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© Mary Ann Liebert, Inc.
DOI: 10.1089/thy.2019.0284
1
Mouse Model of Thyroid Cancer Progression and Dedifferentiation
Mouse Model of Thyroid Cancer Progression and Dedifferentiation Driven by STRN‐ALK Expression and Loss of p53: Evidence for the Existence of Two Types of Poorly Differentiated Carcinoma (DOI:
Driven by STRN‐ALK Expression and Loss of p53: Evidence for the
This paper has been peer‐reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
Existence of Two Types of Poorly Differentiated Carcinoma
Alyaksandr V. Nikitski1, Susan L. Rominski1, Vincenzo Condello1, Cihan Kaya1, Mamta
Wankhede2, *, Federica Panebianco1, Hong Yang3, ⁑, Daniel L. Altschuler2, and Yuri E.
Nikiforov1
1
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Department of Pathology, University of Pittsburgh, Pittsburgh, PA, USA
2
Department of Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh,
PA, USA
3
Division of Experimental Pathology, Department of Pathology, University of Pittsburgh,
Pittsburgh, PA, USA
10.1089/thy.2019.0284)
*
Current affiliation: School for Engineering of Matter, Transport, and Energy, Arizona State
Thyroid
University, Tempe, AZ 85287 USA
⁑
Current affiliation: Department of Medical Ultrasonics, The First Affiliated Hospital of
Guangxi Medical University, Nanning, Guangxi, China
Alyaksandr V. Nikitski, MD, PhD
Department of Pathology, University of Pittsburgh, Scaife Hall A‐721, 3350 Terrace St.,
Pittsburgh, PA 15213; E‐mail: nikitski.alyaksandr@pitt.edu
Susan L. Rominski, B.S.
Department of Pathology, University of Pittsburgh, Scaife Hall A‐721, 3350 Terrace St.,
Pittsburgh, PA 15213; E‐mail: rominskisl@upmc.edu
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Vincenzo Condello, PhD
Mouse Model of Thyroid Cancer Progression and Dedifferentiation Driven by STRN‐ALK Expression and Loss of p53: Evidence for the Existence of Two Types of Poorly Differentiated Carcinoma (DOI:
Department of Pathology, University of Pittsburgh, Scaife Hall A‐721, 3350 Terrace St.,
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Pittsburgh, PA 15213; E‐mail: Vic40@pitt.edu
Cihan Kaya, PhD
Department of Pathology, University of Pittsburgh Medical Center, CLB Room 8029, 3477
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Euler Way, Pittsburgh, PA 15213; E‐mail: kayac@upmc.edu
Mamta Wankhede, PhD
School for Engineering of Matter, Transport, and Energy, Arizona State University, 651 E
10.1089/thy.2019.0284)
Tyler Mall, Tempe, AZ 85281; E‐mail: Mamta.wankhede@asu.edu
Thyroid
Federica Panebianco, PhD
Department of Pathology, University of Pittsburgh, Scaife Hall A‐721, 3350 Terrace St.,
Pittsburgh, PA 15213; E‐mail: panebiancof@upmc.edu
Hong Yang, M.D, PhD
Department of Medical Ultrasonics, The First Affiliated Hospital of Guangxi Medical
University, No.6. Shuangyong Rd, Nanning, Guangxi 530021, P.R. China; E‐mail:
yanghonggx@163.com
Daniel L. Altschuler, PhD
Department of Pharmacology and Chemical Biology, University of Pittsburgh, W1316
BSTWR, 200 Lothrop St., Pittsburgh, PA 15213; E‐mail: altschul@pitt.edu
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Thyroid
Mouse Model of Thyroid Cancer Progression and Dedifferentiation Driven by STRN‐ALK Expression and Loss of p53: Evidence for the Existence of Two Types of Poorly Differentiated Carcinoma (DOI:
10.1089/thy.2019.0284)
This paper has been peer‐reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
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carcinoma
Yuri E. Nikiforov, MD, PhD
Pittsburgh, PA, 15213; E‐mail: nikiforovye@upmc.edu
Running title: Mouse model of thyroid cancer dedifferentiation
Key words: STRN‐ALK, p53, thyroid cancer, dedifferentiation, poorly differentiated
Department of Pathology, University of Pittsburgh, CLB Room 8031, 3477 Euler Way,
3
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Abstract
Mouse Model of Thyroid Cancer Progression and Dedifferentiation Driven by STRN‐ALK Expression and Loss of p53: Evidence for the Existence of Two Types of Poorly Differentiated Carcinoma (DOI:
Background: Thyroid tumor progression from well‐differentiated cancer to poorly
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differentiated thyroid carcinoma (PDTC) and anaplastic thyroid carcinoma (ATC) involves
step‐wise dedifferentiation associated with loss of iodine avidity and poor outcomes. ALK
fusions, typically STRN‐ALK, are found with higher incidence in human PDTC compared to
well‐differentiated cancer and, as we previously have shown, can drive the development
of murine PDTC. The aim of this study was to evaluate thyroid cancer initiation and
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progression in mice with concomitant expression of STRN‐ALK and inactivation of the
tumor suppressor p53 (Trp53) in thyroid follicular cells.
Methods: Transgenic mice with thyroid‐specific expression of STRN‐ALK and biallelic p53
loss were generated and aged on a regular diet or with methimazole and sodium
perchlorate goitrogen treatment. Development and progression of thyroid tumors were
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monitored using ultrasound imaging, followed by detailed histological and
Thyroid
immunohistochemical evaluation. Gene expression analysis was performed on selected
tumor samples using RNA‐Seq and qRT‐PCR.
Results: In mice treated with goitrogen, first thyroid cancers appeared at 6 months of age
reaching 86% penetrance by the age of 12 months, while a similar rate (71%) of tumor
occurrence in mice on regular diet was observed by 18 months of age. Histological
examination revealed well‐differentiated papillary thyroid carcinomas (PTC) (n=26), PDTC
(n=21) and ATC (n=8) that frequently co‐existed in the same thyroid gland. The tumors
were frequently lethal and associated with the development of lung metastasis in 24% of
cases. Histological and immunohistochemical characteristics of these cancers recapitulated
tumors seen in humans. Detailed analysis of PDTC revealed two tumor types with distinct
cell morphology and immunohistochemical characteristics, designated as PDTC type 1
(PDTC1) and type 2 (PDTC2). Gene expression analysis showed that PDTC1 tumors
retained higher expression of thyroid differentiation genes including Tg and Slc5a5 (Nis) as
compared to PDTC2 tumors.
