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.
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 combined-type 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.


CONCLUSIONS
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.
Division of Medical Genetics, Department of Biomedical Sciences and Human Oncology (DIMO), University of Bari 'Aldo Moro', Bari, Italy.
Daria C
DC
Loconte
Division of Medical Genetics, Department of Biomedical Sciences and Human Oncology (DIMO), University of Bari 'Aldo Moro', Bari, Italy; National Cancer Institute, IRCCS Oncologico Giovanni Paolo II, Bari, Italy.
Valentina
V
Grossi
Department of Clinical and Molecular Medicine, "Sapienza" University of Rome, Rome, Italy.
Cristina
C
Bozzao
Cancer Genetics Laboratory, IRCCS "S. de Bellis", Castellana Grotte, Italy.
Giovanna
G
Forte
Division of Medical Genetics, Department of Biomedical Sciences and Human Oncology (DIMO), University of Bari 'Aldo Moro', Bari, Italy.
Rosanna
R
Bagnulo
Division of Medical Genetics, Department of Biomedical Sciences and Human Oncology (DIMO), University of Bari 'Aldo Moro', Bari, Italy.
Alessandro
A
Stella
Center for Rare Diseases-Internal Medicine "C. Frugoni", University Hospital of Bari, Bari, Italy.
Patrizia
P
Lastella
US Dermatologia Pediatrica, Ospedale dell'Angelo Ulss 12 Mestre, Venezia, Italy.
Mario
M
Cutrone
Genetic Counseling Service, Department of Pediatrics, Regional Hospital of Bolzano, Bolzano, Italy.
Francesco
F
Benedicenti
Division of Medical Genetics, Department of Biomedical Sciences and Human Oncology (DIMO), University of Bari 'Aldo Moro', Bari, Italy.
Francesco C
FC
Susca
Division of Medical Genetics, Department of Biomedical Sciences and Human Oncology (DIMO), University of Bari 'Aldo Moro', Bari, Italy.
Margherita
M
Patruno
Division of Medical Genetics, Department of Biomedical Sciences and Human Oncology (DIMO), University of Bari 'Aldo Moro', Bari, Italy.
Dora
D
Varvara
Division of Medical Genetics, Department of Biomedical Sciences and Human Oncology (DIMO), University of Bari 'Aldo Moro', Bari, Italy.
Aldo
A
Germani
Department of Clinical and Molecular Medicine, "Sapienza" University of Rome, Rome, Italy.
Luciana
L
Chessa
Neonatology and NICU Section, Department of Biomedical Sciences and Human Oncology (DIMO), University of Bari 'Aldo Moro', Bari, Italy.
Nicola
N
Laforgia
University of Padova, Padova, Italy.
Romano
R
Tenconi
Division of Medical Genetics, Department of Biomedical Sciences and Human Oncology (DIMO), University of Bari 'Aldo Moro', Bari, Italy; National Cancer Institute, IRCCS Oncologico Giovanni Paolo II, Bari, Italy.
Cristiano
C
Simone
Division of Medical Genetics, Department of Biomedical Sciences and Human Oncology (DIMO), University of Bari 'Aldo Moro', Bari, Italy.
Nicoletta
N
Resta
D000273:Adipose Tissue / Q000378:metabolism / Q000473:pathology; D002478:Cells, Cultured; D002648:Child; D058534:Class I Phosphatidylinositol 3-Kinases; D000013:Congenital Abnormalities / Q000175:diagnosis / Q000235:genetics*; D003238:Connective Tissue / Q000378:metabolism / Q000473:pathology; D005260:Female; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D007223:Infant; D008297:Male; D009154:Mutation; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics* / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D015398:Signal Transduction; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism*; D015053:Zygote / Q000378:metabolism
D058534:Class I Phosphatidylinositol 3-Kinases; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D047428:Protein Kinase Inhibitors; D051057:Proto-Oncogene Proteins c-akt; D058570:TOR Serine-Threonine Kinases; C484760:PIK3CA protein, human