Osteopontin expression is correlated with differentiation and good prognosis in medullary thyroid carcinoma

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  • 1 Instituto de Investigação e Inovacão em Saúde, Institute of Molecular Pathology and Immunology of the University of Porto (Ipatimup) – Cancer Biology, Medical Faculty, Unidade de Investigação em Patobiologia Molecular (UIPM), Molecular Pathology Service of the Portuguese Institute of Oncology of Coimbra FG, Department of Pathology, Research Coordination, Natural Sciences Department, Universidade do Porto, 4200-135 Porto, Portugal

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Background

Osteopontin (OPN) or secreted phosphoprotein 1 (SPP1) is a matricellular glycoprotein whose expression is elevated in various types of cancer and has been shown to be involved in tumourigenesis and metastasis in many malignancies, including follicular cell-derived thyroid carcinomas. Its role in C-cell-derived thyroid lesions and tumours remains to be established.

Objective

The objective of this study is to clarify the role of OPN expression in the development of medullary thyroid carcinoma (MTC).

Methods

OPN expression was analysed in a series of 116 MTCs by immunohistochemistry and by qPCR mRNA quantification of the 3 OPN isoforms (OPNa, OPNb and OPNc) in six cases from which fresh frozen tissue was available. Statistical tests were used to evaluate the relationship of OPN expression and the clinicopathological and molecular characteristics of patients and tumours.

Results

OPN expression was detected in 91 of 116 (78.4%) of the MTC. We also observed high OPN expression in C-cell hyperplasia as well as in C-cells scattered in the thyroid parenchyma adjacent to the tumours. OPN expression was significantly associated with smaller tumour size, PTEN nuclear expression and RAS status, and suggestively associated with non-invasive tumours. OPNa isoform was expressed significantly at higher levels in tumours than in non-tumour samples. OPNb and OPNc presented similar levels of expression in all samples. Furthermore, OPNa isoform overexpression was significantly associated with reduced growth and viability in the MTC-derived cell line (TT).

Conclusion

The expression of OPN in normal C-cells and C-cell hyperplasia suggests that OPN is a differentiation marker of C-cells, rather than a marker of biological aggressiveness in this setting. At variance with other cancers, OPN expression is associated with good prognostic features in MTC.

Abstract

Background

Osteopontin (OPN) or secreted phosphoprotein 1 (SPP1) is a matricellular glycoprotein whose expression is elevated in various types of cancer and has been shown to be involved in tumourigenesis and metastasis in many malignancies, including follicular cell-derived thyroid carcinomas. Its role in C-cell-derived thyroid lesions and tumours remains to be established.

Objective

The objective of this study is to clarify the role of OPN expression in the development of medullary thyroid carcinoma (MTC).

Methods

OPN expression was analysed in a series of 116 MTCs by immunohistochemistry and by qPCR mRNA quantification of the 3 OPN isoforms (OPNa, OPNb and OPNc) in six cases from which fresh frozen tissue was available. Statistical tests were used to evaluate the relationship of OPN expression and the clinicopathological and molecular characteristics of patients and tumours.

Results

OPN expression was detected in 91 of 116 (78.4%) of the MTC. We also observed high OPN expression in C-cell hyperplasia as well as in C-cells scattered in the thyroid parenchyma adjacent to the tumours. OPN expression was significantly associated with smaller tumour size, PTEN nuclear expression and RAS status, and suggestively associated with non-invasive tumours. OPNa isoform was expressed significantly at higher levels in tumours than in non-tumour samples. OPNb and OPNc presented similar levels of expression in all samples. Furthermore, OPNa isoform overexpression was significantly associated with reduced growth and viability in the MTC-derived cell line (TT).

Conclusion

The expression of OPN in normal C-cells and C-cell hyperplasia suggests that OPN is a differentiation marker of C-cells, rather than a marker of biological aggressiveness in this setting. At variance with other cancers, OPN expression is associated with good prognostic features in MTC.

Introduction

Thyroid tumours are the most common malignancies of the endocrine system. About 1% of the thyroid cells correspond to (parafollicular) C-cells that are located in the basal layer of thyroid follicles (1). Medullary thyroid carcinoma (MTC) is the thyroid tumour that arises from the neural crest-derived C-cells of the thyroid gland. MTCs account for 5–10% of the clinically evident thyroid cancers and are associated to a higher incidence of distant metastases and poorer prognosis, when compared with follicular cell-derived well-differentiated thyroid carcinomas (2, 3). In total, 70–80% of MTCs are sporadic and the remaining 20–30% are hereditary (1).