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Conclusions: In this study, we generated a new mouse model of multi‐step thyroid cancer
Mouse Model of Thyroid Cancer Progression and Dedifferentiation Driven by STRN‐ALK Expression and Loss of p53: Evidence for the Existence of Two Types of Poorly Differentiated Carcinoma (DOI:
dedifferentiation with evidence of progression from PTC to PDTC and ATC. Further, PDTC
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in these mice showed two distinct histologic appearances correlated with levels of
expression of thyroid differentiation and iodine metabolism genes, suggesting a possibility
of existence of two PDTC types with different functional characteristics and potential
implication for therapeutic approaches to these tumors.
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10.1089/thy.2019.0284)
Thyroid
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INTRODUCTION
Mouse Model of Thyroid Cancer Progression and Dedifferentiation Driven by STRN‐ALK Expression and Loss of p53: Evidence for the Existence of Two Types of Poorly Differentiated Carcinoma (DOI:
Thyroid cancer is the most common malignancy of the endocrine system and showed a
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steady rise in incidence worldwide in recent decades (1, 2). Most thyroid cancers are well‐
differentiated papillary thyroid carcinomas (PTC), which overall have a more indolent
clinical behavior and 5‐year survival rates >95%; less common, poorly differentiated
thyroid carcinomas (PDTC) and anaplastic thyroid carcinomas (ATC) are aggressive cancers
with a 5‐year mortality of 30‐50% and >90%, respectively (3‐9). Despite the positive effect
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of surgical treatment and other therapeutic modalities on survival, PDTC and ATC are still
frequently lethal malignancies due to their rapid infiltrative growth, propensity for regional
and distant spread, and progressive loss of the ability to concentrate radioiodine (RAI) (4,
8‐14).
PDTC and ATC frequently develop as a result of step‐wise dedifferentiation of
preexisting well‐differentiated carcinomas (8, 10, 14‐19). PDTC is a particularly interesting
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group of tumors that have an intermediate position between well‐differentiated thyroid
Thyroid
cancers and ATC with respect to histopathologic features, molecular characteristics and
clinical outcomes (20, 21). The diagnostic criteria for PDTC adopted by the WHO
classification of endocrine tumors are based on the Turin consensus proposal (20). They
include the characteristic solid/trabecular/insular architecture, absence of nuclear features
of papillary carcinoma, and presence of a high mitotic rate, convoluted thyroid cell nuclei
or tumor necrosis. Although proposals for calling PDTC based exclusively on high‐grade
features (tumor necrosis and high mitotic count) exist (22), the Turin‐based WHO criteria
offer a standardized, reproducible, and clinically relevant approach to diagnosing these
tumors (21).
The molecular pathogenesis of PDTC involves the accumulation of genetic
alterations that encompass early driver mutations, also shared by well‐differentiated
thyroid cancers, and late driver mutations, which are characteristic of advanced thyroid
cancers including PDTC and ATC (23, 24). The most common early driver mutations are
point mutations in BRAF and RAS, but gene fusions affecting the ALK, RET, and PPARG
genes are also found. Late driver mutations commonly affect TERT and TP53, and less
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frequently genes encoding the members of the PI3K/AKT signaling pathway, SWI/SNF
Mouse Model of Thyroid Cancer Progression and Dedifferentiation Driven by STRN‐ALK Expression and Loss of p53: Evidence for the Existence of Two Types of Poorly Differentiated Carcinoma (DOI:
subunits, and histone methyltransferases (23, 24).
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PDTC also occupy an intermediate position with respect to expressing the markers
of thyroid epithelial differentiation. When studied by immunohistochemistry, PDTC
typically preserve immunoreactivity for the transcription factors TTF‐1 and PAX8;
moreover, they express cytokeratins but can lose the expression of thyroglobulin and
epithelial markers such as E‐cadherin. Similarly, on gene expression analysis, these tumors
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have decreased or lost expression of thyroid restricted genes such as SLC5A5 (sodium
iodine symporter, NIS) and TG (thyroglobulin), which are essential for thyroid hormone
synthesis, and generally preserve the expression of the key thyroid transcription factors
PAX8, TTF‐1 and FOXE1, as well as cytokeratins (24‐30). The expression and functional
activity of the NIS protein are directly related to the ability of thyroid cells to trap
radioiodine (31‐33), which is the most efficient targeted treatment of those thyroid
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carcinomas that retain iodine avidity (34).
Thyroid
Animal models of thyroid cancer are an important tool for dissecting the molecular
mechanisms of cancer progression and exploring new therapeutic approaches in
preclinical studies. Several mouse models of tumor dedifferentiation were based on
introducing BRAF V600E and other genes affected by point mutations combined with a late
event such as p53 inactivation (35‐41), and many of them showed rapid progression to
ATC, often bypassing the stage of PDTC (42‐45). In this respect, thyroid carcinogenesis
driven by ALK fusions could represent an attractive model to study PDTC because ALK
fusions, most commonly STRN‐ALK, are found in different types of human thyroid cancers
including well‐differentiated papillary carcinoma, PDTC, and ATC, with the highest
incidence (4‐9%) in PDTC tumors (24, 29, 46‐48). Furthermore, we have generated a
mouse model of thyroid‐specific expression of STRN‐ALK and showed that this fusion alone
can drive the development of PDTC with histopathological features closely recapitulating
those of PDTC in humans (49). The aim of this study was to expand this observation and
investigate the development of thyroid cancer in mice with thyroid‐specific expression of
STRN‐ALK and inactivation of p53.
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MATERIALS AND METHODS
Mouse Model of Thyroid Cancer Progression and Dedifferentiation Driven by STRN‐ALK Expression and Loss of p53: Evidence for the Existence of Two Types of Poorly Differentiated Carcinoma (DOI:
Generation of STRN‐ALK/p53KO mice and experimental design
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Experimental triple transgenic Tg‐Luc‐tdT‐STRN‐ALK+/‐;Tg‐Cre+/‐;Trp53LoxP/LoxP (STRN‐
ALK;p53KO) mice with a mixed genetic background (C57BL/6J;FVB/N) were created by
crossing already established and characterized Tg‐Luc‐tdT‐STRN‐ALK (49), Tg‐Cre (50) and
Trp53Loxp (51) mouse lines. Experimental animals were arranged into 6‐, 12‐ and 18‐month‐
age groups and aged in standard conditions (noG) or treated with goitrogen (G) (2 months
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on and 1 month off). For goitrogen treatment mice were supplied with drinking water
containing 0.5 g/L methimazole (M8506; Sigma, St. Louis, MO, USA) and 5 g/L sodium
perchlorate (410241; Sigma) starting from 5 weeks of age. For control purposes, the
C57BL/6J;FVB/N wild type and double transgenic Tg‐Cre+/‐;Trp53LoxP/LoxP (p53KO) mice
were also generated and aged. Animal care and all experimental procedures were
performed in accordance with federal guidelines and institutional policies.
