Limits...
The modifier role of RET-G691S polymorphism in hereditary medullary thyroid carcinoma: functional characterization and expression/penetrance studies.

Colombo C, Minna E, Rizzetti MG, Romeo P, Lecis D, Persani L, Mondellini P, Pierotti MA, Greco A, Fugazzola L, Borrello MG - Orphanet J Rare Dis (2015)

Bottom Line: Moreover, among the 25 RET-S891A carriers, a trend towards an earlier age of diagnosis was found in the MTC patients harboring RET-S891A in association with RET-G691S.We demonstrate that the RET-G691S non-synonymous polymorphism enhances in vitro the oncogenic activity of RET-S891A.Moreover, an effect on the phenotype was observed in the RET-G691S/S891A patients, thus suggesting that the analysis of this polymorphism could contribute to the decision on the more appropriate clinical and follow-up management.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Sciences and Community Health, University of Milan, and Endocrine Unit, Fondazione IRCCS Ca' Granda, Milan, Italy. carla.colombo1@unimi.it.

ABSTRACT

Background: Hereditary medullary thyroid carcinoma (MTC) is caused by germ-line gain of function mutations in the RET proto-oncogene, and a phenotypic variability among carriers of the same mutation has been reported. We recently observed this phenomenon in a large familial MTC (FMTC) family carrying the RET-S891A mutation. Among genetic modifiers affecting RET-driven MTC, a role has been hypothesized for RET-G691S non-synonymous polymorphism, though the issue remains controversial. Aim of this study was to define the in vitro contribution of RET-G691S to the oncogenic potential of the RET-S891A, previously shown to harbour low transforming activity.

Methods: The RET-S891A and RET-G691S/S891A mutants were generated by site-directed mutagenesis, transiently transfected in HEK293T cells and stably expressed in NIH3T3 cells. Their oncogenic potential was defined by assessing the migration ability by wound healing assay and the anchorage-independent growth by soft agar assay in NIH3T3 cells stably expressing either the single or the double mutants. Two RET-S891A families were characterised for the presence of RET-G691S.

Results: The functional studies demonstrated that RET-G691S/S891A double mutant displays a higher oncogenic potential than RET-S891A single mutant, assessed by focus formation and migration ability. Moreover, among the 25 RET-S891A carriers, a trend towards an earlier age of diagnosis was found in the MTC patients harboring RET-S891A in association with RET-G691S.

Conclusions: We demonstrate that the RET-G691S non-synonymous polymorphism enhances in vitro the oncogenic activity of RET-S891A. Moreover, an effect on the phenotype was observed in the RET-G691S/S891A patients, thus suggesting that the analysis of this polymorphism could contribute to the decision on the more appropriate clinical and follow-up management.

Show MeSH

Related in: MedlinePlus

RET-S891A and RET-G691S/S891A stable mutants anchorage-independent growth ability by soft-agar assay. A. Agar colonies in NIH3T3 cells stably expressing either RET-S891A or RET-G691S/S891A. NIH3T3 cells and RET-M918T-expressing cells are included as negative and positive controls. The mean ± SD of duplicate counts is reported. For RET-S891A and RET-G691S/S891A mutants two independent clones were analyzed and the mean between clones is reported. Data from one representative of two independent soft-agar assays are shown. Below representative images of the plates scored for agar colonies number and the corresponding pictures of agar colonies the day before fixing and staining (scale bar 200 um). B. Agar colonies mean diameter determined by Image-Pro Plus 7.0.1 software. Data are presented as mean diameter ± SD for each sample. For RET-S891A and RET-G691S/S891A two independent clones were analyzed and the mean between clones is reported. ***P < 0.0001 statistical significance determined via Student’s t-test. Below representative images of agar colonies used for diameter measurement (LEICA inverted microscope, scale bar 1000 μm).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4373282&req=5

Fig3: RET-S891A and RET-G691S/S891A stable mutants anchorage-independent growth ability by soft-agar assay. A. Agar colonies in NIH3T3 cells stably expressing either RET-S891A or RET-G691S/S891A. NIH3T3 cells and RET-M918T-expressing cells are included as negative and positive controls. The mean ± SD of duplicate counts is reported. For RET-S891A and RET-G691S/S891A mutants two independent clones were analyzed and the mean between clones is reported. Data from one representative of two independent soft-agar assays are shown. Below representative images of the plates scored for agar colonies number and the corresponding pictures of agar colonies the day before fixing and staining (scale bar 200 um). B. Agar colonies mean diameter determined by Image-Pro Plus 7.0.1 software. Data are presented as mean diameter ± SD for each sample. For RET-S891A and RET-G691S/S891A two independent clones were analyzed and the mean between clones is reported. ***P < 0.0001 statistical significance determined via Student’s t-test. Below representative images of agar colonies used for diameter measurement (LEICA inverted microscope, scale bar 1000 μm).

Mentions: Moreover, the anchorage-independent growth ability was assessed by soft agar assay in cells expressing the single and double mutants (Figure 3). On average RET-G691S/S891A double mutants give rise to a statistically significant higher number of agar colonies than RET-S891A single mutants (Figure 3A). In addition, the size of agar colonies originated by RET-G691S/S891A double mutants results statistically significant higher than those generated by RET-S891A single mutants (Figure 3B). To be noted, RET-M918T mutant generates agar colonies bigger than RET-G691S/S891A double mutants in agreement with its reported high oncogenic potential. These results indicate that in RET mutants the presence of the G691S polymorphism enhances the oncogenic properties, such as the migration and the agar colony formation ability.Figure 3


The modifier role of RET-G691S polymorphism in hereditary medullary thyroid carcinoma: functional characterization and expression/penetrance studies.

