
compared with frequent BRAF mutation in CPTC. As shown
in Figure 6, the SPRY1 and SPRY4 expression is reduced in
normal thyroids. The expression of SPRY2 and SPRY3 tends
to increase in normal thyroids and is variable in both CPTC
and FVPTC. The SPRY1 expression is also variable in CPTC
and FVPTC. In CPTC, increased SPRY1 expression is present
in 36.6% (162/409) samples, decreased expression in 40.6%
(166/409) and unchanged in 19.8% (81/409) samples. In
FVPTC, 40.6% (43/106) samples have increased expression,
36.8% (39/106) have decreased expression, and 22.6%
(24/106) unchanged. The expression of SPRY4 is consistently
increased in FVPTC: 66.1% (72/109) have increased expres-
sion and 13.8% (15/109) have decreased expression. These
data suggest that the SPRY family of proteins is involved in
the regulation of thyroid tumorigenesis. The exact role of each
protein remains to be determined.
Figure 1 Activation of KRAS
G12D
in thyroid induces mild thyroid enlargement and hyperplasia. (a) Genotyping of TPO-Cre mediated recombination of
LSL-KRAS
G12D
(A) and LSL-BRAF
V600E
(B). The PCR product was run on a 1.5% agarose gel. The activation of KRAS
G12D
and BRAF
V600E
occurred only in the
thyroid as a result of Cre-mediated deletion of a floxed transcriptional stop sequence. (b) Mild thyroid enlargement and hyperplasia were observed in
TPO-KRAS
G12D
mice. Thyroids of a TPO-KRAS
WT
mouse (A) and a TPO-KRAS
G12D
mouse (B) at the age of 8 months were shown at the top panel. Histology
of thyroids from the same TPO-KRAS
WT
mice (C, × 20) and TPO-KRAS
G12D
mice (D, × 20) was shown at the bottom panel.
TSH mediated RAS transformation of thyroid follicular cells
M Zou et al
1272 Laboratory Investigation | Volume 95 November 2015 | www.laboratoryinvestigation.org
Figure 3 TSH cooperates with KRAS
G12D
to induce transformation of thyroid follicular cells into FTC. (a) Thyroid of a TPO-KRAS
G12D
mouse with FTC
following 14-month PTU treatment (A). FTC with neck muscle invasion as indicated by arrows (B, × 10) and blood vessel invasion (indicated by arrows,
C, × 20; D, × 40). (b) Pulmonary metastasis of FTC from the same mice. Multiple foci of metastasis can be seen from gross examination of the lung (A)
and microscopic examination (B, × 10; C, × 20, D, × 40).
Figure 2 TSH cooperates with KRAS
G12D
to induce massive thyroid hyperplasia. (a) Thyroids of a TPO-KRAS
wt
mouse (A) and a TPO-KRAS
G12D
mouse (B) at
16 months old were shown. The mice were treated with PTU in drinking water for 14 months. Goiters were present in both mice and significant thyroid
enlargement was observed in TPO-KRAS
G12D
mice. (b) Thyroid weight in PTU-treated mice. Thyroid was collected from 10 TPO-KRAS
G12D
and 10 TPO-KRAS
WT
mice treated with PTU for 14 months (at the end of experiment). Thyroid weight was measured and data were expressed as mean ± s.e.m., *Po0.001. (c)
Histology of thyroids from the same TPO-KRAS
WT
and TPO-KRAS
G12D
mice was shown. Follicular architecture was replaced by papillary structure in
hyperplastic goiters from a TPO-KRAS
WT
mouse (A, × 4; C, × 20) whereas in the goiter of a TPO-KRAS
G12D
mouse, the follicular architecture was intact (B, × 4,
D, × 20). (d) Thyroid hormone levels in PTU-treated mice. Serum was collected from six TPO-KRAS mice treated with PTU for 1 month and from six mice of
same age group without PTU treatment. The samples were assayed individually in triplicates. The data are expressed as mean ± s.em., *Po0.05.