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Thyroid
PCR genotyping and confirmation of thyroid‐specific Trp53 ex2‐10 deletion
DNA was extracted from tail tissues using QuickExtract™ DNA Extraction Solution
(QE09050, Epicentre, Madison, Wi, USA). Genotyping was performed by multiplex PCR
using HotStarTaq DNA polymerase (203025, Qiagen, Germantown, MD, USA) and three
pairs of primers for simultaneous detection of Tg‐Luc‐tdT‐STRN‐ALK, Tg‐Cre, Trp53LoxP and
wild type Trp53 alleles (Supplementary Table S1). For validation of the thyroid‐specific
Trp53loxP ex2‐10 deletion, DNA was extracted from snap‐frozen thyroid and spleen tissues
using a QIAmp DNA Mini Kit (51306, Qiagen) and PCR was performed as described above
with primers covering intron 1 and 10 of the Trp53 (Supplementary Table S1).
Ultrasound imaging
To monitor tumor development and growth, ultrasound screening of thyroids in
experimental mice was performed monthly using a preclinical Vevo 3100 micro ultrasound
imaging system (FUJIFILM VisualSonics, Toronto, ON, Canada). For high‐frequency analysis,
40 MHz B‐Mode imaging with the MX550D transducer with an axial resolution of 40 μm
was used.
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Thyroid tumor samples and serum collection
Mouse Model of Thyroid Cancer Progression and Dedifferentiation Driven by STRN‐ALK Expression and Loss of p53: Evidence for the Existence of Two Types of Poorly Differentiated Carcinoma (DOI:
Following euthanasia, blood serum was collected by cardiac puncture and stored at ‐80°C
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before measurement of serum TSH and free T4 as described previously (52). Guided by the
pre‐sacrifice ultrasound imaging and tumor‐specific red fluorescence from tdTomato,
tissue samples from areas of primary tumors with different morphological appearance
were dissected and snap‐frozen in liquid nitrogen. Remaining tumor tissues were fixed
with 10% buffered FA at 4°C overnight and embedded in paraffin.
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Histological and immunohistochemical analysis
Formalin‐fixed paraffin‐embedded (FFPE) tissues were sectioned at a thickness of 4 μm,
deparaffinized and stained with hematoxylin and eosin (H&E) or immunohistochemically
(IHC). For IHC, antigen retrieval and non‐specific blocking were done as described
previously (49). Endogenous biotin was blocked with streptavidin (Thermo Fisher Scientific,
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Waltham, MA, USA) followed by incubation with biotin (Thermo Fisher Scientific)
Thyroid
according to the manufacturer instructions. Primary antibodies (Supplementary Table S2)
were applied overnight at 4°C. Secondary biotinylated anti‐rabbit antibodies (1:1000; BA‐
1000; Thermo Fisher Scientific) were applied for 1 hour at room temperature. Following
the incubation with complexes of biotinylated peroxidase (Thermo Fisher Scientific) and
streptavidin for 20 minutes at room temperature, the visualization of the
immunoreactivity was done using DAB Peroxidase Substrate Kit (Vector Laboratories,
Burlingame, CA) according to manufacturer instructions.
RNA extraction, quantitative RT‐PCR, RNA sequencing and gene expression analysis
Tumor samples with verified distinct histology were subjected to RNA extraction. Total
RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA) from frozen thyroid
tissues, followed by treatment with DNAse I (Invitrogen) and purification with RNeasy Mini
Kit (Qiagen). RNA from FFPE tumor samples was extracted using an RNeasy FFPE kit
(Qiagen) after H&E‐guided microdissection of tumor regions of interest.
For quantitative RT‐ PCR (qRT‐PCR), the purified RNA was reverse transcribed using
a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA).
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Samples were tested in triplicates using a 7500 Real‐Time PCR System (Applied Biosystems,
Mouse Model of Thyroid Cancer Progression and Dedifferentiation Driven by STRN‐ALK Expression and Loss of p53: Evidence for the Existence of Two Types of Poorly Differentiated Carcinoma (DOI:
Foster City, CA, USA) with QuantiTect SYBR® Green PCR Kit (Qiagen) and primers listed in
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Supplementary Table S1. Expression levels of each gene relative to β‐actin (Actb) were
calculated using Q‐Gene software (53). To minimize biases associated with goitrogen
treatment, all gene expression data from tumor samples were normalized to the average
expression levels of corresponding genes in non‐treated (n=5) or goitrogen‐treated (n=5)
benign thyroid samples.
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For RNA sequencing, RNA extracted from frozen tissues was subjected to library
preparation using NEBNext® Ultra™ II RNA Library Prep Kit for Illumina (NEB, Ipswich, MA,
USA) and paired‐end sequenced on the Illumina NovaSeq 6000 S4 flow cell (20M PE150
reads) by Novogene (Novogene Corporation, Sacramento, CA, USA). Raw sequencing data
were imported into CLC Biomedical Genomics Workbench 12 (Qiagen), high‐quality
trimmed paired reads were aligned to the mouse genome (Ensembl v80) and the number
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of reads mapped to each gene was normalized using TPM. The principal component
Thyroid
analysis (PCA) was performed on whole‐transcriptome data. For differential gene
expression (DEG) analysis, gene expression data from type 1 PDTC (n=3) and type 2 PDTC
(n=3) tumor samples were compared to gene expression in benign (noG=2, G=2) thyroid
samples while controlling for goitrogen treatment. Genes with an absolute fold change >3
and FDR p‐value of <0.05 were selected as differentially expressed genes and used for
building the Venn diagram. Heatmap of the expression of 15 preselected thyroid
differentiation genes was generated using complete‐linkage clustering (Manhattan
distance). ERK‐score calculation was performed as previously described (47). Gene
expression datasets were additionally analyzed using GSEA (MSidDB database,
http://software.broadinstitute.org/gsea/index.jsp) and Ingenuity Pathway Analysis
(Qiagen) software.
Statistical analysis
Statistical analysis was performed using IBM SPSS Statistics version 21 (IBM, Armonk, NY).
P‐values were 2‐sided and considered significant if ≤0.05. DEG analysis was performed
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using multi‐factorial statistics based on a negative binominal generalized linear model (by
Mouse Model of Thyroid Cancer Progression and Dedifferentiation Driven by STRN‐ALK Expression and Loss of p53: Evidence for the Existence of Two Types of Poorly Differentiated Carcinoma (DOI:
default in CLC Biomedical Genomics Workbench 12).
