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Zarkesh Maryam [email protected],Zadeh-Vakili [email protected],Azizi Fereidoun [email protected],fanaie seyed [email protected],Foroughi Forough [email protected],Hedayati Dr. Mehdi*[email protected]

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#: The association of the BRAF V600E mutation wi... Revision 0

Journal: International Journal... of 25 25 June 2017 10:32:14

The Editors,� International Journal of Endocrinology and Metabolism�

Dear Modem/Sir,

We intend to publish an article entitled �The association of the BRAF V600E mutation with tissue inhibitor of metalloproteinase-3 expression and clinicopathological features in papillary thyroid cancer� in your esteemed journal as an original article. On behalf of all the contributors I will act and guarantor and will correspond with the journal from this point onward.

The authors hereby affirm that the manuscript is original, that all statements asserted as factsare based on author�s careful investigation and accuracy, that the manuscript has not beenpublished in total or in part elsewhere. The authors have full power authority to enter into thiscopyright assignment and to make the grants herein content. Each author acknowledges he/she has participated in the work in a substantive way and isprepared to take public responsibility for the work.

Yours� sincerely,

Corresponding contributor:Mehdi Hedayati (PhD.)Associate Professor,Cellular and Molecular Endocrine Research Center (CMERC), Research Institute for Endocrine Sciences of Shahid Beheshti University of Medical Sciences,Tehran, IranPo Box: 19395-4763Tel: +98(21)22432500Fax: +98(21)22416264Email: [email protected]



Title: The association of the BRAF V600E mutation with tissue inhibitor of metalloproteinase-3 expression and clinicopathological features in papillary thyroid cancer

Authors

1. Maryam Zarkesh: Cellular and Molecular Endocrine Research Center, Research Institute forEndocrine Sciences, Shahid Beheshti University of Medical Sciences, Tehran, Iran

Email: [email protected]. Azita Zadeh-Vakili: Cellular and Molecular Endocrine Research Center, Research Institute for

Endocrine Sciences, Shahid Beheshti University of Medical Sciences, Tehran, IranEmail: [email protected]

3. Fereidoun Azizi: Endocrine Research Center, Research Institute for Endocrine Sciences, ShahidBeheshti University of Medical Science, Tehran, Iran

Email: [email protected]. Ahmad S. Fanaei: Association Professor of General Surgery, Erfan Hospital, Tehran, Iran

Email: [email protected]. Forough Foroughi: Department of Pathology, Shahid Beheshti University of Medical Sciences,

Tehran, IranEmail: [email protected]

6. Mehdi Hedayati: Cellular and Molecular Endocrine Research Center, Research Institute forEndocrine Sciences, Shahid Beheshti University of Medical Sciences, Tehran, Iran

Email: [email protected]

Running title: BRAF V600E mutation & TIMP3 expression in PTC

Corresponding author

Mehdi Hedayati (Ph.D.)Associate Professor,Cellular and Molecular Endocrine Research Center (CMERC),Research Institute for Endocrine Sciences of Shahid Beheshti University of Medical Sciences, Tehran, IranPo Box: 19395-4763

1



Tel: +98(21)22432500Fax: +98(21)22416264Email: [email protected]

2



Title: The association of the BRAF V600E mutation with tissue inhibitor of metalloproteinase-3 expression and clinicopathological features in papillary thyroid cancer

Running title: BRAF V600E mutation & TIMP3 expression in PTC

1



Abstract

Background: Papillary thyroid cancer (PTC) is the most common endocrine malignancy. The

aim of this study was to investigate the association of tissue inhibitor of metalloproteinase-3

(TIMP3) expression in thyroid tissue, based on BRAF V600E status with the clinicopathologic

characteristics of PTC.

Methods: Sixty fresh frozen tissues of PTC patients (15 male and 45 female) were collected

during thyroidectomy surgery. All clinicopathological information was obtained, and samples

were reviewed by a pathologist and confirmed. Exon 15 of the BRAF gene was genotyped by

sequencing; TIMP3 gene expression was assessed using SYBR-Green Real-Time PCR, and

TIMP3 protein expression was measured using ELISA.

