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. 2005 Dec 20;102(51):18443-8.
doi: 10.1073/pnas.0508988102. Epub 2005 Dec 8.

The oncogenic properties of mutant p110alpha and p110beta phosphatidylinositol 3-kinases in human mammary epithelial cells

Jean J Zhao  1 , Zhenning Liu, Li Wang, Eyoung Shin, Massimo F Loda, Thomas M Roberts
Affiliations

The oncogenic properties of mutant p110alpha and p110beta phosphatidylinositol 3-kinases in human mammary epithelial cells

Jean J Zhao et al. Proc Natl Acad Sci U S A. .

Abstract

The PIK3CA gene encoding the p110alpha subunit of Class IA phosphatidylinositol 3-kinases (PI3Ks) is frequently mutated in human tumors. Mutations in the PIK3CB gene encoding p110beta, the only other widely expressed Class IA PI3K, have not been reported. We compared the biochemical activity and transforming potential of mutant forms of p110alpha and p110beta in a human mammary epithelial cell system. The two most common tumor-derived alleles of p110alpha, H1047R and E545K, potently activated PI3K signaling. Human mammary epithelial cells expressing these alleles grew efficiently in soft agar and as orthotopic tumors in nude mice. We also examined a third class of mutations in p110alpha, those in the p85-binding domain. A representative tumor-derived p85-binding-domain mutant R38H showed modestly reduced p85 binding and weakly activated PI3K/Akt signaling. In contrast, a deletion mutant lacking the entire p85-binding domain efficiently activated PI3K signaling. When we constructed in p110beta a mutation homologous to the E545K allele of p110alpha, the resulting p110beta mutant was only weakly activated and allowed minimal soft-agar growth. However, a gene fusion of p110beta with the membrane anchor from c-Src was highly active and transforming in both soft-agar and orthotopic nude mouse assays. Thus, although introduction of activating mutations from p110alpha at the corresponding sites in p110beta failed to render the enzyme oncogenic in human cells, the possibility remains that other mutations might activate the beta isoform.

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Figures

Fig. 1.
Fig. 1.
Substitution of SV40 LT with a dominant negative allele of p53 in the transformation of HMECs. p53DD sufficed to replace SV40 LT and promoted soft-agar colony growth of HMEC in combination with hTERT, elevated c-myc, and myristoylated bovine p110α. The assay was carried out as described in Materials and Methods, and the means (±SD) for three experiments are shown.
Fig. 2.
Fig. 2.
Soft-agar colony formation of HMECs expressing mutant alleles of human p110 isoforms. (A) Relative expression levels of hp110α mutant alleles and their activation on Akt signaling in HMECs. Cells were starved in basal medium, and lysates were prepared as described in Materials and Methods for immunoblot analysis with an anti-HA antibody and anti-phospho-Akt(Ser-473) or total Akt antibodies. (B) Expression of hp110β mutant alleles and their activation on Akt signaling in HMECs were performed as described in A. (C) Soft-agar colony growth of HMEC-hTERT-p53DD expressing various alleles of hp110α or hp110β. The assay was carried out as described in Materials and Methods, and the means (±SD) for three experiments are shown.
Fig. 3.
Fig. 3.
Orthotopic tumor formation of HMECs expressing mutant alleles of human p110α.(A) HMECs expressing E545K or H1047R hp110α in combination with hTERT, p53DD, and HrasV12 were injected into axial mammary fat pads as described in Materials and Methods. Orthotopic tumors developed and reached a 1-cm diameter within 8 wks after injection. (B) Histology of orthotopic tumors derived from HMECs expressing E545K or H1047R mutant hp110α. All tissues were stained with hematoxylin and eosin. The tumors were poorly differentiated and infiltrated through adipocytes and muscle tissue layers (indicated by arrows).
Fig. 4.
Fig. 4.
Effects of p85-binding-domain mutations on p110α activity. (Upper) The p85-binding activities of various alleles of hp110α, as indicated. The normalized level of p85-binding activity of each allele was determined by dividing the amount of p85 bound by the amount of each hp110α allele expressed. (Lower) The specific activity of each hp110α allele, determined by normalizing the level of phospho-Akt to the expression level of each hp110α allele, as indicated. Each column reflects the average of three independent experiments, with error bars indicating SD.
Fig. 5.
Fig. 5.
Orthotopic tumor formation by HMECs expressing myristoylated human p110β. HMECs expressing myr-hp110β in combination with hTERT, p53DD, and HrasV12 were injected into axial mammary fat pads as described in Materials and Methods. The tumors were examined as described in Fig. 4. The tumors were also poorly differentiated but showed considerable size heterogeneity. The large tumor showed infiltration through bone and adipocyte muscle tissue layers, and a lymphoma was identified adjacent to the small tumor located in the contralateral axial mammary fat pad, as indicated.
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