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RESULTS
Generation of mice with thyroid‐specific expression of STRN‐ALK and loss of p53
In order to investigate the effect of STRN‐ALK expression combined with a common late
event, inactivation of p53 function, we created triple transgenic mice by crossing the
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existing Tg‐Luc‐tdT‐STRN‐ALK, Tg‐Cre and Trp53Loxp mouse lines (Fig. 1A, B). Final Tg‐Luc‐
tdT‐STRN‐ALK+/‐;Tg‐Cre+/‐;Trp53LoxP/LoxP (STRN‐ALK;p53KO) experimental animals had
thyroid‐specific expression of the HA‐tagged STRN‐ALK oncoprotein driven by the
transgenic thyroglobulin promoter (Supplementary Fig. S1) and Cre‐recombinase‐mediated
thyroid‐specific deletion of LoxP‐flanked exons 2‐10 of Trp53LoxP alleles (Fig. 1C),
introduced in a homozygous state.
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STRN‐ALK;p53KO mice develop metastatic and lethal thyroid tumors with high
Thyroid
penetrance
STRN‐ALK;p53KO mice were divided into 6‐, 12‐ and 18‐month‐age groups (n=11‐16 per
age/treatment group) and housed on normal diet or on goitrogen treatment, which led to
a ~ 3‐fold decrease in FT4 and a ~100‐fold rise in TSH levels (Supplementary Fig. S2).
Thyroid tumor development and progression in these mice were monitored using
ultrasonographic screening. Ultrasound imaging allowed early detection of ~1 mm thyroid
nodules from the age of 7 and 5 months in mice on regular diet (noG) or treated with
goitrogen (G), respectively. Upon initial detection, continuous monitoring of tumor
nodules showed their gradual increase in volume and, in many cases, transition to a phase
of rapid growth accompanied by changes in ultrasonographic patterns. Subsequent
histological examination of such cases revealed areas of PDTC growing adjacent to PTC
(Fig. 2A, B). Visual and manual examination of the mice generally revealed large hard
cervical masses fixed to the surrounding tissues.
Thyroid cancers were first detected on histological analysis of 6‐month old mice on
goitrogen treatment (Table 1). With continued aging, about half of tumor‐bearing animals
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(noG, n=7; G, n=10) were removed from the experiment before the planned sacrifice date
Mouse Model of Thyroid Cancer Progression and Dedifferentiation Driven by STRN‐ALK Expression and Loss of p53: Evidence for the Existence of Two Types of Poorly Differentiated Carcinoma (DOI:
due to the development of life‐threatening conditions. By the age of 12 months, 48% of
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mice on regular diet and 86% mice treated with goitrogen developed histologically
confirmed thyroid cancers. In mice treated with goitrogen, the distribution of histological
cancer types was shifted to dedifferentiated tumors (Table 1). Among age‐matched
controls, wild type (noG, n=17; G, n=11) and p53KO (noG, n=22; G, n=10) mice developed
no cancer.
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Irrespective of goitrogen treatment, thyroid cancers in these mice showed
histological features of aggressive behavior including extrathyroidal growth and invasion
into trachea and blood vessels (Fig. 2C). Lung metastases were found in 8 (24%) tumor‐
bearing mice and were presented as multiple small nodules (Fig. 2D,E and Table 1). Most
metastatic tumors had either PDTC histology or contained areas with features of PTC and
PDTC.
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Thyroid tumors in STRN‐ALK;p53KO mice undergo step‐wise dedifferentiation
Thyroid
Thyroid tumors developed in these mice included well‐differentiated PTC (n=26), PDTC
(n=21) and ATC (n=8), which frequently co‐existed in the same thyroid gland, although
typically grew as well‐defined tumor nodules (Table 2; Fig. 3). The co‐occurrence of PTC
and PDTC was the most common (n=11), but all other combinations of PTC, PDTC, and ATC
were also observed (Table 2).
PTCs had microscopic features of different tumor variants including follicular
variant, classic type of papillary carcinoma, and hobnail variant. Although PTC was overall
the most common tumor type found, in many mice it was seen as a smaller component,
whereas PDTC tumors were typically most dominant, occupying the majority of the thyroid
volume and reaching a size of 8‐15 mm (Fig. 3). Histologically, all PDTC tumors had
microscopic characteristics similar to those seen in PDTC in humans as defined by the Turin
criteria. They commonly showed a solid growth pattern, although insular and trabecular
architecture were also observed. Nuclear features of papillary carcinoma were lost, and
high mitotic activity and tumor necrosis were frequently seen. Eight cases showed
anaplastic transformation, however the anaplastic component was not prominent and was
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present as small distinct foci (maximum size of 2‐3 mm) of ATC adjacent to PDTC (6 out of
Mouse Model of Thyroid Cancer Progression and Dedifferentiation Driven by STRN‐ALK Expression and Loss of p53: Evidence for the Existence of Two Types of Poorly Differentiated Carcinoma (DOI:
8 ATC) or next to PTC (2 out of 8 ATC). The ATC tumors typically had a pleomorphic cell
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appearance, but spindle cell and squamoid patterns were also seen.
Immunohistochemical analysis showed that PTC and PDTC tumors had strong
expression of HA‐tagged STRN‐ALK whereas cells in ATC generally had focal weak or no
expression of the transgene (Fig. 3). All PTCs and PDTCs preserved the expression of the
transcription factors TTF‐1 and PAX8, with typically higher levels in PTCs. All PTCs and
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approximately half of PDTCs preserved immunoreactivity for thyroglobulin. ATC lost
expression of all of these thyroid differentiation markers. Expression of epithelial markers
cytokeratins 17/19 and E‐cadherin showed a gradual decrease and complete loss with
tumor dedifferentiation, with the opposite trend for vimentin, a mesenchymal marker.
PDTC tumors in STRN‐ALK;p53KO mice show two subtypes with distinct histological and
immunohistochemical features
10.1089/thy.2019.0284)
Thyroid
Further histological analysis of the PDTC tumors led us to identify two distinct microscopic
tumor appearances which correlated with the expression of thyroid differentiation
markers detectable by immunohistochemistry (Fig. 4). Although all of these PDTC tumors
fully met the Turin histological criteria of PDTC, 12 tumor nodules were composed of solid
sheets of cells that retained a significant amount of cytoplasm and had smaller‐sized nuclei
with dense, evenly distributed chromatin; we defined these tumors as poorly
differentiated carcinomas of type 1 (PDTC1) (Fig. 4D). We also found 13 PDTC tumor
nodules composed of solid sheets of cells with a reduced amount of cytoplasm and larger,
vesicular nuclei that had prominent and frequently multiple nucleoli; these tumors were
designated as poorly differentiated carcinomas type 2 (PDTC2) (Fig. 4D). In the majority of
mice, the PDTC tumors had histological features of either type 1 (n=8) or type 2 (n=9),
whereas in 4 cases the mouse thyroid glands had nodules with PDTC1 and PDTC2
appearance co‐existing as distinct, well‐delineated nodules located next to each other (Fig.