Results: The BRAF mutation was found in 24 (40%) of the 60 PTC cases. BRAF (+) PTC had

significantly larger tumor size than BRAF (-) PTC (P= 0.039). Lymph node metastasis frequency

was significantly higher in BRAF (+) compared to the BRAF (-) group (P=0.030). No significant

reduction was seen in the tumoral tissues of the TIMP3 gene expression in BRAF (+) compared

to BRAF (-) PTC samples. However, mean TIMP3 protein level was significantly lower in

tumoral tissues compared to matched non-tumoral tissues in BRAF (+) PTC (P=0.003); TIMP3

protein level significantly was lower in tumoral tissues compare to matched non-tumoral in

BRAF (+) in subjects who had not lymph node metastasis and also subjects with lymph node

metastasis in both BRAF positive and negative.

Conclusion: The lower TIMP3 expression associated with the lymph node metastasis in PTC

patients. Our results showed that BRAF mutation was associated with larger tumor size, higher

frequency of lymph node metastasis, and lower TIMP3 protein expression.

Keywords: BRAF V600E, TIMP3 expression, papillary thyroid cancer

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Introduction

Papillary thyroid cancer (PTC), a follicular epithelial cell-derived thyroid cancer, is the most

common endocrine system malignancy, which accounts for approximately 85% of thyroid

cancers (1).

According to data from the Surveillance, Epidemiology, and End Results (SEER) the incidence

of PTC increased 2.9 fold from 1973 to 2002 among the American populations aged over 65

years; with a rapid increase in PTC incidence from 0 to 29.7 per 100,000 (2, 3). The prevalence

of PTC differs in various countries, and particularly among Asian populations, in whom it is

higher than other ethnicities (4). Although most of the patients with PTC have a good survival

rate, they may be prone to recurrence with increased morbidity and mortality (5, 6).

Existing evidence shows new aspect of molecular mechanisms such as genetic alterations opens

up a number of research avenues to determine pathways of progression and aggressiveness of

PTC. One of the most well-known oncogenic genetic alterations occurring in over 45% of cases

of PTC is the B-type raf proto-oncogene (BRAF) point mutation (7-9). Mutation in BRAF

(V600E) is associated with an invasive clinical period and poor clinicopathologic properties,

such as extrathyroidal invasion, lymph node metastasis, and advanced TNM stage (10), the TNM

system considers tumor size and local extent (T), lymph node metastases (N), and distant

metastases (M). However, the molecular events whereby mutation in the BRAF gene contribute

to development and progression of PTC remains unknown.

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Recently, regulations in the expression of various tumor suppressor genes that might be affected

by mutations in the specific gene have been investigated (11, 12). Hu et al, (12) showed that

methylation of various tumor suppressor genes such as tissue inhibitor of metalloproteinase-3

(TIMP3) may mediate progression and aggressiveness of PTC in response to a mutation in the

BRAF gene. TIMP3 is a member of tissue inhibitors of metalloproteinases that play an important

role in inhibiting growth, angiogenesis, invasion, and metastasis of various human cancers,

including thyroid cancer. It is of particular interest for therapeutic purposes due to its

characteristic of inhibiting different aspects of tumor development, mainly because it is a potent

angiogenesis inhibitor. Hu et al, (12) reported a significant association between BRAF mutation

and aberrant methylation of TIMP3. In addition hypermethylation of the TIMP3 gene is related

to thyroid cancer (13) and correlated with high-risk clinicopathological characteristics of PTC,

including extrathyroid invasion, lymph node metastasis, and advanced disease stages (III and IV)

(12). However, based on our knowledge, no study has apparently investigated the changes in

TIMP3 gene expression and protein levels in papillary thyroid tissues in relation to the BRAF

mutation and clinicopathologic features among PTC patients.

The purpose of this study was primarily to compare the association of TIMP3 gene expression

and protein levels with BRAF mutation in PTC patients. Secondly, we evaluated the association

of TIMP3 gene expression and protein levels with the clinicopathologic characteristics in PTC.