TSH mediated RAS transformation of thyroid follicular cells
M Zou et al
www.laboratoryinvestigation.org | Laboratory Investigation | Volume 95 November 2015 1273
DISCUSSION
In the present study, we have demonstrated that KRAS
G12D
mutation alone is not sufficient to induce oncogenic
transformation of thyroid follicular cells. With long-term
TSH stimulation, KRAS
G12D
can promote thyroid growth and
hyperplasia, and eventually result in oncogenic transforma-
tion into follicular thyroid cancer. This demonstrates the
significant oncogenic role of TSH in thyroid tumorigenesis:
without TSH stimulation, thyroid cancer would not be able to
develop or progress even in the presence of KRAS
G12D
or
BRAF
V600E
.25
The animal model in the current study is very similar to
congenital hypothyroidism in humans, which is caused by
defects in thyroid hormone synthesis.26 High levels of TSH
are often present in patients with congenital hypothyroidism
due to poor management or patients’non-compliance to
treatment. We and others have reported thyroid cancer
development as a result of long-term TSH stimulation.27,28
Although both PTC and FTC were reported in those cases,
BRAF mutation was found only in patients with PTC.29 RAS
mutations have been reported in both benign thyroid goiters
and cancers,30 indicating that additional factors are required
to initiate malignant transformation. The current study has
demonstrated that chronic TSH stimulation is one of the
factors required for RAS-mediated carcinogenesis.
TSH stimulates growth and differentiation of thyroid
follicular cells through its G protein-coupled receptor. TSH
can activate both cAMP-protein kinase A (PKA) pathway for
differentiation31 and RAS-PI3K pathway for proliferation.32,33
RAS allows growth promoting signals through both MAPK
(RAF-MEK-ERK) and PIK3CA/AKT pathways. Previous
studies have shown that cAMP can inhibit RAS-mediated
signals to MAPK by blocking RAF-1 activation via RAP1.34,35
Under these circumstances, PI3K signaling is the main
mediator of RAS effects. RAP1 is a member of the RAS
family of small G proteins that transmit signals from cAMP-
Figure 4 SPRY1 expression in thyroid cancer. SPRY1 mRNA from KRAS
G12D
-induced FTC (a) and BRAF
V600E
-induced PTC (b) was analyzed by qRT-PCR. RNA
from TPO-KRAS
WT
mouse thyroids and TPO-BRAF
WT
mouse thyroids was used as a control. Data were expressed as mean ± s.e.m. of three separate
experiments. (c) Immunohistochemistry staining of SPRY1, p-AKT, and p-ERK. Normal thyroid (A, D, and G, × 40), FTC from a TPO-KRAS
G12D
mouse
(B, E, and H, × 40), and PTC from a TPO-BRAF
V600E
mouse were stained with SPRY1 antibody (A, B, and C, 1:50 dilution), p-AKT antibody (D, E, and F, 1:50
dilution) and p-ERK antibody (G, H, and I, 1:50 dilution). Increased expression of SPRY1 and p-AKT was seen in FTC. p-ERK was not activated in FTC
whereas both p-AKT and p-ERK were activated in PTC. (d) Western blot analysis of SPRY1 expression in a PTC tumor. Decreased SPRY1 expression is
observed in PTC as compared with normal thyroid.