C-cells are dispersed as individual cells or in small groups in the spaces among thyroid follicles, and their function is to secret a whole series of regulatory peptides and growth factors (4). Hyperplasia of C-cells corresponds to an abnormal increase in the number of these cells and is recognised as a precursor of medullary carcinoma (especially in the setting of multiple endocrine neoplasia types IIA and IIB) and associates with a number of other conditions, including advanced age, hypercalcemia, hypergastrinemia due to Zollinger–Ellison syndrome, Hashimoto disease and chronic lymphocytic thyroiditis, follicular and papillary thyroid neoplasms, exogenous estrogen administration and cimetidine treatment. Yet, patients with germ-line mutations of the RET oncogene present age-related progression of C-cell lesions from C-cell hyperplasia (CCH) towards MTC (5). In sporadic cases, the putative role of CCH in carcinogenesis is controversial.

Thyroid carcinomas frequently present aberrant expression of different gene products (6, 7, 8), and it was advanced that such overexpression can be related to cancer progression. Castellone et al. (9) demonstrated that OPN is one of the most overexpressed molecules in papillary thyroid carcinoma (PTC) and showed that its up-regulation promoted proliferation, migration and spreading of thyroid cancer-derived cell lines (9).

OPN is a secreted multifunctional phosphorylated glycoprotein constitutively expressed in epithelial cells of the gastrointestinal, reproductive and urinary tracts, sweat ducts, lung bronchi, pancreas, lactating breast, salivary glands and, notably, in the gallbladder (10). OPN mRNA and/or protein expression levels were reported to be increased in several types of human cancers, such as prostate carcinoma (11); melanoma (12); and ovary (13), lung and gastric carcinoma (14). OPN overexpression was associated with poor prognosis in some malignancies, namely, in PTC (15). The overexpression of OPN in many types of tumours and its frequent association with invasive properties and aggressive clinicopathological features (16, 17) suggest that OPN may be involved in tumourigenesis, tumour progression and in metastasis formation (18, 19). In contrast to this, OPN expression has been related with better prognostic clinical features in osteosarcoma (20). To date, it has not been described the expression of OPN in neuroendocrine tumours if one excludes a case report of Kuroda et al. (21) showing the expression of OPN in neoplastic cells of a large-cell neuroendocrine carcinoma (LCNEC) of the lung. Kuroda et al. (21) observed that part of the LCNEC neoplastic cells as well as the calcified foci were positive for OPN, suggesting that in addition to dystrophic calcification, OPN may be involved in the mechanism of calcification within necrotic areas of LCNEC.

OPN mRNA is subjected to alternative splicing that generates three protein variant isoforms, designated as OPNa, OPNb and OPNc (22). The three OPN isoforms have been shown to play specific roles in different types of tumours; for example, in breast and ovarian cancer cells, OPNc isoform activates invasion and adhesion properties (13, 23). In prostate cancer cells, OPNb and OPNc stimulate proliferation, migration and invasion (11), and in hepatocellular carcinoma cells, OPNa and OPNb promote migration (24).

To the best of our knowledge, expression of total OPN and of OPN splicing variants has not been systematically assessed so far in MTC samples. The objective of this study was therefore to evaluate the expression profile of OPN in MTC and to relate such expression with molecular and clinicopathological data.

Subjects and methods

Human MTC tissue samples

The study involved 116 cases of MTC (diagnosed in three Institutes from 1974 to 2011). Formalin-fixed, paraffin-embedded tissue and clinical data were retrieved from the files of Centro Hospitalar São João (CHSJ)/Medical Faculty of Porto (FMUP)/Ipatimup (56 cases), Portuguese Institute of Oncology, Coimbra (IPO-C) (19 cases), and Portuguese Institute of Oncology, Lisboa (IPO-L) (41 cases). The diagnosis of MTC was revised by two pathologists (CE and MSS) and confirmed by calcitonin immunostaining. Clinicopathological and follow-up data were obtained from the surgical pathology reports and patients' records of the Department of Pathology and Oncology of CHSJ and from IPO databases (Table 1). RET and RAS genetic characterisation of the series had been done previously and reported in two publications (25, 26). The study was approved by the Hospital Ethical Committees, and National Ethical rules were followed in every procedure.

Table 1

Summary of the clinical, pathological and molecular data of the MTC cases.

MTC
Gender (n=97)*
 Female53 (54.6%)
 Male44 (45.4%)
 Tumour size (cm)* (n=80; mean±s.d.)3.00 (±2.24)
Stroma (n=112)*
 Absent31 (27.7%)
 Present (hyaline)81 (72.3%)
Amyloid deposits (n=42)*
 Absent16 (38.1%)
 Present26 (61.9%)
Extrathyroidal extension (n=64)*
 Absent28 (43.8%)
 Present36 (56.3%)
Metastasis (lymph node and/or distance) (n=73)*
 Absent27 (36.9%)
 Present46 (63.1%)
Invasion (vascular and/or capsular) (n=34)*
 Absent6 (17.6%)
 Present28 (82.4%)
RET mutation (n=116)
 Absent49 (42.2%)
 Present67 (57.8%)
RAS mutation (n=116)
 Absent97 (83.6%)
 Present19 (16.4%)
PTEN nuclear score (n=75)*
 Low 48 (64.0%)
 High27 (36.0%)
PTEN cytoplasmic score (n=75)*
 Low14 (18.7%)
 High61 (81.3%)
pS6 intensity score (n=75)*
 Low33 (44.0%)
 High42 (56.0%)

*Clinicopathological data from all the cases included in the series was impossible to obtain.

