PIK3R1 (p85α) is somatically mutated at high frequency in primary endometrial cancer

Abstract

Phosphoinositide 3-kinase (PI3K) is an important therapeutic target. Mutations in PIK3CA, which encodes p110α, the catalytic subunit of PI3K, occur in endometrioid endometrial cancers (EEC) and nonendometrioid endometrial cancers (NEEC). The goal of this study was to determine whether PIK3R1, which encodes p85α, the inhibitory subunit of PI3K, is mutated in endometrial carcinoma. We carried out exonic sequencing of PIK3R1 from 42 EECs and 66 NEECs. The pattern of PIK3R1 mutations was compared with the patterns of PIK3CA, PTEN, and KRAS mutations. The biochemical effect of seven PIK3R1 mutations was examined by stable expression in U2OS cells, followed by coimmunoprecipitation analysis of p110α, and Western blotting of phospho-AKT(Ser473) (p-AKT(Ser473)). We found that PIK3R1 was somatically mutated in 43% of EECs and 12% of NEECs. The majority of mutations (93.3%) were localized to the p85α-nSH2 and -iSH2 domains. Several mutations were recurrent. PIK3R1 mutations were significantly (P = 0.0015) more frequent in PIK3CA-wild type EECs (70%) than in PIK3CA mutant EECs (18%). Introduction of wild-type p85α into U2OS cells reduced the level of p-AKT(Ser473) compared with the vector control. Five p85α mutants, p85αdelH450-E451, p85αdelK459, p85αdelY463-L466, p85αdelR574-T576, and the p85αN564D positive control, were shown to bind p110α and led to increased levels of p-AKT(Ser473). The p85αR348X and p85αK511VfsX2 mutants did not bind p110α and showed no appreciable change in p-AKT(Ser473) levels. In conclusion, our study has revealed a new mode of PI3K alteration in primary endometrial tumors and warrants future studies to determine whether PIK3R1 mutations correlate with clinical outcome to targeted therapies directed against the PI3K pathway in EEC and NEEC.

Figure 1. p85α (PIK3R1) mutations in primary endometrial carcinomas

(Top panel) Schematic representation of the p85α protein, showing positions of somatic mutations relative to functional domains. Each arrowhead represents a single mutation: nonsense mutations and frameshift mutations (red arrowheads); in-frame insertions and deletions (green arrowheads); missense mutations (blue arrowheads); frameshift mutation that extends the protein (yellow arrowhead). (Bottom panel) Overlapping somatic in-frame deletions (dashed lines) within the proximal iSH2 domain. Three shortest regions of overlap (SRO) between deletions are indicated (black bars).

Figure 2. PIK3R1, PIK3CA, PTEN and KRAS mutational status in primary endometrial carcinomas

The mutation pattern is shown for (A) 42 EECs, and (B) 66 NEECs. Columns represent individual tumors (T). Somatically mutated tumors (yellow bars) are distinguished from tumors with no detectable somatic mutation (gray bars). The mutation frequency for individual genes is shown (at right).

Figure 3. Increased phosphorylation on AKT^Ser473 following exogenous expression of p85α mutants in U2OS cells

(A) Coimmunoprecipitation of p110α with p85α mutants. All mutants bound p110α except p85αR348X and p85αK511VfsX2. (B) Western blots of U2OS osteosarcoma cells stably transfected with MYC-tagged expression constructs encoding either vector only, wild type p85α, or mutant forms of p85α found in endometrial tumors. The p85αN564D mutant served as a positive control because it promotes increased p-AKT^Ser473 levels compared to wild type p85α (10).