Molecular and functional characterization of three different postzygotic mutations in PIK3CA-related overgrowth spectrum (PROS) patients: Effects on PI3K/AKT/mTOR signaling and sensitivity to PIK3 inhibitors

Daria C. Loconte, Valentina Grossi, Cristina Bozzao, Giovanna Forte, Rosanna Bagnulo, Alessandro Stella, Patrizia Lastella, Mario Cutrone, Francesco Benedicenti, Francesco C. Susca, Margherita Patruno, Dora Varvara, Aldo Germani, Luciana Chessa, Nicola Laforgia, Romano Tenconi, Cristiano Simone, Nicoletta Resta

Research output: Contribution to journalArticle

Abstract

Background: PIK3CA-related overgrowth spectrum (PROS) include a group of disorders that affect only the terminal portion of a limb, such as type I macrodactyly, and conditions like fibroadipose overgrowth (FAO), megalencephaly-capillary malformation (MCAP) syndrome, congenital lipomatous asymmetric overgrowth of the trunk, lymphatic, capillary, venous, and combinedtype vascular malformations, epidermal nevi, skeletal and spinal anomalies (CLOVES) syndrome and Hemihyperplasia Multiple Lipomatosis (HHML). Heterozygous postzygotic PIK3CA mutations are frequently identified in these syndromes, while timing and tissue specificity of the mutational event are likely responsible for the extreme phenotypic variability observed. Methods: We carried out a combination of Sanger sequencing and targeted deep sequencing of genes involved in the PI3K/AKT/mTOR pathway in three patients (1 MCAP and 2 FAO) to identify causative mutations, and performed immunoblot analyses to assay the phosphorylation status of AKT and P70S6K in affected dermal fibroblasts. In addition, we evaluated their ability to grow in the absence of serum and their response to the PI3K inhibitors wortmannin and LY294002 in vitro. Results and Conclusion: Our data indicate that patients ' cells showed constitutive activation of the PI3K/Akt pathway. Of note, PI3K pharmacological blockade resulted in a significant reduction of the proliferation rate in culture, suggesting that inhibition of PI3K might prove beneficial in future therapies for PROS patients.

Original languageEnglish
Article numbere0123092
JournalPLoS One
Volume10
Issue number4
DOIs
Publication statusPublished - Apr 27 2015

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phosphatidylinositol 3-kinase
Phosphatidylinositol 3-Kinases
mutation
Mutation
Megalencephaly
70-kDa Ribosomal Protein S6 Kinases
High-Throughput Nucleotide Sequencing
Organ Specificity
Vascular Malformations
Phosphorylation
2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one
Lymphatic Vessels
Lipoma
Fibroblasts
blood vessels
fibroblasts
Assays
phosphorylation
Extremities
Genes

ASJC Scopus subject areas

  • Agricultural and Biological Sciences(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Medicine(all)

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Molecular and functional characterization of three different postzygotic mutations in PIK3CA-related overgrowth spectrum (PROS) patients : Effects on PI3K/AKT/mTOR signaling and sensitivity to PIK3 inhibitors. / Loconte, Daria C.; Grossi, Valentina; Bozzao, Cristina; Forte, Giovanna; Bagnulo, Rosanna; Stella, Alessandro; Lastella, Patrizia; Cutrone, Mario; Benedicenti, Francesco; Susca, Francesco C.; Patruno, Margherita; Varvara, Dora; Germani, Aldo; Chessa, Luciana; Laforgia, Nicola; Tenconi, Romano; Simone, Cristiano; Resta, Nicoletta.

In: PLoS One, Vol. 10, No. 4, e0123092, 27.04.2015.

Research output: Contribution to journalArticle

Loconte, DC, Grossi, V, Bozzao, C, Forte, G, Bagnulo, R, Stella, A, Lastella, P, Cutrone, M, Benedicenti, F, Susca, FC, Patruno, M, Varvara, D, Germani, A, Chessa, L, Laforgia, N, Tenconi, R, Simone, C & Resta, N 2015, 'Molecular and functional characterization of three different postzygotic mutations in PIK3CA-related overgrowth spectrum (PROS) patients: Effects on PI3K/AKT/mTOR signaling and sensitivity to PIK3 inhibitors', PLoS One, vol. 10, no. 4, e0123092. https://doi.org/10.1371/journal.pone.0123092
Loconte, Daria C. ; Grossi, Valentina ; Bozzao, Cristina ; Forte, Giovanna ; Bagnulo, Rosanna ; Stella, Alessandro ; Lastella, Patrizia ; Cutrone, Mario ; Benedicenti, Francesco ; Susca, Francesco C. ; Patruno, Margherita ; Varvara, Dora ; Germani, Aldo ; Chessa, Luciana ; Laforgia, Nicola ; Tenconi, Romano ; Simone, Cristiano ; Resta, Nicoletta. / Molecular and functional characterization of three different postzygotic mutations in PIK3CA-related overgrowth spectrum (PROS) patients : Effects on PI3K/AKT/mTOR signaling and sensitivity to PIK3 inhibitors. In: PLoS One. 2015 ; Vol. 10, No. 4.
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T1 - Molecular and functional characterization of three different postzygotic mutations in PIK3CA-related overgrowth spectrum (PROS) patients

T2 - Effects on PI3K/AKT/mTOR signaling and sensitivity to PIK3 inhibitors

AU - Loconte, Daria C.

AU - Grossi, Valentina

AU - Bozzao, Cristina

AU - Forte, Giovanna

AU - Bagnulo, Rosanna

AU - Stella, Alessandro

AU - Lastella, Patrizia

AU - Cutrone, Mario

AU - Benedicenti, Francesco

AU - Susca, Francesco C.

AU - Patruno, Margherita

AU - Varvara, Dora

AU - Germani, Aldo

AU - Chessa, Luciana

AU - Laforgia, Nicola

AU - Tenconi, Romano

AU - Simone, Cristiano

AU - Resta, Nicoletta

PY - 2015/4/27

Y1 - 2015/4/27

N2 - Background: PIK3CA-related overgrowth spectrum (PROS) include a group of disorders that affect only the terminal portion of a limb, such as type I macrodactyly, and conditions like fibroadipose overgrowth (FAO), megalencephaly-capillary malformation (MCAP) syndrome, congenital lipomatous asymmetric overgrowth of the trunk, lymphatic, capillary, venous, and combinedtype vascular malformations, epidermal nevi, skeletal and spinal anomalies (CLOVES) syndrome and Hemihyperplasia Multiple Lipomatosis (HHML). Heterozygous postzygotic PIK3CA mutations are frequently identified in these syndromes, while timing and tissue specificity of the mutational event are likely responsible for the extreme phenotypic variability observed. Methods: We carried out a combination of Sanger sequencing and targeted deep sequencing of genes involved in the PI3K/AKT/mTOR pathway in three patients (1 MCAP and 2 FAO) to identify causative mutations, and performed immunoblot analyses to assay the phosphorylation status of AKT and P70S6K in affected dermal fibroblasts. In addition, we evaluated their ability to grow in the absence of serum and their response to the PI3K inhibitors wortmannin and LY294002 in vitro. Results and Conclusion: Our data indicate that patients ' cells showed constitutive activation of the PI3K/Akt pathway. Of note, PI3K pharmacological blockade resulted in a significant reduction of the proliferation rate in culture, suggesting that inhibition of PI3K might prove beneficial in future therapies for PROS patients.

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