Rare mutations of the PIK3CA gene in malignancies of the hematopoietic system as well as endometrium, ovary, prostate and osteosarcomas, and discovery of a PIK3CA pseudogene
Introduction
Phosphatidylinositol 3-kinase (PI3K) is a lipid kinase that is a known regulator of cellular growth and proliferation. The PI3K pathway plays a critical role in the development of cancer. Four different classes (I–IV) of the PI3K family are known in mammals [1], [2]. Initially, Samuels et al. [3] screened 16 members of the PI3K family for mutations in colorectal cancer. Class IA p110-alpha catalytic subunit (PIK3CA) was the only member that was found with somatic mutations [3]. Between 19% and 32% of colon cancers have PIK3CA mutations [3], [4]. Other tumors were also examined by several investigators for PIK3CA mutations in primary samples and cell lines including gastric (4–25% samples mutated) [3], [5], [6], breast (8–40%) [3], [4], [6], [7], lung (1–4%) [3], [6], ovary (6%) [4], liver (36%) [6], glioblastomas (5–27%) [3], [8], oligodendrogliomas (14%) [8], medulloblastomas (5%) [8], astrocytomas (3%) [8] and acute myeloid leukemia (AML) plus acute lymphoblastic leukemia (1%) [6]. The mutations clustered in two ‘hot spots’ located in the helical (exon 9) and kinase (exon 20) domains.
We performed mutational screening analysis of PIK3CA in several so far uninvestigated cancers including myelodysplastic syndromes (MDS), non-Hodgkin lymphomas (NHL), endometrial and prostate cancers, as well as AML and ovarian cancers. Cell lines and patient samples were examined by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and nucleotide sequencing of the two ‘hot spots’.
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Cell lines
Human tumor cell lines were purchased from American Type Culture Collection (ATCC, Manassas, VA) (Table 1). Genomic DNA (gDNA) was extracted using phenol-chloroform and ethanol precipitation.
Patients
Samples were obtained from patients with endometrial cancer (28 samples), NHL (43 samples), MDS (20 samples), AML (26 samples), osteosarcoma (40 samples), prostate cancer (12 samples), ovarian cancer (18 samples) and normal peripheral blood of 92 individuals who underwent routine blood-drawing; all samples
Mutational analysis of eight colorectal cell lines as positive controls
The PIK3CA gene was analyzed for variation in the coding region in eight colorectal cancer cell lines, which were used as positive controls since PIK3CA mutations were previously identified in several of these cell lines (Table 1). We examined exons 9 and 20, since it was reported previously that >75% of the mutations found in a large number of cancers occurred in these regions. Variation in migration patterns was examined by SSCP analysis after PCR amplification. Abberantly shifted bands were
Discussion
Mutational analysis of a variety of human cancers discovered mutations in the PIK3CA gene, which encodes the catalytic subunit p110α of PI3-kinase [3], [4], [5], [6], [7], [8]. Functional significance of these mutations was suggested because: (i) they cluster in the critical exon 9 (helical domain) and exon 20 (kinase domain) regions; (ii) they are mostly nonsynonymous; (iii) highly conserved residues are primarily affected; (iv) mutant protein showed increased kinase activity [3]; and (v)
Acknowledgements
We thank Dr. Bert Vogelstein (Johns Hopkins University, Baltimore) for his helpful suggestions and sharing of reagents.
We thank the Inger Foundation, Parker Hughes Trust, The King Harbor Yacht Club Tom Collier Memorial Fund and an NIH grant. HPK is a member of the Jonsson Cancer Center, the Molecular Biology Institute of UCLA and has the endowed Mark Goodson Chair of Oncology Research at Cedars-Sinai Medical Center/UCLA School of Medicine. This work was moreover supported in part by a
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