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

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 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.MethodsWe 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 ConclusionOur 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.

The PI3K Pathway in Human Disease

“…Patients with PIK3CA -dependent vascular anomalies and other tissue overgrowths may achieve even greater benefit from treatment with a selective p110α inhibitor, which would block mTORC1-independent functions of p110α not covered by rapamycin. As expected, PI3K inhibitors strongly suppress the proliferation of CLOVES syndrome cells in culture (Loconte et al, 2015). We expect that p110α inhibitors will be tested in CLOVES syndrome patients in the near future, and, toxicity issues notwithstanding, it is hoped that these drugs will offer the possibility of complete remissions not achievable with rapalog therapy.…”

Somatic PIK3CA mutations in seven patients with PIK3CA‐related overgrowth spectrum

“…In order to characterize the mutation spectrum of patients with congenital/early childhood onset PROS without overgrowth of the brain, we performed an extensive literature search, and have presented the results together with the data of the seven patients described here [Kurek et al, ; Lindhurst et al, ; Rios et al, ; Castiglioni et al, ; Cohen et al, ; Emrick et al, ; Keppler‐Noreuil et al, ; Maclellan et al, ; Rasmussen et al, ; Couto et al, ; Limaye et al, ; Loconte et al, ; Luks et al, ; Osborn et al, ; Castillo et al, ; Hucthagowder et al, ; Mirzaa et al, ; Tripolszki et al, ]. We found that the three highly recurrent cancer‐associated mutations, p.(Glu542Lys), p.(Glu545Lys), and p.(His1047Arg) [Mirzaa et al, ], can be seen in about 70% of patients with PROS; whereas p.(Cys420Arg) and p.(His1047Leu) accounts for nearly 20% of the patients (Fig.…”

Klippel–Trenaunay syndrome belongs to the PIK3CA‐related overgrowth spectrum (PROS)

“…As a result of these activities, rapamycin has antiproliferative effects and could prevent tissue overgrowth in patients with PIK3CA mutations . Furthermore, specific inhibitors for other components of the PI3K‐AKT‐mTOR and other interconnected signalling pathways have been developed . Consequently, precise knowledge of the mutated gene and specific mutations is a prerequisite for ascertainment of treatment options in the future as part of personalized healthcare delivery.…”