4B‐C). Foci of anaplastic transformation were found adjacent to PDTC2 tumors in five cases
and adjacent to a PDTC1 tumor in one case. The distribution of PDTC1 and PDTC2 tumors
was similar in mice with and without goitrogen treatment.
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Although immunohistochemical analysis revealed that both PDTC types had
Mouse Model of Thyroid Cancer Progression and Dedifferentiation Driven by STRN‐ALK Expression and Loss of p53: Evidence for the Existence of Two Types of Poorly Differentiated Carcinoma (DOI:
comparable levels of STRN‐ALK transgene expression, evaluated by staining with the anti‐
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HA tag antibody, the immunoreactivity for several thyroid cell markers showed significant
differences between these PDTC types (Fig. 4E). Specifically, PDTC1 tumors consistently
retained typically diffuse and moderately strong or weak staining for thyroglobulin and E‐
cadherin, while both stainings were frequently lost in PDTC2 tumors. TTF‐1 and PAX8
immunoreactivity was preserved in both PDTC types, although in some PDTC2 tumors it
was weaker.
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Review of ultrasound images corresponding to histologically diagnosed PDTC1 and
PDTC2 nodules obtained during different time points allowed to trace back the appearance
and growth of these tumors. In those cases where both tumor types were present in one
thyroid gland, the analysis revealed that PDTC1 tumors appeared at earlier age as
compared to PDTC2 (Fig. 4A‐C).
10.1089/thy.2019.0284)
Type 1 and type 2 PDTC tumors have different expression levels of thyroid
Thyroid
differentiation genes
To further explore potential differences between PDTC tumors with type 1 and type 2
morphology, we investigated gene expression profiles of these tumors using RNA‐Seq. We
generated gene expression data for six PDTC tumors (PDTC1, n=3; PDTC2, n=3) as wells as
two well‐differentiated PTC and four benign thyroids (noG, n=2; G, n=2). The unsupervised
principal component analysis (PCA) of tumor transcriptional profiles showed that PDTC and
PTC were clustered separately from each other and from benign thyroids. Moreover, type
1 and type 2 PDTC tumors also appeared to cluster separately (Fig. 5A).
Gene expression analysis in PDTC1 and PDTC2 tumors was performed in
comparison to benign thyroid tissues (absolute fold change >3; FDR p‐value ≤ 0.05)
(Supplementary Table S3A). The analysis revealed significant differences in gene
expression profiles between type 1 and type 2 PDTC tumors (Fig. 5B). Further analysis of
gene expression sets from these tumors showed that PDTC2 tumors had a significant
upregulation of pathways related to cell cycle control and mitosis, downregulation of
genes associated with the epithelial phenotype (Supplementary Fig S3 A, B), and
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15
enrichment in other pathways including thyroid hormone biosynthesis (Supplementary
Mouse Model of Thyroid Cancer Progression and Dedifferentiation Driven by STRN‐ALK Expression and Loss of p53: Evidence for the Existence of Two Types of Poorly Differentiated Carcinoma (DOI:
Table S4A‐C).
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Next, we focused on the analysis of genes involved in thyroid differentiation, iodine
uptake and metabolism. As compared to benign thyroid tissues, the expression levels of
several thyroid genes involved in iodide metabolism and thyroid hormone synthesis such
as Tg, Tpo, Ano1, and Duox2 were significantly decreased in PDTC2 but not in PDTC1
(Supplementary Table S3B). Both types of PDTC had significantly lower expression of
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Slc5a5 (Nis) as compared to benign thyroid tissue, but the average expression was
approximately two‐fold lower in PDTC2. Some of other thyroid differentiation genes (Tshr,
Foxe1, and Glis3) showed a tendency for a lower expression in PDTC2, and in clustering
analysis of thyroid differentiation genes PDTC1 and PDTC2 tumors formed two distinct
clusters (Fig. 5C).To test if the observed downregulation of thyroid differentiation genes
expression is associated with an increased MAPK pathway activity, we calculated the ERK‐
10.1089/thy.2019.0284)
score, which showed a tendency to be higher in PDTC2 tumors, although the number of
Thyroid
studied samples was low (Supplementary Fig. S3C).
To validate the RNA‐Seq data, we performed qRT‐PCR analysis to compare the
expression of key thyroid follicular cell differentiation and iodide metabolism genes
between five PDTC1 and five PDTC2 tumors. The analysis showed that PDTC2 tumors had
significantly lower expression of the Slc5a5 (Nis), Tg, Tpo, and Duox2 genes, whereas the
expression of Tshr, Nkx2‐1 (Ttf‐1), Pax8 and Foxe1 in these tumors displayed a tendency to
decrease, which however did not reach statistical significance (Fig. 5D). These findings
provide further evidence supporting the existence of two types of PDTC in STRN‐
ALK;p53KO mice, which have not only distinct histological and immunohistochemical
characteristics but also different transcriptomic profiles, including genes that are of
relevance for a differentiated thyroid phenotype.
DISCUSSION
In this study, we generated a new mouse model of thyroid‐specific expression of STRN‐ALK
and inactivation of p53 and show that these animals develop metastatic and frequently
lethal thyroid cancers with a step‐wise progression from PTC to PDTC and ATC and
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phenotypical characteristics recapitulating thyroid cancers in humans. Further, we
Mouse Model of Thyroid Cancer Progression and Dedifferentiation Driven by STRN‐ALK Expression and Loss of p53: Evidence for the Existence of Two Types of Poorly Differentiated Carcinoma (DOI:
demonstrate that in this mouse model the PDTC tumors have two distinct morphological
This paper has been peer‐reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
appearances that correspond to different levels of expression of thyroid differentiation
genes.
Fusions of the ALK oncogene are found in various types of thyroid cancers, with a
higher incidence in dedifferentiated tumors and particularly in PDTC as compared to well‐
differentiated thyroid PTC (24, 46‐48). In a recent study, we have confirmed the oncogenic
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properties of the most common type of ALK fusion, STRN‐ALK, and demonstrated that
mice with thyroid‐specific expression of STRN‐ALK develop PDTC (49). Based on the fact
that human PDTC and ATC frequently harbor additional late mutations such as alterations
of TP53 (23, 24), we expanded our mouse model of STRN‐ALK‐driven PDTC by adding a
thyroid‐specific biallelic inactivation of p53. Loss of p53 function in these mice lead to a
higher penetrance with respect to generation of PDTC and progression to complete tumor
10.1089/thy.2019.0284)
dedifferentiation with formation of ATC.