Immunohistochemistry

Semi-quantitative immunohistochemistry (IHC) analysis of OPN expression was performed in representative 116 MTC tumour tissue sections using an antibody that recognises all OPN isoforms (anti-total OPN). To demonstrate specificity of the antibody to human OPN, we used normal gallbladder as positive control, because it has been previously reported to strongly express OPN in both supranuclear and luminal contents (10) (Fig. 1A). The following was the IHC procedure: briefly, deparaffinised and rehydrated sections were subjected to microwave treatment in 10 mM sodium citrate buffer, pH 6.0, for antigen retrieval. After blocking, the sections were incubated overnight at 4 °C in a humidified chamber with a primary antibody anti-OPN (polyclonal, goat, 1:500; R&D Systems, Minneapolis, MN, USA). The detection was performed with a labelled streptavidin–biotin immunoperoxidase detection system (Thermo Scientific/Lab Vision, Fremont, USA), and the IHC staining was developed with 3,3′-diaminobenzidine substrate. Negative control consisted in the omission of the primary antibody incubation. Semi-quantitative OPN expression was evaluated using a staining score, which has been independently established by two observers (CE and LF). Proportion of positive-stained cells was scored as <5%=0, 5–25%=1, 25–50%=2, 50–75%=3 and >75%=4, while staining intensity values were classified as absent=0, faint=1, moderate=2 or strong=3 (Table 2). The OPN staining score (range from 0 to 7) corresponds to the sum of staining intensity and the proportion of positive-stained cells (Table 3). OPN staining score was correlated with data previously obtained by our group in this tumour series with regards to PTEN and pS6 IHC expression (25).

Figure 1
Figure 1

OPN cytoplasmic intracellular immunoexpression. (A) Staining in normal gallbladder used as positive control. (B) Normal thyroid gland showing no OPN staining. (C) Normal thyroid tissue showing OPN staining in scattered C-cells. (D) ‘Normal’ thyroid tissue adjacent to MTC; note the OPN positive scattered C-cells (in the inset calcitonin staining of the same area). (E) C-cell hyperplasia showing OPN staining (F) The same area of C-cell hyperplasia stained for calcitonin. (G) A case of MTC showing OPN staining in many neoplastic cells, classified as score: 6 (proportion of positive cells: 50–75%+ staining intensity: 3+). (H) The same case stained for calcitonin showing positivity of almost every neoplastic cells. A full colour version of this figure is available at http://dx.doi.org/10.1530/EJE-15-0577.

Citation: European Journal of Endocrinology 174, 4; 10.1530/EJE-15-0577

Table 2

Staining intensity and proportion of positive stained cells of OPN IHC in 116 MTC cases.

Frequency (n)Percentage (%)
Staining intensity
 Absent2521.6
 Faint3126.7
 Moderate2521.6
 Strong3530.1
 Total116100
Proportion of positive stained cells
 <5%3126.7
 5–25%1210.3
 25–50%1916.4
 50–75%1210.3
 75–100%4236.2
 Total116100
Table 3

Staining score of OPN IHC in 116 MTC cases.

OPN staining score*Frequency (n)Percentage (%)
02521.6
110.9
232.6
31412.1
41613.8
52723.3
61815.5
71210.3
Total116100%

*Staining intensity+proportion of positive-stained cells.

Cell culture and generation of medullary cancer cells overexpressing OPN isoforms

As a model to examine the putative roles of OPN isoforms in MTC, we used a MTC-derived cell line (TT). TT cell line was cultured in RPMI 1640 supplemented with 20% of foetal bovine serum, 100 IU/ml penicillin and 100 mg/ml streptomycin in a humidified environment containing 5% CO2 at 37 °C. The open reading frame of OPN splicing variants, OPNa, OPNb and OPNc, were cloned into pCR3.1 mammalian expression vector as previously described (23), and the DNA constructs were used for transfection into TT cells. Transfections were carried out using Lipofectamine 2000 (Invitrogen). The OPN isoforms/pCR3.1 plasmids or the vector alone (empty vector (EV)) were transfected into TT cells, and the stably expressing cells were selected with 600 μg/ml of G418 in the culture medium. The cells overexpressing each OPN isoform were tested individually on functional assays, as indicated below.

RNA extraction and reverse transcription

Total RNA was extracted from frozen specimens of MTC (n=6), adjacent normal tissue specimens (n=6), two MTC-derived cell lines (TT and MZ-CRC-1) and from TT cells overexpressing OPNa, OPNb, OPNc and EV using a Trizol commercial kit (Life Technologies, GIBCO BRL), according to the manufacturer's protocol. RNA was quantified by spectrophotometry, and its quality was checked by analysis of 260/280 and 260/230 nm ratios. For cDNA preparation, 1 μg of total RNA was reverse transcribed using the RevertAid first-strand cDNA synthesis kit (Fermentas, Burlington, ON, Canada).

