Background
Medullary Thyroid Cancer (MTC) is a neuroendocrine neoplasia arising from thyroid parafollicular C cells. Hereditary forms account for 25% of cases and include multiple endocrine neoplasia syndromes type 2A (MEN2A), type 2B (MEN2B) and familial MTC (FMTC), caused by mutations in the rearranged during transfection (
RET) proto-oncogene. The
RET gene, which encodes a tyrosine kinase receptor with a crucial role in development, comprises 21 exons and generates a transcript subjected to alternative splicing leading to two main isoforms: a protein of 1114 residues displaying 51 C-terminal-specific amino acids (RET51) and a shorter protein of 1072 residues displaying nine unrelated C-terminal-specific amino acids (RET9) [
1].
Beyond the known role of
RET gain-of-function mutations in MTC, in recent years several authors investigated whether the presence of single nucleotide polymorphisms (SNPs) could be associated with susceptibility for the development or progression of MTC [
2-
5]. Among
RET polymorphisms,
RET-G691S, localized in exon 11 and reported in general population with an allele frequency of 11–33% (ARUP database “MEN2 and RET”, displaying sequence variation and clinical information [
6]), is a non-synonymous variant [
7-
9], and could potentially affect protein function. Moreover,
RET-G691S has been suggested to be a genetic modifier in MEN2A, related to an earlier age at presentation [
7,
9,
10] and has been associated to the susceptibility to sporadic MTC [
8,
11-
13], though controversial data have been reported about this issue [
14].
Interestingly,
RET-G691S has been also hypothesized to play a functional role on tumor growth and aggressiveness in pancreatic cancers and cutaneous melanoma, where it works as a genetic modifier or even as a low penetrance gene [
15,
16]. In particular,
RET-G691S was reported to enhance the activation of the downstream ERK pathway compared to
RET-wt [
15], though this variant was not found to be oncogenic
per se by focus formation assays [
5,
17].
Consistently with the potential modifier role of
RET-G691S in MTC, Vandenbosch et al. observed that this polymorphism is able to enhance
RET oncogenicity of mutations affecting RET codon 666, by increasing its penetrance in the clinical onset [
18]. Accordingly, we firstly demonstrated the
in vitro effect of the
RET-G691S variant as enhancer of the ERK1/2 activation and of the transforming activity of the MTC-associated
RET-K666E mutant [
17].
In the present study, we investigated more deeply the potential role of this non synonymous
RET polymorphism as a modifier of the phenotypic expression in familial MTC. To this purpose, we focused on the potential contribution of
RET-G691S to the oncogenicity of
RET-S891A, a
RET mutant previously reported to be associated with low transforming activity. In particular, this gain-of-function mutation, located in the second intracellular tyrosine kinase domain of the
RET proto-oncogene, accounts for less than 5% of all
RET mutated patients, and can cause FMTC or the phenotype associated to MEN2A [
19-
23].
Since the functional characterization of RET mutants adds useful information to the genotyping of patients/families optimizing the diagnostic and clinical management, we both evaluated the in vitro biological activity of the RET-G691S polymorphism on the oncogenic potential of RET-S891A and carried out expression/penetrance studies in two unrelated RET-S891A FMTC families.
Methods
In vitro analyses
Construction of the RET mutants
All
RET mutants were obtained by mutagenesis of
RET51-wt construct (pCDNA3 vector expressing the proto-
RET gene long isoform).
RET51-G691S,
RET51-C634R (containing an MEN2A causing mutation), and
RET51-M918T (containing the main MEN2B causing mutation) used as controls are described elsewhere [
17].
RET51-S891A and RET51-G691S/S891A were obtained by site-directed mutagenesis of RET51-WT using an in vitro oligonucleotide mutagenesis system (Quik-Change XL site-directed mutagenesis; Stratagene, La Jolla, CA, USA). The transition c.2071G > A leading to G691S polymorphism (SNIP rs1799939) and the transversion c.2671 T > G leading to S891A mutation were verified by DNA direct sequencing and then the mutant clones were entirely sequenced to exclude possible additional mutations. Plasmid DNA was extracted using the MAXI PREP Kit (Qiagen) as suggested by the supplier.
Cell culture, transfections and focus formation assay
Human HEK293T cells were maintained in DMEM with 10% FCS. The recombinant plasmids were transiently transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions.