TSH mediated RAS transformation of thyroid follicular cells
M Zou et al
1274 Laboratory Investigation | Volume 95 November 2015 | www.laboratoryinvestigation.org
PKA pathway to MAPK pathway by interaction with BRAF.36
Without BRAF, GTP-loaded RAP1 blocks RAS activation of
RAF-1, thereby inhibiting MAPK signaling.37 In a mouse
model with constitutive RAP1 activation in thyroid, FTC was
not developed until given 12 months goitrogen treatment,
indicating that TSH signaling is required and is probably via
RAS-PI3K pathway for FTC initiation.38 In two different FTC
mouse models, TSH signaling is constantly activated with
simultaneous activation of either PI3K/AKT39,40 or phospho-
lipase C pathway.41 FTC was developed in all mice with PI3K/
AKT activation, suggesting that PI3K is an important
mediator of RAS effects in TSH-induced FTC. FTC can
develop rapidly without TSH signaling when both RAS and
PI3K/AKT pathways are constitutively activated.42 Although
MAPK is activated in these mice,42 PI3K signaling probably
predominates over MAPK, resulting in FTC phenotype. Since
serum TSH is very low in these mice, MAPK activation may
reflect the loss of cAMP-mediated inhibition of RAS signals to
MAPK.43
TSH signaling promotes the tumorigenesis of PTC,13
indicating a cross-talk between TSH-cAMP-PKA pathway
and MAPK pathway.37 As discussed above, RAP1 may be one
of the molecular switches to control TSH-cAMP signaling
through either RAS-PI3K to initiate FTC or RAS-MAPK to
induce PTC. In the current study, we have identified that
SPRY1 is another molecular switch to regulate RAS-MAPK
signaling. SPRY1 has been reported as a candidate tumor-
suppressor gene in medullary thyroid carcinoma44 and its
expression is decreased in human prostate cancer.45 We also
found reduced SPRY1 expression in human PTC with
BRAF
V600E
(data will be presented elsewhere). Concomitant
Figure 5 Regulation of TSH signaling by SPRY1. BVE cell line was
transfected with mouse SPRY1 cDNA and cultured in the presence or
absence of 10 mU/ml bovine TSH for 48 h. The expression of SPRY1,
phospho-ERK, and phospho-AKT was determined by western blot analysis.
Reduction of p-ERK is observed by increased SPRY1 expression, and TSH
stimulation only activates p-ATK in the presence of increased SPRY1
expression. TSH can increase both p-ERK and p-ATK expression when
SPRY1 expression is reduced.
Figure 6 The gene expression profile of SPRY family of proteins in human thyroid cancer. The TCGA thyroid cancer gene expression data set of 572
thyroid cancer samples was used to examine gene expression profile among normal thyroid, PTC, classic PTC (CPTC), tall cell PTC, and follicular variant
PTC (FVPTC). The gene expression profile is presented as a heatmap of red (upregulated), black (no change), and green (downregulated). The SPRY1 and
SPRY4 expression is downregulated in normal thyroids. The expression of SPRY4 is consistently increased in FVPTC.
TSH mediated RAS transformation of thyroid follicular cells
M Zou et al
www.laboratoryinvestigation.org | Laboratory Investigation | Volume 95 November 2015 1275
loss of SPRY1 and SPRY2 results in hyperactive MAPK
signaling and low-grade prostatic intraepithelial neoplasia.46
However, when SPRY1 and SPRY2 loss-of-function occurs in
the context of loss of one Pten allele, aberrant activation of
AKT and invasive neoplasia occur, suggesting that Sprouty
genes can negatively regulate the PI3K/AKT pathway as well.46
Indeed, PI3K/AKT activation has been reported in PTC
patients with BRAF
V600E
.47 Our data also demonstrate the
activation of both MAPK and PI3K/AKT pathways in
BRAF
V600E
-induced PTC when SPRY1 expression is reduced.
In KRAS
G12D
-induced lung cancer, SPRY2 is upregulated via
negative feedback loop to inhibit MAPK signaling.48 However,
we only found SPRY1 upregulation in KRAS
G12D
-induced FTC
and SPRY2 expression was not changed (data not shown). The
differential SPRY1 expression in TPO-KRAS
G12D
and TPO-
BRAF
V600E
mice indicates that SPRY1 can selectively regulate
TSH-mediated RAS signaling pathways in thyroid: favoring
MAPK pathway and PTC initiation when its expression is
downregulated and PI3K/AKT pathway and FTC initiation
when its expression is upregulated. It has been reported that
supraphysiologic expression of a mutant RAS can result in
PTC or mixed papillary–follicular features in early transgenic
mice studies,8,10 which appears to be contradictory to our
hypothesis. However, overexpressed mutant RAS beyond
physiological level may escape SPRY1-mediated feedback
inhibition of MAPK signaling and induce PTC.
ACKNOWLEDGMENTS
The study is supported by KACST grants (11-BIO1434-20 and PL-10-0051). We
would like to thank Dr Mario Encinas for critical discussions.
DISCLOSURE/CONFLICT OF INTEREST
The authors declare no conflict of interest.
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