Real-time PCR

The amplification of fragments corresponding to each OPN isoform was performed using the following oligonucleotide pairs: OPNa: 5′-ATC TCC TAG CCC CAC AGA AT-3′ (forward) and 5′-TTC TCC ATG GTG GTG AAG ACG CCA-3′ (reverse); OPNb: 5′-CTC CTA GCC CCA CAG ACC CT-3′ (forward) and 5′-TAT CAC CTC GGC CAT CAT ATG-3′ (reverse); OPNc: 5′-CTG AGG AAA AGC AGA ATG-3′ (forward) and 5′-AAT GGA GTC CTG GCT GT-3′ (reverse). GAPDH was amplified with primers 5′-TCC CAT CAC CAT CTT CCA GGA GCG-3′(forward) and 5′- TGG CGT CTT CAC CAC CAT GGA GAA-3′(reverse) and was used as an internal control to normalise the expression and to verify integrity of the cDNA. All qRT-PCRs were conducted using SYBR Green (Applied Biosystems). Gene expression of the target gene was calculated by using the ΔCT method.

Immunocytochemistry

Upon being plated on coverslips, cells were fixed in 4% paraformaldehyde for 20 min at room temperature (RT). Cells were emerged in NH4Cl 50 mM in PBS during 10 min, and then, cells were permeabilised in 0.2% Triton X-100 and blocked in 5% BSA in PBS for 30 min at RT. Primary antibodies were diluted in PBS containing 5% BSA and incubated overnight at 4 °C as follows: rabbit polyclonal against OPN (Rockland, Limerick, PA, USA, diluted 1:500). Coverslips were washed in 0.1% Triton X-100 prepared in PBS (PBT) and incubated with goat anti-rabbit IgG secondary antibodies conjugated with Alexa Fluor 594 (Invitrogen; diluted 1:300 in 5% BSA–PBT) for 1 h at RT. Nuclei were stained with 0.1 mg/ml diamino phenylindole (DAPI; Sigma–Aldrich). Images were taken by a Zeiss fluorescence microscope with ApoTome attachment (Axio Imager Z1 stand).

Cell growth and viability assays

Cell growth was analysed by MTT assay. Cells overexpressing OPNa, OPNb, OPNc and EV were seeded in 96-well plates at 5×104 cells density per well. After 48 h for adherence of the cells, 5 mg/ml of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) were added to each well. Cells stayed in the incubator at 37 °C, 5% CO2 for 4 h, and then dimethyl sulphoxide was added in the cells for solubilisation of MTT. Absorbance was measured at 570 nm. Viability assay was performed by Trypan blue exclusion after 48 h of plating the cells. Three independent experiments were carried out using triplicates in each experiment. Data are expressed as mean±s.e.m. and analysed using Mann–Whitney test. Significance level was fixed in 0.05.

Statistical analysis

Statistical analysis was performed using 22.0 SPSS statistical package (IBM, 2014). χ2 and independent samples t-test were performed to verify if there was any association(s) between OPN expression and clinicopathological data. For in vitro experiments, data are expressed as mean±s.e.m. and analysed using Mann–Whitney test. Values of P≤0.05 were considered statistically significant.

Results

OPN immunoexpression in MTC, C-cell hyperplasia and C-cells

IHC studies were performed in samples of the 116 MTC cases. Stromal areas of MTC, when present, were virtually negative for OPN staining (Fig. 1B). OPN protein expression was found in 91 of 116 MTCs (78.4%). In the positive cases, the staining intensity was faint in 26.7%, moderate in 21.6% and strong in 30.1% of the cases (Table 2); on average, more than 50% of the cells were positive (Table 2). Among the positive cases, staining score was 1 in 0.9%, 2 in 2.6%, 3 in 12.1%, 4 in 13.8%, 5 in 23.3%, 6 in 15.5% and 7 in 10.3% of the cases (Table 3).

OPN staining was mainly localised in the cytoplasm, i.e. intracellular. Representative regions of OPN and calcitonin staining are depicted in Fig. 1G and H. We also observed strong OPN staining in dispersed C-cells (Fig. 1C and 1D), as well as in C-cell hyperplasia foci detected in the adjacent thyroid tissue of some samples (Fig. 1E), which was confirmed by calcitonin positive staining (Fig. 1F).