NIH3T3 cells were cultured in DMEM supplemented with 10% serum (Colorado Company, CO). Stable transfection was performed by the CaPO4 method [
24] using 100 ng plasmid DNA together with 10 μg NIH3T3-derived DNA carrier. Transfected cells were grown in DMEM with 10% serum and selected in the presence of G418 antibiotic (650 μg/ml) to obtain G418-resistant colonies indicative of transfection efficiency; transformed foci were obtained in DMEM with 5% serum in the presence of chronic stimulation with the RET ligand GDNF at a concentration of 10 ng/ml (AlomoneLabs, Jerusalem, Israel). Both G418-resistant colonies and transformed foci were fixed and counted to determine the transforming activity, calculated as foci number/colonies number ratio. NIH3T3 cell lines stably expressing
RET mutants were derived from transformation foci picked from parallel original plates of NIH3T3 transfected cells and cultured in the presence of chronic stimulation of GDNF (10 ng/ml).
Western blot analysis
Cells were lysed in ice-cold RIPA buffer (20 mM Tris pH 7.4, 137 mM NaCl, 10% glycerol, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100, 2 mM EDTA pH 8.0) containing protease and phosphatase inhibitors.
Proteins were quantified using a modified Bradford assay (Bio-Rad). Protein samples were boiled in NuPAGE LDS sample buffer (Invitrogen), separated by NuPAGE Novex Gels with the appropriate running buffer (Invitrogen), then transferred onto nitrocellulose filters, and immunoblotted with the indicated antibodies. Anti-RET (C-20), anti-RET (H300) and anti-phospho-RET are from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-MAP kinase (ERK1/2), anti-MAP kinase activated (pERK1/2), anti-vinculin and anti-β-tubulin are from Sigma Aldrich (Saint Louis, Missouri, USA).
Densitometric analyses were performed by the Quantity One 4.6.6 software (Bio-Rad, Hercules, CA).
Wound healing assay
NIH3T3 cells stably expressing RET mutants were seeded at 30000 cells/chamber in a culture insert (Ibidi #80209) placed in 24-well plate and incubated overnight. Inserts were then removed to generate a 500 μm gap between cells. Following PBS wash and fresh medium supplement, plates were placed in the Cell-IQ SLF instrument (CM Technology Oy, Tampere, Finland) and cultured at 37°C, 5% CO2. Two images for well were taken each hour for 24 h using Cell-IQ Imagen software (CM Technology Oy) to monitor the gap closure. Images were then analyzed with the Scratch wound measurement tool of the Cell-IQ Analyser software (CM Technology Oy), to evaluate the % of closed area calculated with the equation: (Start wound - wound [μm2])/Start wound × 100. Data and graphs were analyzed using GraphPad Prism 5.02.
Soft-agar assay
For anchorage-independent growth assay, NIH3T3 cells stably expressing RET mutants were suspended in DMEM containing 0.33% agar, 10% serum and GDFN 10 ng/ml (50000 cells/1.5 ml medium) and added into a layer of medium containing 0.5% agar and 10% serum in 60 mm dishes. After 3 weeks incubation, plates were analyzed for colonies number and size. Colony number was determined in plates stained with p-iodonitrotetrazolium chloride violet (Sigma Aldrich).
Agar colonies size, calculated as mean diameter, was determined by the count/size tool in Image-Pro Plus 7.0.1 software analyzing the agar colonies images the day before staining. A cutoff area of 250 pixel was manually set on NIH3T3 control cells. Only foci surpassing the cut-off area were automatically selected and scored by the software. Diameter data relative to the total foci analyzed per each field were exported and the mean diameter for each sample was calculated.
Patients: clinical and molecular characterization
RET-G691S polymorphism expression/penetrance was assessed in two FMTC families carrying the germline activating mutation
RET-S891A. Family 1 is an extremely large family with members distributed in several cities of the North of Italy, with a common ancestor [
25]. Data related to some members of Family 1 have been partially reported in a previous study [
23]. Family 2, from Southern Italy, is composed by 2 generations for a total of 3 subjects. Clinical and molecular features, including age at diagnosis, gender, TNM staging [
26] and the disease’s outcome after a mean follow-up time of 60 months were obtained for both families. Patients were diagnosed and treated according to the American guidelines for the management of MTC [
27,
28]; in particular,
RET gene carriers were screened by means of yearly basal and stimulated CT measurement and neck ultrasound. Prophylactic surgery was performed at the first evidence of a positive provocative test.
Genomic DNA was extracted from peripheral blood leucocytes of FMTC families members and the presence of
RET-G691S polymorphism was assessed by PCR and direct sequencing as previously described [
23]. All patients gave their informed consent, approved by the local Ethical Committee, to the genetic characterization and to the analysis of data.