Thyroid
The observed patterns of co‐occurrence of tumors with different degrees of
differentiation and a shift to more dedifferentiated tumors in older animals, especially
after treatment with goitrogen, recapitulate the accepted paradigm of step‐wise
dedifferentiation of thyroid cancer in humans. In includes the progression of well‐
differentiated cancer like PTC to less differentiated PDTC and fully dedifferentiated ATC, a
process that is accompanied by progressive accumulation of driver mutations and a loss of
tumor suppressors (54, 55). This progression model is supported by the co‐occurrence of
well‐differentiated and/or poorly differentiated cancer areas in 30‐80% of ATC in humans
(12, 16, 56‐61). Additional and more direct evidence is offered by animal models of thyroid
cancer driven by an early event such as activating BRAF or RAS mutations or inactivation of
Pten combined with the loss of p53 or Nf2 tumor suppressor genes or activating mutation
in PIK3CA. These animals develop thyroid cancer with either rapid anaplastic
transformation (42‐45) or show cancer dedifferentiation with a well‐defined step of PDTC
(35‐41).
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The tyroid tumors present in this mouse model of thyroid‐specific STRN‐ALK
Mouse Model of Thyroid Cancer Progression and Dedifferentiation Driven by STRN‐ALK Expression and Loss of p53: Evidence for the Existence of Two Types of Poorly Differentiated Carcinoma (DOI:
expression coupled with p53 inactivation are morphologically and functionally similar to
This paper has been peer‐reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
the respective cancer types seen in humans. Interestingly, among PTC tumors, we
observed classical papillary type, follicular variant, as well as hobnail variant tumors. The
latter resembled the hobnail variant of PTC in humans that frequently carry mutations in
TP53 and show progression to PDTC (62‐66). In many of these mice, PDTC was the
dominant tumor type found on histological examination. These tumors had all
morphologic characteristics defined for human PDTC by the Turin criteria which have been
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adopted by the WHO classification of endocrine tumors (20, 21). Similar to human tumors,
PDTC in these mice show a variable decrease or loss of immunoreactivity for thyroglobulin
and epithelial markers such as E‐cadherin and preserved PAX8 and TTF‐1 thyroid
differentiation markers, consistent with an intermediate immunophenotype between well‐
differentiated cancer and ATC observed in humans (25, 26, 28, 29, 67, 68).
10.1089/thy.2019.0284)
However, despite fully meeting the Turin diagnostic criteria, PDTC tumors in STRN‐
Thyroid
ALK;p53KO mice revealed two distinct tumor cell appearances with respect to the amount
of cytoplasm, size of nuclei and appearance of chromatin, and nucleus to cytoplasm (N:C)
ratio. Tumors designated as PDTC1 are composed of cells with smaller hyperchromatic
nuclei, a larger amount of cytoplasm, and an overall lower N:C ratio. This cellular
appearance is more reminiscent of well‐differentiated thyroid cancer cells, and indeed,
these tumors preserve stronger immunoreactivity for thyroglobulin and E‐cadherin and
higher levels of expression of mRNA of genes related to thyroid differentiation and iodine
metabolism. In contrast, PDTC2 tumors are composed of cells with larger nuclei, vesicular
chromatin with prominent and typically multiple nuclei, a smaller volume of cytoplasm,
and a higher N:C ratio. These cells have virtually no resemblance to differentiated thyroid
cancer cells, but they retain a monomorphic appearance of the nuclei without the marked
atypia seen in AC cells. Compared to PDTC1, PDTC2 tumors show a more pronounced loss
of markers of thyroid differentiation on immunohistochemistry and gene expression
analysis. However, both types of PDTC found in this study do not differ with respect to
predominantly solid growth pattern, high mitotic activity, and presence of coagulative
necrosis, which are diagnostic features of PDTC in humans.
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Most tumor‐bearing thyroid glands contained either PDTC type 1 or type 2 tumors,
Mouse Model of Thyroid Cancer Progression and Dedifferentiation Driven by STRN‐ALK Expression and Loss of p53: Evidence for the Existence of Two Types of Poorly Differentiated Carcinoma (DOI:
which frequently were located adjacent to areas of well‐differentiated PTC. However, in
This paper has been peer‐reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
four cases, both PDTC types were found co‐existing in the same thyroid. Reconstruction of
tumor nodule growth using ultrasound images taken at different time intervals suggests
the possibility of progression from PDTC1 to PDTC2. Furthermore, in most cases, areas of
ATC were found adjacent to type 2 PDTC. These findings point towards the possibility of
progression from PDTC1 to PDTC2 and then to ATC. Nevertheless, in two cases, the areas
of ATC were seen adjacent to PTC without appreciable PDTC tumor present, which
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suggests that a direct transition from well differentiated PTC to ATC may also occur.
Overall, based on the frequencies of various tumor types coexisting in the same thyroid, it
appears that dedifferentiation of thyroid cancer driven by activated ALK kinase coupled
with loss of p53 may often involve a PTC → PDTC1 → PDTC2 → ATC progression, although
deviations from such linearity or more direct dedifferentiation pathways are also possible
(Fig. 6).
10.1089/thy.2019.0284)
Thyroid
Differences in expression of Nis and other genes involved in thyroidal iodide uptake
and organification found between the two PDTC types in these mice are intriguing and
potentially relevant. In humans, PDTCs are known to have significant variability in iodine
uptake and response to RAI. In fact, most studies showed that approximately half of PDTCs
in humans retain substantial and occasionally a strong ability to concentrate RAI, whereas
the other half are RAI‐refractory (4, 69, 70). The results of this study raise at least a
theoretical possibility that the group of PDTC defined by the current diagnostic criteria is
not homogeneous and, while still occupying an intermediate position in terms of thyroid
differentiation between well‐differentiated cancer and ATC, may include two tumor types
with different degrees of expression of iodide‐metabolizing genes and responses to RAI.
However, it remains unclear whether the same PDTC types can be found in humans and
whether or not the differences in expression of thyroid differentiation genes would
translate into variable abilities of these tumor cells to concentrate and retain radioiodine.
We observed that goitrogen treatment accelerated thyroid tumor development
and progression from PTC to PDTC and ATC. This is likely caused by the elevated TSH levels
and, surprisingly, preserved expression of Tshr in the majority of mouse PDTC tumors. This
Page 19 of 41
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observation is consistent with other studies showing that elevated levels of TSH play a role
Mouse Model of Thyroid Cancer Progression and Dedifferentiation Driven by STRN‐ALK Expression and Loss of p53: Evidence for the Existence of Two Types of Poorly Differentiated Carcinoma (DOI:
in initiation and progression of thyroid cancers and are associated with a more severe
This paper has been peer‐reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
tumor phenotype in mice (71, 72) and humans (73‐75).