Association of OPN immunoexpression with clinicopathological and molecular features of MTC

We observed significant differences of OPN staining score with regards to tumour size, PTEN nuclear expression and RAS status. Tumours larger than 2 cm presented a lower average OPN staining score than tumours smaller than 2 cm (2.8 vs 4.6 respectively; P=0.001). Tumours displaying PTEN nuclear expression showed a higher average of OPN staining score than tumours without PTEN nuclear expression (5.3 vs 3.9 respectively; P=0.003). Finally, tumours WT for RAS presented a higher average staining score for OPN than tumours harbouring a RAS mutation (4.0 vs 2.6 respectively; P=0.043). There was a suggestively significant association with invasion (capsular and/or vascular): tumours without evidence of invasion presented a higher OPN staining score than tumours presenting invasion (4.8 vs 3.5 respectively; P=0.083). No significant or suggestive associations were observed between OPN staining score and gender or age of the patients, presence of stroma, lymph node and/or distant metastasis, presence of amyloid, extrathyroidal extension, RET mutation, PTEN cytoplasmic expression and pS6 expression (Table 4).

Table 4

Clinicopathological and molecular associations with OPN expression. Data is presented as mean±s.d.

ClinicopathologicalnOPN immunoexpressionP value
Gender0.196
 Female 53(3.9±2.3)
 Male44(3.3±2.4)
Tumour size (cm)0.001
 <230(4.6±1.8)
 ≥250(2.8±2.4)
Stroma0.492
 Absent31(3.6±2.4)
 Present (hyaline)81(4.0±2.2)
Amyloid deposits0.239
 Absent16(4.8±2.0)
 Present26(4.0±2.2)
Extrathyroidal extension0.620
 Absent28(3.4±2.2)
 Present36(3.1±2.5)
Metastasis0.434
 Absent27(3.0±2.3)
 Present46(3.4±2.5)
Invasion (vascular and/or capsular)0.083
 Absent6(3.5±2.4)
 Present28(4.8±1.3)
RET0.883
 Normal49(3.7±2.3)
 Mutated67(3.8±2.3)
RAS0.043
 Normal97(4.0±2.2)
 Mutated19(2.6±2.5)
PTEN nuclear score 0.003
 Low48(3.9±2.2)
 High27(5.3±1.4)
PTEN cytoplasmic score 0.716
 Low14(4.2±2.4)
 High61(4.4±2.0)
pS6 intensity score 0.744
 Low33(4.5±1.8)
 High42(4.3±2.3)

Expression of OPN isoforms in MTC

In order to further investigate the OPN expression profile in MTC samples, we advanced that determining OPN splicing isoform expression could provide evidence regarding the contribution of each OPN variant within the total OPN expression evaluation. To achieve this objective, we evaluated mRNA levels of OPNa, OPNb and OPNc splice variants in six MTC cases with their corresponding matched non-tumoural tissues, from which frozen samples were available. OPNa isoform expression is significantly higher in the MTC tumour samples than in non-tumoural specimens (P=0.032). Conversely, OPNb and OPNc isoforms have similar expression levels in tumour and in non-tumour tissue samples (Fig. 2A). In addition, the expressions of these three splice variants were also analysed in two MTC-derived cell lines (TT and MZ-CRC-1). Both MTC cell lines expressed the three OPN splicing isoforms. Similarly to tumour tissue specimens, OPNa expression levels are higher than OPNb and OPNc expression levels (OPNa >OPNb >OPNc), being the expression higher in TT cell line than in MZ-CRC-1 (Fig. 2B).

Figure 2
Figure 2

Expression of OPN mRNA splicing isoforms in thyroid tissues and in MTC cell lines. (A) Expression of OPNa, OPNb and OPNc splicing isoforms by qRT-PCR in MTC specimens and in the adjacent thyroid. (B) Expression of OPNa, OPNb and OPNc splicing isoforms in TT and in MZ-CRC-1 cell lines by qRT-PCR. Expression of OPN isoforms is presented relative to GAPDH gene expression. A full colour version of this figure is available at http://dx.doi.org/10.1530/EJE-15-0577.

Citation: European Journal of Endocrinology 174, 4; 10.1530/EJE-15-0577

Overexpression of OPN isoforms in TT cell line, decrease cell growth and viability

To examine the contributions of each OPN isoform in medullary thyroid cancer, we used in vitro gain-of-function experiments. We chose the approach to stable overexpress each of these splicing isoforms in TT cells once this cell line is a well-established model for medullary thyroid cancer functional studies (27). Analysis by real-time PCR demonstrated the overexpression of OPN isoforms in TT cell lines transfected by OPN isoforms, showing higher mRNA levels of the corresponding isoform in relation to their levels in EV-transfected cells (Fig. 3A). The overexpression of OPN protein in TT cell lines transfected with OPNa, OPNb and OPNc isoforms was confirmed through immunocytochemistry analyses using OPN antibody (Fig. 3B). By contrast, OPN protein expression was poorly identified in control cells (EV) (Fig. 3B). Additionally, we evaluated cell proliferation by MTT assay, and we observed that TT cells overexpressing OPNa, OPNb and OPNc significantly reduced growth when compared with control cells (EV) (Fig. 3C). We also confirmed the viability of these cells using Trypan blue exclusion, and we found that OPNa isoform is able to significantly reduce the viability when compared with the control (EV) (Fig. 3D).