Statistical analysis
Statistical analysis and graphs were generated using GraphPad Prism version 5.02. Comparisons between two groups were performed by two-tailed Student’s t-test with unequal variance. A value of P < 0.05 was considered statistically significant.
Discussion
In this study we demonstrated for the first time that the functional polymorphic variant RET-G691S, not oncogenic per se, enhances the in vitro oncogenic potential of the RET-S891A, a mutant causing hereditary MTC. Furthermore, we showed in two FMTC families that carriers of RET-S891A mutation associated with the RET-G691S polymorphic variant display a trend towards an earlier age at diagnosis of MTC and the adrenal medulla involvement.
RET mutations in hereditary MTC are a paradigmatic example of clinical decision based on molecular diagnosis. Nevertheless, even within families and patients with the same
RET mutation, the age of disease onset and the phenotypes are unpredictable, suggesting the existence of modifier loci influencing the expression and severity of the disease whose identification could drive the diagnostic and therapeutic management. Accordingly, though
RET-S891A mutation has been associated to isolated FMTC for several years after its first identification in 1997 [
29], a MEN2 clinical spectrum has been more recently reported in around 4% of cases [
19]. Interestingly, a recent case report paper identify bilateral pheochromocytoma as the first manifestation of MEN2A disease in a patient of a family carrying the
RET-S891A mutation [
30].
Among the potential genetic modifiers,
RET gene polymorphisms have been associated with susceptibility and/or disease progression [
2-
5], and the attention has been mainly focused on the non-synonymous variant
RET-G691S. Although debated,
RET-G691S has been suggested to have a role in MTC susceptibility and in the modulation of the age of onset [
7-
9,
13,
31]. In this context we recently showed that
RET-G691S, not oncogenic
per se, enhances
in vitro the oncogenic potential of the rare germline mutation
RET-K666E [
5,
17], accordingly with the observation of Vandenbosch et al. that this polymorphism is able to increase the penetrance in the clinical onset of mutations affecting
RET 666 codon [
18].
In the present work, to get more insights and to confirm the possible role of
RET-G691S in the modulation of the oncogenic capacity and phenotype expression of
RET mutants, we focused on the
RET-S891A. This intermediated risk mutation displays
in vitro low transforming activity [
21] that may be enhanced by GDNF treatment (data not shown), as previously reported for other
RET mutations localized in the tyrosine kinase domain, such as
RET-M918T causally related to MEN2B [
32]. In agreement with our initial hypothesis, we showed in this study that also for
RET-S891A mutation the association with
RET-G691S polymorphism correlates
in vitro with enhanced tumorigenic properties such as transforming activity and migratory and clonogenic ability. These findings, indicating a possible role of genetic modifier for this polymorphism, are concordant with the expression/penetrance pattern of the two analyzed
RET-S891A FMTC families, that include members harboring the
RET-S891A mutation associated or not with the
RET-G691S polymorphism. Though not statistically significant, an earlier age at diagnosis of MTC was observed in
RET-G691S/S891A carriers compared with
RET-S891A carriers, suggesting a possible role in the modulation of the age at disease onset.
Our
in vitro studies, mimicking the
cis presentation of
RET-S891A mutation and
RET-G691S polymorphism observed in the Family 2, demonstrate the oncogenic enhancer role of the polymorphism in the case of
cis presentation. Moreover, the clinical data from both families suggest a trend towards an earlier age at diagnosis of MTC in the
RET-G691S/S891A carriers irrespectively of the
cis or
trans presentation of the
RET-S891A mutation and the
RET-G691S polymorphism (Additional file
1: Figure S1B). Therefore, it is conceivable to hypothesize that the
RET-G691S polymorphism may act as genetic modifier also in the case of
trans presentation with
RET-S891A mutation. However, further studies are necessary to demonstrate that
RET-
G691S polymorphism may enhance the oncogenic activity of
RET-S891A, or of other
RET mutants, also in trans.
Moreover, clinical data from Family 2 indicate that two out of three RET-G691/S891A patients had elevated normetanephrine levels, suggesting that the full expression of MEN2A phenotype could be related to the association with this non synonymous polymporphism, especially in the case of cis presentation with RET-S891A mutation.
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
CC, LF, MGB conceived the study and participated in its design and coordination. CC, MGR, EM, PR performed in vitro studies, analyzed the data and carried out statistical analysis. DL performed and analyzed wound healing assay. CC, LP, LF, MGB contributed to clinical and genetic evaluation of the patients. CC, LF, MGB wrote the manuscript. EM, PR, LP, AG participated in manuscript writing. All authors read and approved the final manuscript.