Materials and methods

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Patients and clinicopathological data collection: In the current study, we collected primary PTC

tumor tissues, matched non-tumoral tissues, and clinicopathological data of sixty patients with

PTC who had undergone total thyroidectomy over a one-year period (2015-2016) from the Erfan

and Atiyeh hospitals in Tehran city. Clinicopathological data collected including information, i.e.

age, gender, the status of extrathyroidal invasion, lymph node metastasis, multifocality, and stage

of the tumor. A pathologist reviewed all samples, confirmed the diagnosis of PTC. To determine

the stage of tumors stage we used American Cancer Society (ACS), American Joint Committee

on Cancer (AJCC) handbook (14). All confirmed thyroid samples were snap-frozen in liquid

nitrogen and stored at -80˚C.

Ethics approval for the study was obtained from the ethical committee of the Research Institute

for Endocrine Sciences of the Shahid Beheshti University of Medical Sciences

(25ECRIES93/10/23). Written informed consent was obtained from all of the patients.

DNA extraction and BRAF mutation detection: Genomic DNA was extracted from collected

fresh frozen thyroid tissue specimens by the TRIzol reagent (Invitrogen U.S. Cat. No. 15596-

026) according to the manufacturer�s instructions after histological control. Isolated DNA was

amplified by the polymerase chain reaction (PCR) that was carried out using following specific

primers for exon 15 of the BRAF gene containing the site for the T1799A (V600E) mutation: 5�-

CTTCATAATGCTTGCTCTGATAGG-3� (Forward) and 5�-

TTCTAGTAACTCAGCAGCATCTCA-3� (Reverse); The PCR reaction was performed in

volume 20µ L with PCR master mix (Kowsar Biotech Co. Cat. No. K1140), forward and reverse

5



primers (10pmol), genomic DNA (100ng/µ L) with annealing 62̊ C. After quality confirmation by

agarose gel electrophoresis, direct DNA sequencing of the 251bp PCR-amplified products was

carried out using the same primers by Applied Biosystems 3130/3130xl Genetic Analyzers. The

results were analyzed using Chromas software version 2.6.

RNA samples and quantitative real-time PCR: Total RNA was extracted from fresh snap-frozen

PTC tissue samples after histological control, using TRIzol reagent (Invitrogen U.S. Cat. No.

15596-026) according to the manufacturer�s instructions. RNA quantity and purity were

measured using the NanoDrop 1000 (Thermo Scientific, Waltham, and Mass). Total RNA (1µ g)

was reverse transcribed with cDNA synthesis kit (Thermo Fisher Scientific, USA) according to

the manufacturer�s protocol and stored at -20°C for future use.

The amplification condition of TIMP3 templates was optimized by quantitative reverse

transcriptase real-time PCR (qRT-PCR) using the Rotor-Gene 6000 (Corbett Research, Sydney,

Australia) with annealing 60°C. Real-time quantification was monitored by measuring the

fluorescence activity. β-actin gene was used as internal control to normalize mRNA levels. All

experiments were repeated twice. Primers were designed from the sequence of the human

cDNAs. The sequences of these primers were as follows: 5�-CTGACAGGTCGCGTCTATGA-

3� (Forward) and 5�- CCGATAGTTCAGCCCCTTGC-3� (Reverse) for TIMP3, 5�-

GATCAAGATCATTGCTCCTCCT-3� (Forward) and 5�- TACTCCTGCTTGCTGATCCA-3�

6



(Reverse) for β-actin. PCR amplification was performed in 25 µL volumes, using the SYBR

Green master mix (Thermo Fisher Scientific, USA).

Enzyme-linked immune sorbent assay (ELISA) analysis of TIMP3 protein expression: Total

protein was extracted from snap-frozen thyroid tissues. The samples were incised and weighted

(totally 100mg tissue/ 1mL buffer). A certain amount of PBS (pH 7.4, 100mM) was added and

homogenized thoroughly using a homogenizer (QIAGEN, Germany), and then, centrifuged (at

4000-6000 RPM) for approximately 10 minutes; supernatants were collected and frozen at -20°C

for later use. ZellBio GmbH assay kit (Cat. No: ZB-11267-H9648, Germany) was used to

quantitatively assay human TIMP3 based on the Biotin double antibody sandwich technology.