Figure 3
Figure 3

Proliferation and viability in TT cells overexpressing OPNa, OPNb, OPNc and EV. (A) Real-time PCR showing relative expression in TT cells overexpressing OPNa, OPNb, OPNc and EV. (B) Immunocytochemistry analyses of OPN expression in control cells (TT cells with EV) and in TT cells overexpressing OPNa, OPNb and OPNc. (C) MTT and (D) Trypan blue assays showing significantly decreased proliferation levels in TT cells overexpressing OPNa, OPNb, OPNc and EV (n≥3; *P<0.05; **P<0.005). A full colour version of this figure is available at http://dx.doi.org/10.1530/EJE-15-0577.

Citation: European Journal of Endocrinology 174, 4; 10.1530/EJE-15-0577

Discussion

In this study, we investigated the expression pattern of the glycoprotein OPN in 116 MTCs and correlated it with clinicopathological features of the cases. We observed that OPN expression is a potential marker of good prognosis for MTCs, and we gathered suggestive evidence that total OPN can be a newly identified differentiation marker of (parafollicular) C-cells.

Besides the aforementioned studies (15, 28) in thyroid cancer, OPN expression has been investigated by other groups (29, 30, 31, 32, 33, 34), but only one study evaluated MTC; in this study, Briese et al. (31) observed an up-regulation of OPN in three of the four MTCs of their series (75%). Our results, obtained in a much large series, confirm such percentage and demonstrate that 78.4% (91/116) of MTCs express OPN. We have also found that OPN expression is significantly associated with smaller, usually non-invasive cancers. In other words, at variance with PTC, in MTCs, the expression of OPN is associated with features of good prognosis and is reduced during tumour progression.

Interestingly, we observed that isolated C-cells scattered in adjacent normal thyroid parenchyma show strong OPN expression. A strong expression of OPN was also observed in clusters of C-cell hyperplasia (both in cases with and without germinal RET mutation – data not shown), indicating that this multifunctional protein may be a differentiation marker for C-cells. This assumption is supported by the observation, using serial sections, of strong calcitonin staining in the C-cells displaying OPN expression (Fig. 1F). In contrast to the strong expression in C-cells, we did not observe any OPN expression in follicular cells of the adjacent ‘normal’ thyroid tissue. Our results suggest that OPN immunostaining may be a useful marker in the study of the physiology and physiopathology of C-cells, C-cell hyperplasia and MTC, but further studies with an in-depth analysis of C-cell characteristics are needed to confirm this assumption.

Often OPN up-regulation in tumours has been associated with poor prognosis (34), but due to its versatility, this protein is also able to play roles that may be related to protective factors and cell differentiation. For instance, OPN expression usually correlates with the intermediate and later stages of osteoblast differentiation from mesenchymal stem cells (35, 36, 37). Furthermore, recent studies support the requirement of OPN expression in maintaining macrophage differentiation and function (38), and Chang et al. (39) reported that OPN is implicated in keratinocyte differentiation. Taken together, these findings and our observations suggest the potential role of OPN in differentiation of specific cell types and, in particular, as we are herein advancing, in C-cells.

Using IHC, OPN staining pattern in MTC is consistent with a predominant intracellular localisation of OPN (iOPN). OPN is usually known as an extracellular protein. There is increasing knowledge that extracellular OPN (eOPN) binds to its main receptors, integrins and CD44, activating their corresponding signalling pathways and mediating different OPN functions in cancer cells (40). However, other reports have also described iOPN, both in the cytoplasm and in the nucleus, in distinct cell contexts (41, 42). Similarly to eOPN, iOPN isoforms have also been associated to intracellular CD44 complex (43) and related to cellular processes such as cytoskeletal relocation and cell motility (42, 44). Some authors have suggested furthermore that iOPN and eOPN can act in concert (41, 42). Our results disclose the predominant intracellular expression of OPN in MTC cells. We cannot rule out that an unbalanced proportion of iOPN and eOPN can be present in C-cells and MTC, and that the aforementioned proportion may also modulate tumour- and tissue-specific OPN functional roles (41, 45). Additional studies should be performed to determine the putative regulation and functional roles of both iOPN and eOPN in C-cells and MTC cells.

Although nuclear PTEN function remains poorly understood, it has been shown that acting as a tumour suppressor, nuclear PTEN might lead to cell cycle arrest and decreased tumour growth (46, 47). It was also recently described the maintenance of chromosomal stability as another function of nuclear PTEN (48). PTEN has been related with OPN regulation, and it was reported that PTEN overexpression suppresses cellular migration stimulated by OPN (49). As higher levels of OPN were significantly associated with higher levels of nuclear PTEN in MTCs, these two molecules can probably act together in the maintenance of the differentiation of C-cells, thus contributing to prevent tumour progression.

