PIK3CA gene is frequently mutated in breast carcinomas and hepatocellular carcinomas

Abstract

A recent report revealed that phosphoinositide-3-kinase, catalytic, alpha (PIK3CA) gene is somatically mutated in several types of human cancer, suggesting the mutated PIK3CA gene as an oncogene in human cancers. However, because the previous report focused the mutational search primarily on colon cancers, the data on PIK3CA mutations in other types of human cancers have been largely unknown. Here, we performed mutational analysis of the PIK3CA gene by polymerase chain reaction-single-strand conformation polymorphism assay in 668 cases of common human cancers, including hepatocellular carcinomas, acute leukemias, gastric carcinomas, breast carcinomas, and non-small-cell lung cancers. We detected PIK3CA somatic mutations in 26 of 73 hepatocellular carcinomas (35.6%), 25 of 93 breast carcinomas (26.9%), 12 of 185 gastric carcinomas (6.5%), one of 88 acute leukemias (1.1%), and three of 229 non-small-cell lung cancers (1.3%). Some of the PIK3CA mutations were detected in the early lesions of breast cancer carcinoma, hepatocellular carcinoma, and gastric carcinomas, suggesting that PIK3CA mutation may occur independent of stage of the tumors. The high incidence and wide distribution of PIK3CA gene mutation in the common human cancers suggest that alterations of lipid kinase pathway by PIK3CA mutations contribute to the development of human cancers.

Main

Protein-tyrosine kinases (PTK) are important regulators of the intracellular signal-transduction pathway. The lipid kinase phosphatidylinositol 3-kinases (PI3Ks) and their targets are connected with PTK signaling, and are considered as important effectors in PTK signaling (Cantley, 2002; Luo et al., 2003). PI3K catalyses the production of the lipid second messenger phosphatidylinositol-3,4,5-triphosphate (PIP3), which in turn contributes to the recruitment and activation of a wide range of downstream targets, including the Akt (Stokoe et al., 1997; Vanhaesebroeck and Alessi, 2000). The PI3K-Akt signaling pathway regulates many normal cellular processes, including cell proliferation, survival, growth, and motility (Vivanco and Sawyers, 2002). Among the PI3Ks, class IA PI3K exists as heterodimers consisting of a unique catalytic subunit (p110α, p110β, or p110δ) along with one of a number of shared regulatory subunits (p85α, p85β, or p55γ) (Ueki et al., 2000).

Evidence exists that perturbation of PI3K-Akt signaling results in deregulated kinase activity and malignant transformation. Increased PI3K activity has been reported in human tumors, including colon, bladder, and ovarian tumors (Phillips et al., 1998; Benistant et al., 2000; Yuan et al., 2000). Somatic mutations of PTEN and PI3K p85α, and amplifications of Akt and PI3K p110α have been identified in human cancers (Bellacosa et al., 1995; Cantley and Neel, 1999; Shayesteh et al., 1999; Samuels et al., 2004). These alterations may explain the activation of PI3K signaling in the cancer cells. Recently, Samuels et al. (2004) identified somatic mutations of phosphoinositide-3-kinase, catalytic, alpha (PIK3CA), encoding p110α of the class IA PI3K, in several types of human tumors. They found that 32% of 74 colorectal tumors, 27% of 15 glioblastomas, 25% of 12 gastric cancers, 8% of 12 breast cancers, and 4% of 24 lung cancers harbored PIK3CA somatic mutations. By contrast, no PIK3CA mutation was identified in 11 pancreatic cancers and 12 medulloblastomas. All of the PIK3CA mutations except one small deletion were missense mutations, and more than 75% of the mutations were observed in two clusters in the helical and kinase domains. Functional assay revealed that one of the hotspot mutant of PIK3CA (H1047R) had an elevated lipid kinase activity compared to the wild-type PIK3CA, suggesting its casual role in tumorigenesis.

Although the screening of PIK3CA mutations in human cancers has been widely performed by the previous study (Samuels et al., 2004), it did not include the data on PIK3CA mutations in some common cancers, including hepatocellular carcinomas and leukemias. Also, because they focused the mutation screening on colon tumors, they analysed small numbers of samples in other cancers. Thus, further studies on PIK3CA mutations with large sample numbers are required. In the present study, to explore the possibility that PIK3CA gene mutations might play a role in the tumorigenesis of human cancers, we analysed somatic mutations of PIK3CA gene in the tissue samples from gastric carcinomas, breast carcinomas, non-small-cell lung cancers, hepatocellular carcinomas, and acute leukemias by polymerase chain reaction (PCR)-based single-strand conformation polymorphism (SSCP) analysis.