Statistical analysis: Normal distribution of quantitative data was determined by Kolmogorov-

Smirnov test. Group comparisons of categorical variables were performed using the x2 test or

linear-by-linear association. Categorical variables were expressed as frequency and percentage.

Comparisons of means were evaluated by the independent samples t-test. The intergroup

differences were assessed with the one-sample paired t-test (tumoral versus matched non-tumoral

tissues). All normal distributed continuous data were expressed as mean ± standard deviation

(SD). All analyses were performed using SPSS software (version 20.0 for Windows; SPSS Inc.,

Chicago, IL). Probability values <0.05 were used as the cutoff for statistical signi� cance. The

relative gene expression was assessed using the relative expression software tool (REST)

software (version 2009).

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Results

BRAF mutation status in relation to clinicopathological characteristic: Mean age of males and

females was 39.6±15.6 and 36.9±11.7 years, respectively; the BRAF V600E mutation was found

in 24 of 60 (40%) of the PTC cases. Figure 1 shows a sample of the sequencing results for a

BRAF (+) and (-). The frequencies of classic, follicular, oncocytic, and sclerosing variants of

PTC were 83.3, 11.7, 1.7, and 1.7%, respectively. There was no significant difference in

frequency of variants in BRAF mutation status (P=0.322); prevalence of unifocal, multifocal,

and no multicentricity tumors was 27.5, 47.5, and 25%, respectively, which did not differ

significantly in BRAF mutation status (P=0.953).

We analyzed the clinicopathological characteristics of the PTC cases with BRAF mutation

(Table 1), and found no extrathyroidal invasion in cases. BRAF (+) PTC had significantly larger

tumor size than BRAF (-) PTC (mean size 1.98±0.82 vs. 1.31±1.35 cm, respectively; P= 0.039).

Lymph node metastasis frequency was significantly higher in the BRAF (+) compared to the

BRAF (-) group (62.5 vs. 34.3%, respectively, P= 0.030).

Expression of TIMP3 in PTC: The expression of TIMP3 gene was lower in tumoral tissues than

those in matched non-tumoral ones (1.79±1.76 vs. 2.42±2.83; P=0.152) (Figure 2a), mean

TIMP3 protein level was significantly lower in tumoral tissues compared to the matched non-

tumoral tissues (7.36±2.79 vs. 10.77±5.57 ng/mL, respectively; P=0.001) (Figure 2b).

Differential expression of TIMP3 in BRAF V600E PTC: Moreover, the expression of TIMP3

gene was lower in BRAF (+) PTC compared to BRAF (-) PTC patients (1.21±1.09 vs.8



1.76±2.13; P=0.274) (Figure 3a). Mean TIMP3 protein level was significantly lower in tumoral

tissues compared to the matched non-tumoral tissues (6.97±2.37 vs. 11.4±5.59 ng/mL,

respectively; P= 0.003) in BRAF (+) PTC; protein level was 7.77±3.19 and 10.0±5.61 ng/mL in

tumoral and matched non-tumoral tissues, respectively in BRAF (-) PTC, which was marginally

significant (P= 0.084) (Figures 3b and 3c).

TIMP3 protein level and lymph node metastasis in BRAF V600E (+) PTC: No association was

found between TIMP3 protein level and clinicopathological features, except with lymph node

metastasis. In all PTC cases the protein level of TIMP3 was lower in tumoral tissues compared to

the matched non-tumoral tissues of subjects with positive lymph node metastasis (7.55±2.63 vs.

12.4±6.78; P= 0.004). After splitting the file into BRAF positive and negative, results showed

that protein level of TIMP3 remained significantly lower in tumoral tissues in BRAF (+) in

subjects without lymph node metastasis and those with lymph node metastasis (Table 2).

Discussion

Data shows that the most frequent genetic variation occurring in PTC is a mutation in the

nucleotide 1796 of the BRAF gene, giving rise to an activated form, BRAF V600E (15).

