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 andRET-G691S/S891A mutants biochemical characterization and transforming activity. A. RET protein domains (CL, cadherin-like; CR, Cysteine-rich; TM, transmembrane; JM, juxtamembrane; TK, tyrosine kinase) and the RET51 isoform C-terminal tail are indicated. B. Western blot analysis of RET protein and associated ERK signaling in HEK293T cells transiently transfected. RET was detected by the antibody RET H300, specific for the RET extracellular portion, and by RET C20, specific for the intracellular portion of the RET long isoform. Vinculin is shown as protein loading control. Protein levels were quantified by densitometric analysis by Quantity One 4.6.6 software. Data are shown as mean expression value ± SD for 2 independent transfections. C. RET-S891A and RET-G691S/S891A mutants transforming activity by focus formation assay in NIH3T3 cells. pCDNA3 empty vector (Mock) and RET-M918T are included as negative and positive controls, respectively. The transforming activity, calculated as foci number/colonies number ratio, is reported as mean ± SD of triplicate counts. Data from one representative of two independent transfections are shown. D. Representative images of foci-derived clones. Scale bar 50um. E. Western blot analysis of RET protein and associated ERK signaling in NIH3T3 cells stably expressing the indicated RET variants: 634, RET-C634R; 918, RET-M918T; 691/891, RET-G691S/S891A; 891, RET-S891A. NIH, NIH3T3 untransfected cells. The partially and fully glycosylated forms of RET are identified in NIH3T3 cells. Vinculin is shown as protein loading control. Three RET-G691S/S891A clones and four RET-S891A clones were analysed. The ratio pERK/ERK/RET was calculated for each clone and the mean among clones ± SD was reported.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig1: RET-S891A andRET-G691S/S891A mutants biochemical characterization and transforming activity. A. RET protein domains (CL, cadherin-like; CR, Cysteine-rich; TM, transmembrane; JM, juxtamembrane; TK, tyrosine kinase) and the RET51 isoform C-terminal tail are indicated. B. Western blot analysis of RET protein and associated ERK signaling in HEK293T cells transiently transfected. RET was detected by the antibody RET H300, specific for the RET extracellular portion, and by RET C20, specific for the intracellular portion of the RET long isoform. Vinculin is shown as protein loading control. Protein levels were quantified by densitometric analysis by Quantity One 4.6.6 software. Data are shown as mean expression value ± SD for 2 independent transfections. C. RET-S891A and RET-G691S/S891A mutants transforming activity by focus formation assay in NIH3T3 cells. pCDNA3 empty vector (Mock) and RET-M918T are included as negative and positive controls, respectively. The transforming activity, calculated as foci number/colonies number ratio, is reported as mean ± SD of triplicate counts. Data from one representative of two independent transfections are shown. D. Representative images of foci-derived clones. Scale bar 50um. E. Western blot analysis of RET protein and associated ERK signaling in NIH3T3 cells stably expressing the indicated RET variants: 634, RET-C634R; 918, RET-M918T; 691/891, RET-G691S/S891A; 891, RET-S891A. NIH, NIH3T3 untransfected cells. The partially and fully glycosylated forms of RET are identified in NIH3T3 cells. Vinculin is shown as protein loading control. Three RET-G691S/S891A clones and four RET-S891A clones were analysed. The ratio pERK/ERK/RET was calculated for each clone and the mean among clones ± SD was reported.

Mentions: To assess the contribution of the RET-G691S polymorphism to the in vitro oncogenic potential of RET-S891A mutant, both localized in the intracellular domains of RET protein (Figure 1A), we generated RET-S891A single and RET-G691S/S891A double mutants by site-directed mutagenesis of recombinant plasmids carrying the RET-wt long isoform.Figure 1


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 mutants biochemical characterization and transforming activity. A. RET protein domains (CL, cadherin-like; CR, Cysteine-rich; TM, transmembrane; JM, juxtamembrane; TK, tyrosine kinase) and the RET51 isoform C-terminal tail are indicated. B. Western blot analysis of RET protein and associated ERK signaling in HEK293T cells transiently transfected. RET was detected by the antibody RET H300, specific for the RET extracellular portion, and by RET C20, specific for the intracellular portion of the RET long isoform. Vinculin is shown as protein loading control. Protein levels were quantified by densitometric analysis by Quantity One 4.6.6 software. Data are shown as mean expression value ± SD for 2 independent transfections. C. RET-S891A and RET-G691S/S891A mutants transforming activity by focus formation assay in NIH3T3 cells. pCDNA3 empty vector (Mock) and RET-M918T are included as negative and positive controls, respectively. The transforming activity, calculated as foci number/colonies number ratio, is reported as mean ± SD of triplicate counts. Data from one representative of two independent transfections are shown. D. Representative images of foci-derived clones. Scale bar 50um. E. Western blot analysis of RET protein and associated ERK signaling in NIH3T3 cells stably expressing the indicated RET variants: 634, RET-C634R; 918, RET-M918T; 691/891, RET-G691S/S891A; 891, RET-S891A. NIH, NIH3T3 untransfected cells. The partially and fully glycosylated forms of RET are identified in NIH3T3 cells. Vinculin is shown as protein loading control. Three RET-G691S/S891A clones and four RET-S891A clones were analysed. The ratio pERK/ERK/RET was calculated for each clone and the mean among clones ± SD was reported.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig1: RET-S891A andRET-G691S/S891A mutants biochemical characterization and transforming activity. A. RET protein domains (CL, cadherin-like; CR, Cysteine-rich; TM, transmembrane; JM, juxtamembrane; TK, tyrosine kinase) and the RET51 isoform C-terminal tail are indicated. B. Western blot analysis of RET protein and associated ERK signaling in HEK293T cells transiently transfected. RET was detected by the antibody RET H300, specific for the RET extracellular portion, and by RET C20, specific for the intracellular portion of the RET long isoform. Vinculin is shown as protein loading control. Protein levels were quantified by densitometric analysis by Quantity One 4.6.6 software. Data are shown as mean expression value ± SD for 2 independent transfections. C. RET-S891A and RET-G691S/S891A mutants transforming activity by focus formation assay in NIH3T3 cells. pCDNA3 empty vector (Mock) and RET-M918T are included as negative and positive controls, respectively. The transforming activity, calculated as foci number/colonies number ratio, is reported as mean ± SD of triplicate counts. Data from one representative of two independent transfections are shown. D. Representative images of foci-derived clones. Scale bar 50um. E. Western blot analysis of RET protein and associated ERK signaling in NIH3T3 cells stably expressing the indicated RET variants: 634, RET-C634R; 918, RET-M918T; 691/891, RET-G691S/S891A; 891, RET-S891A. NIH, NIH3T3 untransfected cells. The partially and fully glycosylated forms of RET are identified in NIH3T3 cells. Vinculin is shown as protein loading control. Three RET-G691S/S891A clones and four RET-S891A clones were analysed. The ratio pERK/ERK/RET was calculated for each clone and the mean among clones ± SD was reported.
Mentions: To assess the contribution of the RET-G691S polymorphism to the in vitro oncogenic potential of RET-S891A mutant, both localized in the intracellular domains of RET protein (Figure 1A), we generated RET-S891A single and RET-G691S/S891A double mutants by site-directed mutagenesis of recombinant plasmids carrying the RET-wt long isoform.Figure 1

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