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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.

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RET-S891A andRET-G691S/S891A stable mutants migration ability by wound healing assay. A. Western blot analysis of RET protein and associated ERK signaling in NIH3T3 cells stably expressing either RET-S891A or RET-G691S/S891A. RET was detected by RET C20 primary antibody. Tubulin is shown as protein loading control. Two clones expressing different levels of RET were analyzed for both variants. NIH3T3 cells and RET-M918T-expressing cells are included as negative and positive controls, respectively. B. Wound-healing assay in RET stable mutants. The wound closure percentage was quantified each hour for 24 h post-wound. Graphs are mean of two independent experiments performed in duplicate. C. Wound closure percentage at 12 h post-wound. Data are shown as mean ± SD of two independent experiments performed in duplicate. Statistical significance determined by Two-tailed Unpaired t test. *p < 0.05. D. Representative images at 0 h and 12 h post-wound (x10).
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Fig2: RET-S891A andRET-G691S/S891A stable mutants migration ability by wound healing assay. A. Western blot analysis of RET protein and associated ERK signaling in NIH3T3 cells stably expressing either RET-S891A or RET-G691S/S891A. RET was detected by RET C20 primary antibody. Tubulin is shown as protein loading control. Two clones expressing different levels of RET were analyzed for both variants. NIH3T3 cells and RET-M918T-expressing cells are included as negative and positive controls, respectively. B. Wound-healing assay in RET stable mutants. The wound closure percentage was quantified each hour for 24 h post-wound. Graphs are mean of two independent experiments performed in duplicate. C. Wound closure percentage at 12 h post-wound. Data are shown as mean ± SD of two independent experiments performed in duplicate. Statistical significance determined by Two-tailed Unpaired t test. *p < 0.05. D. Representative images at 0 h and 12 h post-wound (x10).

Mentions: To better characterize the tumorigenic properties of RET-S891A single and RET-G691S/S891A double mutants, we performed the wound healing assay (Figure 2). The wound closure capacity, indicative of migration ability, was assessed for both mutants in two representative clones expressing different levels of RET protein (Figure 2A). Data indicate that RET-S891A single mutants display a statistically significant reduced capacity to close the wound compared with the RET-G691S/S891A double mutants. Of note, the presence of the 691S variant enhances the wound closure ability in clones expressing both high levels of RET (691/891 clone 3 vs. 891 clone 5) and low levels of RET (691/891 clone 1 vs. 891 clone 4).Figure 2


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 andRET-G691S/S891A stable mutants migration ability by wound healing assay. A. Western blot analysis of RET protein and associated ERK signaling in NIH3T3 cells stably expressing either RET-S891A or RET-G691S/S891A. RET was detected by RET C20 primary antibody. Tubulin is shown as protein loading control. Two clones expressing different levels of RET were analyzed for both variants. NIH3T3 cells and RET-M918T-expressing cells are included as negative and positive controls, respectively. B. Wound-healing assay in RET stable mutants. The wound closure percentage was quantified each hour for 24 h post-wound. Graphs are mean of two independent experiments performed in duplicate. C. Wound closure percentage at 12 h post-wound. Data are shown as mean ± SD of two independent experiments performed in duplicate. Statistical significance determined by Two-tailed Unpaired t test. *p < 0.05. D. Representative images at 0 h and 12 h post-wound (x10).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig2: RET-S891A andRET-G691S/S891A stable mutants migration ability by wound healing assay. A. Western blot analysis of RET protein and associated ERK signaling in NIH3T3 cells stably expressing either RET-S891A or RET-G691S/S891A. RET was detected by RET C20 primary antibody. Tubulin is shown as protein loading control. Two clones expressing different levels of RET were analyzed for both variants. NIH3T3 cells and RET-M918T-expressing cells are included as negative and positive controls, respectively. B. Wound-healing assay in RET stable mutants. The wound closure percentage was quantified each hour for 24 h post-wound. Graphs are mean of two independent experiments performed in duplicate. C. Wound closure percentage at 12 h post-wound. Data are shown as mean ± SD of two independent experiments performed in duplicate. Statistical significance determined by Two-tailed Unpaired t test. *p < 0.05. D. Representative images at 0 h and 12 h post-wound (x10).
Mentions: To better characterize the tumorigenic properties of RET-S891A single and RET-G691S/S891A double mutants, we performed the wound healing assay (Figure 2). The wound closure capacity, indicative of migration ability, was assessed for both mutants in two representative clones expressing different levels of RET protein (Figure 2A). Data indicate that RET-S891A single mutants display a statistically significant reduced capacity to close the wound compared with the RET-G691S/S891A double mutants. Of note, the presence of the 691S variant enhances the wound closure ability in clones expressing both high levels of RET (691/891 clone 3 vs. 891 clone 5) and low levels of RET (691/891 clone 1 vs. 891 clone 4).Figure 2

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