Colombo C, Minna E, Rizzetti MG, Romeo P, Lecis D, Persani L, Mondellini P, Pierotti MA, Greco A, Fugazzola L, Borrello MG - Orphanet J Rare Dis (2015)

RET-S891A and RET-G691S/S891A stable mutants anchorage-independent growth ability by soft-agar assay. A. Agar colonies in NIH3T3 cells stably expressing either RET-S891A or RET-G691S/S891A. NIH3T3 cells and RET-M918T-expressing cells are included as negative and positive controls. The mean ± SD of duplicate counts is reported. For RET-S891A and RET-G691S/S891A mutants two independent clones were analyzed and the mean between clones is reported. Data from one representative of two independent soft-agar assays are shown. Below representative images of the plates scored for agar colonies number and the corresponding pictures of agar colonies the day before fixing and staining (scale bar 200 um). B. Agar colonies mean diameter determined by Image-Pro Plus 7.0.1 software. Data are presented as mean diameter ± SD for each sample. For RET-S891A and RET-G691S/S891A two independent clones were analyzed and the mean between clones is reported. ***P < 0.0001 statistical significance determined via Student’s t-test. Below representative images of agar colonies used for diameter measurement (LEICA inverted microscope, scale bar 1000 μm).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4373282&req=5

Fig3: RET-S891A and RET-G691S/S891A stable mutants anchorage-independent growth ability by soft-agar assay. A. Agar colonies in NIH3T3 cells stably expressing either RET-S891A or RET-G691S/S891A. NIH3T3 cells and RET-M918T-expressing cells are included as negative and positive controls. The mean ± SD of duplicate counts is reported. For RET-S891A and RET-G691S/S891A mutants two independent clones were analyzed and the mean between clones is reported. Data from one representative of two independent soft-agar assays are shown. Below representative images of the plates scored for agar colonies number and the corresponding pictures of agar colonies the day before fixing and staining (scale bar 200 um). B. Agar colonies mean diameter determined by Image-Pro Plus 7.0.1 software. Data are presented as mean diameter ± SD for each sample. For RET-S891A and RET-G691S/S891A two independent clones were analyzed and the mean between clones is reported. ***P < 0.0001 statistical significance determined via Student’s t-test. Below representative images of agar colonies used for diameter measurement (LEICA inverted microscope, scale bar 1000 μm).
Mentions: Moreover, the anchorage-independent growth ability was assessed by soft agar assay in cells expressing the single and double mutants (Figure 3). On average RET-G691S/S891A double mutants give rise to a statistically significant higher number of agar colonies than RET-S891A single mutants (Figure 3A). In addition, the size of agar colonies originated by RET-G691S/S891A double mutants results statistically significant higher than those generated by RET-S891A single mutants (Figure 3B). To be noted, RET-M918T mutant generates agar colonies bigger than RET-G691S/S891A double mutants in agreement with its reported high oncogenic potential. These results indicate that in RET mutants the presence of the G691S polymorphism enhances the oncogenic properties, such as the migration and the agar colony formation ability.Figure 3

Bottom Line: Moreover, among the 25 RET-S891A carriers, a trend towards an earlier age of diagnosis was found in the MTC patients harboring RET-S891A in association with RET-G691S.We demonstrate that the RET-G691S non-synonymous polymorphism enhances in vitro the oncogenic activity of RET-S891A.Moreover, an effect on the phenotype was observed in the RET-G691S/S891A patients, thus suggesting that the analysis of this polymorphism could contribute to the decision on the more appropriate clinical and follow-up management.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Sciences and Community Health, University of Milan, and Endocrine Unit, Fondazione IRCCS Ca' Granda, Milan, Italy. carla.colombo1@unimi.it.

ABSTRACT

Background: Hereditary medullary thyroid carcinoma (MTC) is caused by germ-line gain of function mutations in the RET proto-oncogene, and a phenotypic variability among carriers of the same mutation has been reported. We recently observed this phenomenon in a large familial MTC (FMTC) family carrying the RET-S891A mutation. Among genetic modifiers affecting RET-driven MTC, a role has been hypothesized for RET-G691S non-synonymous polymorphism, though the issue remains controversial. Aim of this study was to define the in vitro contribution of RET-G691S to the oncogenic potential of the RET-S891A, previously shown to harbour low transforming activity.

Methods: The RET-S891A and RET-G691S/S891A mutants were generated by site-directed mutagenesis, transiently transfected in HEK293T cells and stably expressed in NIH3T3 cells. Their oncogenic potential was defined by assessing the migration ability by wound healing assay and the anchorage-independent growth by soft agar assay in NIH3T3 cells stably expressing either the single or the double mutants. Two RET-S891A families were characterised for the presence of RET-G691S.

Results: The functional studies demonstrated that RET-G691S/S891A double mutant displays a higher oncogenic potential than RET-S891A single mutant, assessed by focus formation and migration ability. Moreover, among the 25 RET-S891A carriers, a trend towards an earlier age of diagnosis was found in the MTC patients harboring RET-S891A in association with RET-G691S.

Conclusions: We demonstrate that the RET-G691S non-synonymous polymorphism enhances in vitro the oncogenic activity of RET-S891A. Moreover, an effect on the phenotype was observed in the RET-G691S/S891A patients, thus suggesting that the analysis of this polymorphism could contribute to the decision on the more appropriate clinical and follow-up management.

Show MeSH
Related in: MedlinePlus