Methacarn-fixed tissues of 185 gastric carcinomas, 93 breast ductal carcinomas, 73 hepatocellular carcinomas, and 229 non-small-cell lung cancers, and nonfixed fresh tissues of 88 acute adult leukemias were selected for the study. We did not include the cancer cell lines in the samples. The gastric carcinomas consisted of 70 diffuse-type, 55 intestinal-type, and 37 mixed-type gastric carcinomas by Lauren's classification, and 33 early and 152 advanced gastric carcinomas according to the depth of invasion. The breast carcinomas consisted of 15 intraductal and 78 invasive ductal carcinomas. The hepatocellular carcinomas consisted of eight grade I, 26 grade II, and 29 grade III cancers by Edmonson's classification (Edmondson and Steiner, 1954). Of the hepatocellular carcinoma tissues, two HCC tissues included low-grade dysplastic nodule (LGDN) lesions and another two included high-grade dysplastic nodule (HGDN) lesions as well. The non-small-cell lung cancers consisted of 111 squamous cell carcinomas, 108 adenocarcinomas, and 10 large-cell carcinomas. The acute leukemia consisted of 12 acute lymphocytic leukemias and 76 acute myelocytic leukemias. We analysed the primary tumors, but not the metastatic lesions. For the solid tumors, tumor cells and corresponding normal cells from the same patients were selectively procured from hematoxylin and eosin-stained slides of the same patients using a 30G1/2 hypodermic needle (Becton Dickinson, Franklin Lakes, NJ, USA) affixed to a micromanipulator, as described previously (Lee et al., 1998). DNA extraction was performed by a modified single-step DNA extraction method, as described previously (Lee et al., 1998). Most of the reported PIK3CA mutations (over 80%) have been detected within the helical domain and the kinase domains (Samuels et al., 2004). Thus, we analysed the PIK3CA mutation in exons 9 and 20, which encode these domains. We repeated the experiments three times, including PCR, SSCP, and sequencing analysis to ensure the specificity of the results.

PCR and subsequent SSCP analysis identified bands aberrantly migrating compared to the wild-type bands (Figure 1). None of the corresponding normal samples from the same patients showed evidence of mutations by SSCP (Figure 1), indicating that the mutations had risen somatically. Enrichment and DNA sequence analysis of the aberrantly migrating bands led us to identify that 67 out of the 668 samples harbored PIK3CA mutations (10.0%) (Table 1). Among the cancers with the mutations, two cancers harbored two mutations each. One of these cancers with double mutations was an advanced gastric carcinoma, which had both G1633A (E545K) and A3062G (Y1021C) mutations. The other was an invasive ductal carcinoma of the breast, which had both G1633A (E545K) and A3140G (H1047R) mutations. The 69 mutations consisted of 55 missense and 14 insertion mutations, and were detected in exons 9 (22 mutations) and 20 (47 mutations). The mutations were identified in 12 of the 185 gastric carcinomas (6.5%), 25 of the 93 breast carcinomas (26.9%), 26 of the 73 hepatocellular carcinomas (35.6%), three of the 229 non-small-cell lung cancers (1.3%), and one of the 88 acute leukemias (1.1%). There was no significant correlation of PIK3CA mutation with the histologic subtypes of the gastric cancers, acute leukemias and non-small-cell lung cancers, and the Edmonson grade of hepatocellular carcinomas (Fisher's exact test, P>0.05).