This mouse model has a limitation related to the expression of STRN‐ALK controlled
by a transgenic thyroglobulin promoter, which results in a significant decrease or
disappearance of transgene expression during anaplastic transformation. This may be
responsible for a lower penetrance with respect to the formation of ATC, and make these
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mice unsuitable to study the effect of targeted ALK kinase inhibition. The generation of a
new doxycycline‐inducible STRN‐ALK mouse line is ongoing, which should overcome the
differentiation‐dependent limitation of the current model. In addition, the number of
tumor samples used for histological and molecular characterization of the two groups of
PDTC tumors was limited and the ability of these tumors to concentrate and retain RAI was
not analyzed in this study. Therefore, it would be important to expand the analysis to a
10.1089/thy.2019.0284)
larger cohort of PDTC samples and evaluate RAI uptake by PDTC type 1 and type 2 tumors
Thyroid
in vivo or using established tumor cell primary cultures.
In summary, the results of our study demonstrate that expression of STRN‐ALK with
simultaneous loss of p53 function in murine thyroid cells initiates a program of thyroid
carcinogenesis and multi‐step dedifferentiation with evidence of progression from PTC to
PDTC and ATC. Furthermore, our findings raise the possibility of the existence of two PDTC
types with different levels of thyroid differentiation and expression of iodine‐metabolizing
genes. These findings may prove to be relevant to human PDTC, which requires further
investigation.
ACKNOWLEDGMENTS
We thank Dr. Satdarshan P. Monga and Dr. Aaron W. Bell (Division of Experimental
Pathology, Department of Pathology, University of Pittsburgh, Pittsburgh, PA, USA) for the
help with mouse ultrasound imaging screening. We also thank Dr. Samuel Refetoff and Dr.
Xia Hui Liao (Departments of Medicine (X‐H.L., S.R.) and Pediatrics and Genetics (S.R), The
University of Chicago, Chicago, IL, USA) for assistance with measurement of mouse TSH
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Thyroid
Mouse Model of Thyroid Cancer Progression and Dedifferentiation Driven by STRN‐ALK Expression and Loss of p53: Evidence for the Existence of Two Types of Poorly Differentiated Carcinoma (DOI:
10.1089/thy.2019.0284)
This paper has been peer‐reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
CORRESPONDENCE
nikiforovye@upmc.edu
DISCLOSURE STATEMENT
No competing financial interests exist
supported by the National Institutes of Health grant CA181150.
8031, 3477 Euler Way, Pittsburgh, PA, 15213; Fax: (412)‐802‐6799; E‐mail:
and FT4 supported by the National Institutes of Health grant DK15070. This work was
Yuri E. Nikiforov, MD, PhD, Department of Pathology, University of Pittsburgh, CLB Room
20
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Table 1. Cancer frequencies in STRN‐ALK;p53KO mice
Mouse Model of Thyroid Cancer Progression and Dedifferentiation Driven by STRN‐ALK Expression and Loss of p53: Evidence for the Existence of Two Types of Poorly Differentiated Carcinoma (DOI:
Cancer type** Lung
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Age Total
Goitr metastases,
group*, number
ogen PTC PDTC ATC Any type % of primary
months of mice
tumors
6 13 ‐ ‐ ‐ ‐ ‐
‐ 12 23 11 (48%) 5 (22%) 3 (13%) 11 (48%) 1 (9%)
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** Some mice had more than one cancer type
Thyroid
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Thyroid
Mouse Model of Thyroid Cancer Progression and Dedifferentiation Driven by STRN‐ALK Expression and Loss of p53: Evidence for the Existence of Two Types of Poorly Differentiated Carcinoma (DOI:
10.1089/thy.2019.0284)
This paper has been peer‐reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
‐
+
n
Goitroge
Total
8
2
10
PTC only
0
4
4
only
PDTC
4
7
11
PDTC
PTC &
2
1
ATC
PTC &
Cancer type
3
3
ATC
PDTC &
Table 2. Thyroid cancer types in all age groups of STRN‐ALK;p53KO mice
3
0
3
ATC
PTC & PDTC &
32
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Page 33 of 41
33
FIGURE LEGENDS
Mouse Model of Thyroid Cancer Progression and Dedifferentiation Driven by STRN‐ALK Expression and Loss of p53: Evidence for the Existence of Two Types of Poorly Differentiated Carcinoma (DOI:
This paper has been peer‐reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
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Figure 1. STRN‐ALK;p53KO mice generation and validation of the genotypes. (A) Simplified
overview of mouse breeding and scheme of transgenic alleles and their interaction
resulting in the STRN‐ALK;p53KO phenotype. tpTg, transgenic thyroglobulin promoter; Cre
encodes transgenic Cre recombinase; Luc‐tdT encodes transgenic fusion protein of
Luciferase (not measured in this study) and tdTomato (red fluorescence was used for
10.1089/thy.2019.0284)
detection of lung metastases during dissection) ; T2A, “self‐cleaving” peptide sequence;
HA‐STRN‐ALK encodes transgenic HA‐tagged STRN‐ALK; epTrp53, endogenous Trp53
Thyroid
promoter; Trp53 encodes endogenous TRP53 (p53). (B) Representative results of multiplex
PCR used for verification of the genotypes during the generation of experimental mice. (C)
PCR confirmation of thyroid‐specific deletion of exons 2‐10 in Trp53loxP alleles in STRN‐
ALK;p53KO mice.
Page 34 of 41
34
Mouse Model of Thyroid Cancer Progression and Dedifferentiation Driven by STRN‐ALK Expression and Loss of p53: Evidence for the Existence of Two Types of Poorly Differentiated Carcinoma (DOI:
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10.1089/thy.2019.0284)
Thyroid
Figure 2. Ultrasonographic monitoring, survival and tumor phenotype of STRN‐ALK;p53KO
mice. (A) Ultrasonography‐based follow up of tumor development and progression. PTC,
papillary thyroid carcinoma; PDTC, poorly differentiated thyroid carcinoma. (A and B)
Correlation of ultrasonographic patterns with histopathological findings. The area of PDTC
shows marked hypoechogenicity compared to PTC. (B) Representative microscopic H&E
images showing the co‐occurrence of PTC and PDTC in the same thyroid lobe. (C)
Representative microscopic H&E images of extrathyroidal extension (ETE) into the larynx,
tracheal (TI) and vascular invasion (VI). (D) Representative gross‐necropsy image showing
cervical tumor mass, dislocation of the trachea (black arrow) and red fluorescence (from
tdTomato encoded by same transgenic allele as HA‐STRN‐ALK) of the primary tumor and
lung metastases (white arrows). (E) Representative image of PDTC lung metastases
positive for thyroglobulin (TG) and HA‐tagged STRN‐ALK (HA).
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Thyroid
Mouse Model of Thyroid Cancer Progression and Dedifferentiation Driven by STRN‐ALK Expression and Loss of p53: Evidence for the Existence of Two Types of Poorly Differentiated Carcinoma (DOI:
10.1089/thy.2019.0284)
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Page 35 of 41
differentiated thyroid carcinoma; ATC, anaplastic thyroid carcinoma.