In sporadic MTC, RAS mutations are often detected in HRAS and KRAS genes (25), representing an alternative genetic event to RET mutation. Casson et al. (50) showed that RAS mutation induces expression of OPN in human esophageal cancer, and other authors reported that OPN promoter have target sequences for RAS binding, inducing OPN expression in both human (51) and mouse (52). At variance with the presence of RET mutations that were not associated with OPN expression, we observed that RAS-mutated MTC cases display a significantly lower expression of OPN. Although in apparent contradiction with the results obtained in other tissue models cited previously (50, 51), our results fit in with the hypothesis that OPN expression is associated with normal differentiation of C-cells. Many studies have demonstrated loss of thyroid-specific gene expression in thyroid cells following the introduction of a variety of transforming oncogenes, in particular of the RAS gene family (53). This issue, as far as we are aware, has not been assessed to date concerning C-cell differentiation.

Another interesting result of the present study relies in the demonstration of the differential expression of the three OPN isoforms in MTC. It is known that OPN splicing variants present different and specific roles depending on the tissue and the tumour type (for a thorough review, see reference (54)). While in some tumour models, an individual OPN splicing isoform can act as a tumour-stimulating factor, in other tumour types, the same variant may present the opposite effect, acting against tumour progression (24, 54, 55, 56). We observed that total OPN, detected by IHC with an antibody that recognises all three OPN isoforms, is expressed in MTCs. Unfortunately, the expression of the different isoforms in paraffin sections could not be evaluated for technical reasons. We were able to evaluate the mRNA expression of OPN isoforms, by real-time PCR, using distinct primers to recognise three different isoforms, in six pairs of tumours and respective adjacent thyroid parenchyma. To the best of our knowledge, this is the first study reporting the mRNA expression of OPN isoforms in MTCs. OPNa mRNA was expressed at significantly higher levels in tumour samples than in non-tumour samples (P=0.032), while OPNb and OPNc presented similar levels of mRNA expression in tumours and respective adjacent tissues. We realised that when analysing the so-called ‘normal adjacent thyroid’, we were evaluating mainly follicular cells and comparing the expression of the isoforms between follicular cells and MTC. Only by analysing isolated C-cells we were able to compare “normal” C-cells and neoplastic MTC cells. Nevertheless, as we observed that OPNb and OPNc had similar expression levels in ‘normal’ adjacent tissue and in MTC, whereas OPNa showed significantly higher levels in MTC, we can conclude that OPNa mRNA is the isoform more expressed in MTC. This conclusion fits with our observation of higher levels of OPNa mRNA in the two MTC-derived cell lines. It remains to be clarified whether the same is true regarding normal and hyperplastic C-cells, i.e. if OPNa mRNA high expression is also present in normal and hyperplastic C-cells, or if it is tumour specific.

Additionally, we examined the functional roles of each OPN splicing isoform in medullary thyroid cancer progression by using an in vitro model. Here we show that TT cells overexpressing OPNa, OPNb and OPNc show a significant decrease in proliferation and viability, being OPNa isoform having the most prominent effect. Although OPN is widely known as implicated in promoting invasive and metastatic progression in many carcinomas (11, 57), in medullary thyroid cancer, the overexpression of OPN isoforms seems to be related with a protective role, as reported in pancreatic adenocarcinoma (58). These results indicate that in MTC, OPN contributes to impair tumour growth, corroborating the hypothesis that OPN can act in the maintenance of the differentiation of C-cells. Further studies, including evaluation of adhesion, invasion and migration, would allow a view of the whole picture and the complete understanding of the role of OPN in C-cell biology and disease.

In summary, the present study shows for the first time that OPN is expressed in MTCs and that such expression is associated with smaller tumour size, less invasive features and overexpression of nuclear PTEN. Yet, our study reports the first description of OPN expression in the specialised neuroendocrine cells of the thyroid, indicating a role for this multifunctional protein in C-cell differentiation. Furthermore, we showed that TT cells overexpressing OPNa isoform show reduced proliferation and viability, indicating that OPN contributes to prevent tumour progression.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding

This study was supported by a CNPq PhD Scholarship (‘National Counsel of Technological and Scientific Development’, Brazil), Science Without Borders, Process n# 237322/2012-9 for L B Ferreira and by FCT, the Portuguese Foundation for Science and Technology, through a PhD grant to CT SFRH/BD/87887/2012 and AP SFRH/BD/110617/2015 and a project CCT FCT/CAPES (Proc 4.4.1.00 CAPES). Further funding was obtained from the project ‘Microenvironment, metabolism and cancer’ that was partially supported by Programa Operacional Regional do Norte (ON.2 – O Novo Norte) under the Quadro de Referência Estratégico Nacional (QREN) and the Fundo Europeu de Desenvolvimento Regional (FEDER). IPATIMUP integrates the i3S Research Unit, which is partially supported by FCT. This study was funded by FEDER funds through the Operational Programme for Competitiveness Factors – COMPETE and National Funds through FCT, under the project ‘PEst-C/SAU/LA0003/2013’.

