The oncogenic properties of mutant p110α and p110β phosphatidylinositol 3-kinases in human mammary epithelial cells

Jean Zhao

Zhenning Liu

Li Wang

Eyoung Shin

Massimo Loda

Thomas Roberts

Departments of ^{Cancer Biology and}^{Medical Oncology, Dana–Farber Cancer Institute, and}^{Department of Pathology, Harvard Medical School, Boston, MA 02115}

^{To whom correspondence may be addressed at: Dana–Farber Cancer Institute, Smith 970, 44 Binney Street, Boston, MA 02115. E-mail:}ude.dravrah.icfd@strebor_samoht or ude.dravrah.icfd@oahz_naej.

^{J.J.Z. and Z.L. contributed equally to this work.}

Communicated by Lewis C. Cantley, Harvard Institutes of Medicine, Boston, MA, October 20, 2005

Received 2005 Sep 6

Abstract

The PIK3CA gene encoding the p110α subunit of Class IA phosphatidylinositol 3-kinases (PI3Ks) is frequently mutated in human tumors. Mutations in the PIK3CB gene encoding p110β, the only other widely expressed Class IA PI3K, have not been reported. We compared the biochemical activity and transforming potential of mutant forms of p110α and p110β in a human mammary epithelial cell system. The two most common tumor-derived alleles of p110α, 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 p110α, 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 p110β a mutation homologous to the E545K allele of p110α, the resulting p110β mutant was only weakly activated and allowed minimal soft-agar growth. However, a gene fusion of p110β 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 p110α at the corresponding sites in p110β failed to render the enzyme oncogenic in human cells, the possibility remains that other mutations might activate the β isoform.

Keywords: orthotopical tumor, PIK3CA, PIK3CB, Akt, oncogene

Abstract

The phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases defined by their ability to phosphorylate the 3′-OH of phosphoinositides. Three classes of mammalian PI3Ks have been characterized and shown to differ in their expression patterns, activation mechanisms, and substrate specificities (1, 2). The Class I PI3Ks have been most widely investigated, because it is these isoforms that are generally coupled to extracellular stimuli. This class is further divided into Class IA enzymes activated by receptor tyrosine kinases (RTKs) and Class IB enzymes regulated by G protein-coupled receptors. Although PI3Ks have been implicated in the regulation of a wide variety of cellular processes, only Class IA enzymes are clearly implicated in human cancers (3).

Class IA PI3Ks consist of a p110 catalytic subunit and a regulatory subunit. Currently, three isoforms (α, β, and δ) exist for the p110 subunit, whereas there are seven known proteins for the regulatory subunit: p85α, p85β, and p55γ and their splicing variants. The p85 subunit (or the shorter isoform) keeps the p110 subunit in a stable but low activity state in quiescent cells (4). Upon stimulation by growth factors, the p85 subunit recruits the catalytic subunit to the membrane through the interaction of its SH2 domains and the phosphotyrosine motifs on activated RTKs. Subsequently, the activated p110 catalytic subunit converts phosphatidylinoditol-4, 5-biphosphate to phosphatidylinoditol-3, 4, 5-triphosphate at the membrane, providing docking sites for signaling proteins with pleckstrin-homology domains. Among these proteins are the protein Ser-Thr kinase Akt (also called protein kinase B or PKB) and the phosphoinositide-dependent kinase 1 (PDK1), which come into proximity with each other through PIP3 association. PDK1 phosphorylates and activates Akt, which subsequently phosphorylates a series of other signaling proteins that affect cell growth, proliferation, survival, and transformation.

Deregulation of the Class IA PI3K–Akt pathway is common in human tumors. PTEN, a lipid phosphatase which dephosphorylates the 3′-OH of phosphoinositides and, thus, counteracts the action of PI3Ks, is mutated or silenced in many human malignancies, including breast cancer, prostate cancer, and glioblastoma (5–7). Akt, the major downstream effector of PI3K, has been found amplified or overexpressed in breast, ovarian, and thyroid cancers (8, 9). Recently, mutations have been identified at a high frequency in the PIK3CA gene encoding the p110α subunit in many types of human cancers (10–13). So far, these revealed mutations are exclusively somatic missense mutations and mostly clustered at hot spots within the helical and catalytic domains. Less frequent mutations within the p85-binding domain have also been identified but not characterized. Interestingly, mutations in the PIK3CB gene, encoding p110β, the only other widely expressed catalytic subunit of Class IA PI3Ks, have not yet been reported. However, the mutational analysis of p110β has been performed only on a very limited number of tumors.

In this article, we compare the biochemical activity and transforming potential for several mutant forms of p110α and p110β by using a genetically engineered human mammary epithelial cell (HMEC) transformation system. Our data suggest that p110α is potently activated by the “hotspot” mutations found in human tumors, whereas tumor mutations in the p85-binding domain are only weakly activating. Furthermore, we demonstrate that it may be more difficult to activate p110β than p110α by missense mutation, but p110β, nonetheless, possesses considerable tumorigenic potential.

For each injection, 4 × 10^{of indicated cell populations mixed with an equal volume of matrigel in a final volume of 100 μl were injected s.c. or orthotopically into axial mammary fat pads of nude mice. Mice were killed when tumor size reached 1.0-1.5 cm in diameter or after 2 months of monitoring. N/D, not determined.}

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Acknowledgments

We thank L. Cantley, W. Sellers, J. D. Iglehart, and A. Miron for helpful discussions; Q. Yu for help on the animal experiments; and Harvard Institute of Proteomics for reagents. This work was supported by National Institutes of Health Grants P01-CA50661 and CA30002 (to T.M.R.), CA089021 (to T.M.R. and M.F.L.), and Specialized Programs of Research Excellence Grant 5P50CA090381-05 (to J.J.Z. and M.F.L.); a Claudia Barr Award and the Friends Woman's Cancer Program Award (to J.J.Z.); and the Prostate Cancer Foundation (M.F.L.). In compliance with Harvard Medical School guidelines, we disclose the consulting relationships: Novartis Pharmaceuticals, Inc. (T.M.R. and M.F.L.) and Upstate Biotechnology (T.M.R.).

Acknowledgments

Notes

Author contributions: J.J.Z., Z.L., M.F.L., and T.M.R. designed research; J.J.Z., Z.L., L.W., and E.S. performed research; J.J.Z. and Z.L. contributed new reagents/analytic tools; J.J.Z., Z.L., M.F.L., and T.M.R. analyzed data; and J.J.Z., Z.L., M.F.L., and T.M.R. wrote the paper.

Conflict of interest statement: No conflicts declared.

Abbreviations: HA, hemagglutinin; HMEC, human mammary epithelial cell; LT, large T antigen; PI3K, phosphatidylinositol 3-kinase.

Notes

Conflict of interest statement: No conflicts declared.

Abbreviations: HA, hemagglutinin; HMEC, human mammary epithelial cell; LT, large T antigen; PI3K, phosphatidylinositol 3-kinase.

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