Overactivation of the RAF/MEK/MAPK signaling pathway meddles with proliferation,

differentiation, and programmed cell death (apoptosis) (16). Furthermore, it has been suggested

that the BRAF mutation is associated with a weak prognosis in PTC patients (17). On the other

hand, there is increasing evidence suggesting that TIMP3 protects against tumor development by

suppressing tumor growth, metastasis, angiogenesis, and apoptosis (18). All genetic alternations

9



found in thyroid cancer need to be evaluated for the pathogenesis of this disease, its accurate

diagnosis, precise prognosis and the appropriate management of these patients.

In this study, we evaluated the most common mutation found in PTC as well as a tumor

suppressor gene in tumoral and matched non-tumoral tissues of PTC patients that had not yet

been tested. Results showed that the BRAF mutation was related to tumor size and lymph node

metastasis. Moreover, the gene expression of TIMP3 was downregulated in tumoral tissues

compared to the matched non-tumoral one. Moreover, protein level was downregulated in

tumoral tissues in BRAF (+) PTCs; in addition, results indicated that TIMP3 protein level was

associated with lymph node metastasis in subjects who had the BRAF V600E (+) mutation.

In agreement with our findings, a review by Leonardi GC et al (19) from 19 studies reported a

positive correlation between the BRAF V600E mutation with the clinicopathological features,

including tumor size, extrathyroidal invasion, lymph node metastasis, and advanced stages in a

number of reports of PTC. However, some studies found no such association (20, 21), an

inconsistency which may be due to various factors, such as different diagnostic criteria and the

small number of cases analyzed in different studies.

Like our results, Anania MC et al (18), presented downregulation of TIMP3 in PTC with respect

to normal thyroid tissues using analysis of gene expression data and in silico analysis; they

demonstrated that TIMP3 exerts a negative regulatory role in thyroid carcinogenesis.

Downregulation of TIMP3 gene has been demonstrated in melanoma, kidney, brain,

choriocarcinoma, renal cell carcinoma, esophageal adenocarcinoma, and other human cancers

10



(22-25). Moreover, TIMP3 promoter hypermethylation is observed in many tumors such as

gastric, esophageal, and thyroid carcinoma, and causes loss of TIMP3 protein expression (26,

27). However, downregulation of TIMP3 protein not resulting from TIMP3 promoter

hypermethylation has also been reported in pancreatic adenocarcinoma, an occurrence also

known to be associated with microRNA and the lack of heterozygosity (28, 29).

In this study, we found no significant reduction of TIMP3 gene expressed in tumoral tissues and

matched non-tumoral tissues but it associated significantly with diminished TIMP3 protein

expression. Consistent with previous studies (30), our results indicate that the expression of

TIMP3 in protein levels was reduced in tumoral tissues compared to matched non-tumoral one.

In this study, the observed decline in TIMP3 gene expression level in tumoral tissues, however

not significant, but was in concordance with reduction in protein expression. May be reduced

protein levels depends on other regulatory mechanisms which are acting during neoplastic

transformations.

The positive association of TIMP3 protein expression with BRAF mutation and lymph node

metastasis confirm that expression of this metastasis suppressor gene may play a role in the

aggressiveness of PTC, conferred by the BRAF mutation. This result reveals the biological

relevance of the positive correlation between BRAF mutation and expression of TIMP3 which is

accompanied by MAPK/ERK signaling pathway (31).

To our knowledge, this is the first report indicating a relationship between TIMP3 expression

and BRAF mutation status in tumoral and matched non-tumoral tissues of PTC patients.

11



However, as a limitation for this cross-sectional study we had not access to thyroid samples from

healthy people (without thyroid carcinoma) to evaluate the probable constitutional changes in

TIMP3 expression.

Even though we found an association between BRAF mutation and TIMP3 expression with PTC

invasiveness, we did not have a follow-up study to investigate their prognostic role and/or

recurrence to give more credibility to the results. BRAF mutation, as the most frequent genetic

irregularity in PTC, is not completely understood into long-term prognosis due to the

contradictory findings documented. In the future, the prognostic importance of BRAF V600E

and low expression of TIMP3 should be evaluated based on long-term outcomes.

In conclusion, our results showed that BRAF mutation was associated with larger tumor size,

higher lymph node metastasis, and lower TIMP3 protein expression. Moreover, lymph node

metastasis was seen in the lower TIMP3 protein expression.