Figure 3. Histological and immunohistochemical features of different stages of thyroid
thyroid carcinoma; hvPTC, hobnail variant of papillary thyroid carcinoma; PDTC, poorly
cancer dedifferentiation seen in STRN‐ALK;p53KO mice. fvPTC, follicular variant of papillary
35
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36
Mouse Model of Thyroid Cancer Progression and Dedifferentiation Driven by STRN‐ALK Expression and Loss of p53: Evidence for the Existence of Two Types of Poorly Differentiated Carcinoma (DOI:
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10.1089/thy.2019.0284)
Thyroid
Figure 4. Ultrasonographic, microscopic and immunohistochemical features of poorly
differentiated thyroid carcinoma type 1 (PDTC 1) and type 2 (PDTC 2) in STRN‐ALK;p53KO
mice. (A) Ultrasonographic image of the thyroid gland with PDTC type 1 and (B)
subsequent appearance of PDTC type 2 nodule within one month. (B and C) Correlation
between ultrasonographic and histological appearances of PDTC1 and PDTC2. Type 2 PDTC
with more solid morphology shows lower echogenicity compared to type 1 PDTC. (D)
Representative microscopic H&E images showing histological differences between type 1
and type 2 of PDTCs. High‐power inserts represent differences in nuclear‐to‐cytoplasm
ratio and nuclear features between two types of PDTC. (E) Representative microscopic
H&E and immunohistochemistry images showing preserved expression of HA‐tagged
STRN‐ALK and thyroid differentiation marker TTF‐1 in both types of PDTC; and loss of
thyroglobulin and E‐cadherin immunoreactivities in PDTC2.
Page 37 of 41
37
Mouse Model of Thyroid Cancer Progression and Dedifferentiation Driven by STRN‐ALK Expression and Loss of p53: Evidence for the Existence of Two Types of Poorly Differentiated Carcinoma (DOI:
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10.1089/thy.2019.0284)
Thyroid
Figure 5. Distinct gene expression profiles of type 1 and type 2 PDTC tumors. (A) Principal
component analysis of whole‐transcriptome data obtained from type 1 PDTC (n=3), type 2
PDTC (n=3) and PTC (n=2) tumor samples and benign (regular diet (BnoG),n=2; goitrogen
(BG), n=2) thyroids. Each dot corresponds to a particular sample. PDTC1, poorly
differentiated thyroid carcinoma of type 1, PDTC2, poorly differentiated thyroid carcinoma
of type 2; PTC, papillary thyroid carcinoma. (B) Venn diagram showing differentially
expressed genes (absolute fold change >3, FDR p‐value of ≤0.05) in PDTC1 (n=3) and PDTC2
(n=3) compared to benign thyroids (n=4). (C), Cluster analysis and heatmap of 15 thyroid
differentiation genes expression in PDTC1 (n=3) and PDTC2 (n=3) tumor samples. (D), Box‐
plots showing relative expression of main thyroid differentiation genes in PDTC1 (n=5) and
PDTC2 (n=5) normalized to benign thyroids (regular diet, n=5; goitrogen, n=5) while
controlling for goitrogen treatment. Data presented as % of corresponding gene
expression in benign thyroid tissues. Boxes represent 25th‐75th percentiles; Whiskers
represent maximum and minimum values. *, P‐value <0.05, **, P‐value <0.005, Mann‐
Whitney test.
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Thyroid
Mouse Model of Thyroid Cancer Progression and Dedifferentiation Driven by STRN‐ALK Expression and Loss of p53: Evidence for the Existence of Two Types of Poorly Differentiated Carcinoma (DOI:
10.1089/thy.2019.0284)
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ALK;p53KO mice. Thickness of arrows represents number of cases.
Figure 6. Putative scheme of progression and dedifferentiation of thyroid cancer in STRN‐
38
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Page 39 of 41
39
Supplementary Table S1. Primers used in the study
Mouse Model of Thyroid Cancer Progression and Dedifferentiation Driven by STRN‐ALK Expression and Loss of p53: Evidence for the Existence of Two Types of Poorly Differentiated Carcinoma (DOI:
This paper has been peer‐reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
Primers used for genotyping
F_Luc CAGTCGTCGTGCTGGAACA
Tg‐Luc‐tdT‐STRN‐ALK 144
R_Luc CAACTTGCCGGTCAGTCCTT
Wild type Trp53 and F_int1_p53 GGTTAAACCCAGCTTGACCA wt Trp53 ‐270;
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Thyroid
Mouse Model of Thyroid Cancer Progression and Dedifferentiation Driven by STRN‐ALK Expression and Loss of p53: Evidence for the Existence of Two Types of Poorly Differentiated Carcinoma (DOI:
10.1089/thy.2019.0284)
This paper has been peer‐reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
Actb
Tshr
Pax8
Foxe1
Nkx2.1(Ttf‐1)
Tshr_F
Tshr_R
Actb_F
Actb_R
Pax8_F
Pax8_R
Foxe1_F
Foxe1_R
Nkx2.1_F
Nkx2.1_R
GGATGCTGCCAGTCTCGTA
CTCTCTTACCCGAGCCACTG
AACCTCACCCTCAACGACTG
CAAGCGCATCTCACGTCTCA
GCTTTCGAACATGTCCTCGG
GGACCCCTCAAGATGTTCAC
GTCAACGCATTGTGGACTTG
TCCAGCCTATCCCATCTGAACT
CTGAACCCTAAGGCCAACCGTG
GGCATACAGGGACAGCACAGCC
101
108
117
103
130
40
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41
Supplementary Table S2. Primary antibodies used in the study
Mouse Model of Thyroid Cancer Progression and Dedifferentiation Driven by STRN‐ALK Expression and Loss of p53: Evidence for the Existence of Two Types of Poorly Differentiated Carcinoma (DOI:
This paper has been peer‐reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
Catalog
Antibody Host Dilution Manufacture
number
Cell Signaling, Danvers, MA,
anti‐HA‐Tag Rabbit 1:400 #3724
USA
Proteintech, Rosemont, IL,
anti‐PAX8 Rabbit 1:500 10336‐1‐AP
USA
Dako, Agilent Technologies,
anti‐Thyroglobulin Rabbit 1:3000 A251
Santa Clara, CA, USA
Cell Signaling, Danvers, MA,
anti‐CK17/19 Rabbit 1:300 #3184
USA
10.1089/thy.2019.0284)
Cell Signaling, Danvers, MA,
anti‐E‐cadherin Rabbit 1:200 #3195
Thyroid
USA
Cell Signaling, Danvers, MA,
anti‐Vimentin Rabbit 1:50 #5741
USA
Thermo Fisher Scientific,
anti‐Ki‐67 Rabbit 1:100 RM‐9106
Waltham, MA, USA