Somatic mutation and gain of copy number of PIK3CA in human breast cancer

Abstract

Introduction: Phosphatidylinositol 3-kinases (PI3Ks) are a group of lipid kinases that regulate signaling pathways involved in cell proliferation, adhesion, survival, and motility. Even though PIK3CA amplification and somatic mutation have been reported previously in various kinds of human cancers, the genetic change in PIK3CA in human breast cancer has not been clearly identified.

Methods: Fifteen breast cancer cell lines and 92 primary breast tumors (33 with matched normal tissue) were used to check somatic mutation and gene copy number of PIK3CA. For the somatic mutation study, we specifically checked exons 1, 9, and 20, which have been reported to be hot spots in colon cancer. For the analysis of the gene copy number, we used quantitative real-time PCR and fluorescence in situ hybridization. We also treated several breast cancer cells with the PIK3CA inhibitor LY294002 and compared the apoptosis status in cells with and without PIK3CA mutation.

Results: We identified a 20.6% (19 of 92) and 33.3% (5 of 15) PIK3CA somatic mutation frequency in primary breast tumors and cell lines, respectively. We also found that 8.7% (8 of 92) of the tumors harbored a gain of PIK3CA gene copy number. Only four cases in this study contained both an increase in the gene copy number and a somatic mutation. In addition, mutation of PIK3CA correlated with the status of Akt phosphorylation in some breast cancer cells and inhibition of PIK3CA-induced increased apoptosis in breast cancer cells with PIK3CA mutation.

Conclusion: Somatic mutation rather than a gain of gene copy number of PIK3CA is the frequent genetic alteration that contributes to human breast cancer progression. The frequent and clustered mutations within PIK3CA make it an attractive molecular marker for early detection and a promising therapeutic target in breast cancer.

Figure 1

Detection of somatic mutation of PIK3CA in breast cancer. In each case, the left sequence chromatogram was obtained from normal control and the right sequence chromatogram was obtained from tumor. Arrows indicate the location of missense mutations. The nucleotide and amino acid alterations are indicated on the left.

Figure 2

Typical real-time PCR curves generated for a cell line, a tumor, and normal tissue. Typical standard curves generated for (a) β-actin and (b) PIK3CA using serial diluted DNA from cell line MCF12A. (c) Representative real-time PCR curves for β-actin, generated using paired normal and tumor DNA from breast tissue of one individual. Each experiment was performed in triplicate and is shown by overlapping amplification curves. ΔRn = (Rn⁺) - (Rn^-), where Rn⁺ is the fluorescence emission intensity of reporter/emission intensity of quencher at any time point, and Rn^- is the initial emission intensity of reporter/emission intensity of quencher in the same reaction vessel before PCR amplification was initiated. Ct, cycle threshold.

Figure 3

Gain of PIK3CA gene copy number in breast cancer. (a)The change in the gene copy number of PIK3CA in 33 paired breast tissue samples. *Cases with significant change in gene copy number. The case number is indicated below the panel. (b) The change in the gene copy number of PIK3CA in 92 breast tumors and 33 normal controls. (c) Eight samples show more than 4 copies of PIK3CA. (d) The change in the gene copy number of PIK3CA in breast cancer cells using real-time PCR. Representative images of fluorescence in situ hybridization (FISH) analysis in various breast cancer cell lines. Green signals represent bacterial artificial chromosome (BAC) 466H15 probe. Red signals represent chromosome 3 centromere probe.

Figure 4

Biological effect of PIK3CA mutations in breast cancer. (a) Phosphorylation (P) of Akt in breast cancer cells. Western blotting showed stronger phosphorylation of Akt in BT20 and MCF7 cells than in MDA 231, MDA361, BT474, or T47D. β-Actin was used as a protein loading control. (b) Summary of the fraction of apoptotic cell in three cell lines with different treatments (3 μM and 10 μM LY294002). [(c) Apoptosis measurement in which cells were stained with Annexin V-FITC and for DNA content with Propidium Iodide and analyzed using flow cytometry. Apoptotic cells appear as a discrete population with elevated FITC fluorescence. FITC, fluorescein isothiocyanate; SS, serum starvation