Sources of funding: This research did not receive any specific grant from funding agencies in

the public, commercial, or not-for-profit sectors.

Disclosure: The authors do not report any relevant conflicts of interest.

Acknowledgements: This work is related to Maryam Zarkesh Ph.D. by research thesis and

supported by Research Institute for Endocrine Sciences, Shahid Beheshti University of Medical

Sciences, Tehran, Iran. The authors wish to express their gratitude Ms. Afsoon Danesh-afrooz,

Ms. Laleh Hoghoughi Rad, Ms. Hoda Ghadaksaz, Dr. Adeleh.S Razavi, Mr. Emad Yuzbashian,

12



and Dr. Zahra Nozhat for data collecting and preparing. The authors wish to acknowledge Ms.

Niloofar Shiva for critical editing of English grammar and syntax of the manuscript.

Authors� contribution

Study concept and design: Hedayati & Zarkesh; acquisition of data: Fanaei & Foroughi; analysis

and interpretation of data: Zarkesh; drafting of the manuscript: Zadeh-Vakili, & Zarkesh; critical

revision of the manuscript for important intellectual content: Azizi & Hedayati; statistical

analysis: Zarkesh; study supervision: Hedayati

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16



Table 1. Clinicopathological characteristics of PTC patients in BRAF mutation

Variables Total

BRAF V600E mutation

Positive NegativeP

value

Age (year) 40.9±13.7 38.8±13.1 36.7±12.4 0.527

Male, n(%)Female, n(%)

15(25.0)45(75.0)

8(33.3)16(66.7)

7(19.4)29(80.6)

0.180

Tumor size (cm) 1.58±1.20 1.98±0.82 1.31±1.35 0.039

Extracapsular invasion 14(23.7) 6(25) 8(22.9) 0.544

Lymph node metastasis 27(49.1) 15(62.5) 12(34.3) 0.030

Lymphovascular invasion 8(13.6) 4(16.7) 4(11.4) 0.418

Tumor stage (AJCC), n (%)IIIIIIIVA

46(79.3)3(5.2)7(12.1)2(3.4)

17(70.8)1(4.2)5(20.8)1(4.2)

29(85.3)2(5.9)2(5.9)1(2.9)

0.153

Data in parentheses are percentage.

17



Table 2. Protein levels of TIMP3 in BRAF V600E and lymph node metastasis

Lymph nodemetastasis status

Samplestatus

TIMP3 protein level (ng/mL) PvalueTumoral Matched non-

tumoral

NoAll PTCs

7.17±3.02* 9.09±3.35 0.086

Yes 7.55±2.63 12.4±6.78 0.004

NoBRAF (-) 7.47±3.16 7.58±2.55 0.939

BRAF (+) 6.87±3.04 10.6±3.49 0.028

YesBRAF (-) 8.08±3.39 12.5±6.84 0.043

BRAF (+) 7.08±1.74 12.2±7.10 0.048*Mean±SD

18



Figure 1. The sequence of BRAF V600E (+) and (-) by Chromas software

19



Figure 2. a) Relative expression of TIMP3 gene and b) Protein level of TIMP3 in tumoral tissuescompare to matched non-tumoral tissues; *P< 0.05

20

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Relat

ive ex

press

ion of

TIM

P3 (F

old ch

ange

)

Matched Non_tumoral Tumoral

Prot

ein le

vels

of T

IMP3

(ng/

mL)

a b

*

*



0.0

0.5

1.0

1.5

2.0

2.5

BRAF-V600E Status

Relat

ive ex

press

ion of

TIM

P3 (F

old ch

ange

)

BRAF Negative BRAF Positive

Figure 3. a) Relative expression of TIMP3 gene in BRAF mutation status, b) Protein level of

TIMP3 in BRAF (+), and c) Protein level of TIMP3 in BRAF (-); *P< 0.05

21

a*

a

b c

Prot

ein le

vels

of T

IMP3

(ng/

mL)

Prot

ein le

vels

of T

IMP3

(ng/

mL)

*

BRAF (+) BRAF (-)



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