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  • View in gallery

    OPN cytoplasmic intracellular immunoexpression. (A) Staining in normal gallbladder used as positive control. (B) Normal thyroid gland showing no OPN staining. (C) Normal thyroid tissue showing OPN staining in scattered C-cells. (D) ‘Normal’ thyroid tissue adjacent to MTC; note the OPN positive scattered C-cells (in the inset calcitonin staining of the same area). (E) C-cell hyperplasia showing OPN staining (F) The same area of C-cell hyperplasia stained for calcitonin. (G) A case of MTC showing OPN staining in many neoplastic cells, classified as score: 6 (proportion of positive cells: 50–75%+ staining intensity: 3+). (H) The same case stained for calcitonin showing positivity of almost every neoplastic cells. A full colour version of this figure is available at http://dx.doi.org/10.1530/EJE-15-0577.

  • View in gallery

    Expression of OPN mRNA splicing isoforms in thyroid tissues and in MTC cell lines. (A) Expression of OPNa, OPNb and OPNc splicing isoforms by qRT-PCR in MTC specimens and in the adjacent thyroid. (B) Expression of OPNa, OPNb and OPNc splicing isoforms in TT and in MZ-CRC-1 cell lines by qRT-PCR. Expression of OPN isoforms is presented relative to GAPDH gene expression. A full colour version of this figure is available at http://dx.doi.org/10.1530/EJE-15-0577.

  • View in gallery

    Proliferation and viability in TT cells overexpressing OPNa, OPNb, OPNc and EV. (A) Real-time PCR showing relative expression in TT cells overexpressing OPNa, OPNb, OPNc and EV. (B) Immunocytochemistry analyses of OPN expression in control cells (TT cells with EV) and in TT cells overexpressing OPNa, OPNb and OPNc. (C) MTT and (D) Trypan blue assays showing significantly decreased proliferation levels in TT cells overexpressing OPNa, OPNb, OPNc and EV (n≥3; *P<0.05; **P<0.005). A full colour version of this figure is available at http://dx.doi.org/10.1530/EJE-15-0577.

  • 1

    Pacini F, Castagna MG, Cipri C, Schlumberger M. Medullary thyroid carcinoma. Clinical Oncology 2010 22 475485. (doi:10.1016/j.clon.2010.05.002).

  • 2

    Bhattacharyya N. A population-based analysis of survival factors in differentiated and medullary thyroid carcinoma. Otolaryngology-Head and Neck Surgery 2003 128 115123. (doi:10.1067/mhn.2003.2).

    • Search Google Scholar
    • Export Citation
  • 3

    Ganeshan D, Paulson E, Duran C, Cabanillas ME, Busaidy NL, Charnsangavej C. Current update on medullary thyroid carcinoma. AJR. American Journal of Roentgenology 2013 201 W867W876. (doi:10.2214/AJR.12.10370).

    • Search Google Scholar
    • Export Citation
  • 4

    Sawicki B. Evaluation of the role of mammalian thyroid parafollicular cells. Acta Histochemica 1995 97 389399. (doi:10.1016/S0065-1281(11)80064-4).

    • Search Google Scholar
    • Export Citation
  • 5

    Machens A, Niccoli-Sire P, Hoegel J, Frank-Raue K, van Vroonhoven TJ, Roeher HD, Wahl RA, Lamesch P, Raue F, Conte-Devolx B et al.. Early malignant progression of hereditary medullary thyroid cancer. New England Journal of Medicine 2003 349 15171525. (doi:10.1056/NEJMoa012915).

    • Search Google Scholar
    • Export Citation
  • 6

    Calangiu CM, Margaritescu C, Cernea D, Stepan A, Simionescu CE. The expression of twist in differentiated thyroid carcinomas. Current Health Sciences Journal 2014 40 184189. (doi:10.12865/CHSJ.40.03.05).

    • Search Google Scholar
    • Export Citation
  • 7

    Waligorska-Stachura J, Andrusiewicz M, Sawicka-Gutaj N, Biczysko M, Jankowska A, Kubiczak M, Czarnywojtek A, Wrotkowska E, Ruchala M. Survivin delta Ex3 overexpression in thyroid malignancies. PLoS ONE 2014 9 e100534. (doi:10.1371/journal.pone.0100534).

    • Search Google Scholar
    • Export Citation
  • 8

    Puppin C, Durante C, Sponziello M, Verrienti A, Pecce V, Lavarone E, Baldan F, Campese AF, Boichard A, Lacroix L et al.. Overexpression of genes involved in miRNA biogenesis in medullary thyroid carcinomas with RET mutation. Endocrine 2014 47 528536. (doi:10.1007/s12020-014-0204-3).

    • Search Google Scholar
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  • 9

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