Figure 1
figure1

PCR-SSCP of PIK3CA gene in human cancers. Genomic DNA each from normal cells or tumor cells from the same patients was amplified with one primer pair covering exon 9 and with two primer pairs covering exon 20 of PIK3CA gene. Radioisotope was incorporated into the PCR products for detection by autoradiogram. The PCR reaction mixture was denatured for 1 min at 94°C and incubated for 30 cycles. Other procedures of PCR and SSCP analysis were performed as described previously (Kim et al., 2003). After SSCP, DNAs showing mobility shifts were cut out from the dried gel, and re-amplified for 30 cycles using the same primer sets. Sequencing of the PCR products was carried out using the cyclic sequencing kit (Perkin-Elmer, Foster City, CA, USA). Representative data of SSCP (a, b) and DNA sequencing analysis (c, d) of PIK3CA DNA from tumors (Lane T) and normal tissues (Lane N). (a, b) SSCPs of DNA from the tumors (T) show wild-type bands and additional aberrant bands (arrows) as compared to SSCP from corresponding normal cells (N). (c) Sequencing analysis from the aberrant band in (a). There is an A to G transition at nucleotide 3140 of PIK3CA (arrows) in tumor tissue as compared to normal tissue. (d) Sequencing analysis from the aberrant band in (b). There is an insertion of A at the nucleotide between nucleotides 2004 and 2005 of PIK3CA (arrows) in tumor tissue as compared to normal tissue. Numbering of cDNA of PIK3CA was carried out with respect to the ATG start codon (GenBank)

Table 1 Summary of PIK3CA mutations in the cancers analysed

In the breast cancers, two mutations were detected in the 15 intraductal breast carcinomas (13.3%), while the other 24 mutations were detected in 78 invasive ductal carcinomas (30.7%). In the gastric carcinomas, one mutation was detected in 33 early gastric carcinomas (3.0%), while the other 12 mutations were detected in 152 advanced gastric carcinomas (7.9%). Of the four hepatocellular carcinomas with DN, one case showed PIK3CA mutations (3204_3205insA) both in the hepatocellular carcinoma and the LGDN (Table 1).

In the previous study, there were two mutational hotspots of PIK3CA, E545K and H1047R, which consisted of 28 and 20% of the PIK3CA mutations in colon cancers, respectively (Samuels et al., 2004). In agreement with the previous data, these two mutations consisted of approximately 50% of the mutations detected in the present study (33 out of total 69 mutations). In breast and gastric cancers, the most common PIK3CA mutation was H1047R (Table 1), which was proven to be an activating mutation by the kinase assay (Samuels et al., 2004), indicating that the H1047R mutation may be a functional alteration affecting key genes underlying the neoplastic process in breast and gastric carcinomas. The E545K mutation was detected in three of the gastric cancers, two breast cancers and one lung cancer. The incidences of the E545K mutation in the gastric cancers, the breast cancers, the lung cancers, the hepatocellular carcinomas, and the leukemias were significantly lower than those in the colon cancers observed in the previous study (Fisher's exact test, P<0.01). In the hepatocellular carcinomas, 13 (50.0%) of the 26 mutations were 3204_3205insA frameshift mutation. The 3204_3205insA mutation is a novel PIK3CA mutation, and would change the last C-terminal amino acid (N1068K) in the PIK3CA protein and create additional three amino acids. Whether the 3204_3205insA mutation has oncogenic activities and how this mutation contributes to tumorigenesis remain to be elucidated.

All gastric cancers presumably begin as early gastric cancers, which develop over time into advanced lesions (Bogomoletz, 1984), and similarly invasive ductal carcinomas of breast begin as intraductal carcinomas (Shay et al., 1993). Also, hepatocellular carcinoma progression is a stepwise process from preneoplastic lesions, including LGDN and HGDN, to advanced hepatocellular carcinoma (International Working Party, 1995). The occurrences of PIK3CA mutations in the early lesions as well as the advanced lesions suggested that PIK3CA mutation may occur independent of the stage of the tumors.

To extend the previous knowledge on the PIK3CA gene mutation to a wider range of common human cancers, we investigated somatic mutation of PIK3CA in a series of 668 tumor tissues from various histological origins. In agreement with the earlier report (Samuels et al., 2004), we found that PIK3CA gene is frequently mutated in breast cancers and hepatocellular carcinomas. By contrast, we also found that PIK3CA is rarely mutated in non-small-cell lung cancers and acute leukemias, suggesting that the involvement of PIK3CA mutation in human cancer pathogenesis may be different according to the types of cancers. The most impressive examples of recent cancer therapies used the kinase inhibitors such as Imanitib (Gleevec), Trastzumab (Herceptin), and Gefitinib (Iressa) (Druker et al., 2001; Arteaga et al., 2002; Wakeling et al., 2002). Thus, research will further focus on evaluating kinases as promising molecular targets for cancer treatment. Since PIK3CA regulates signaling pathways important for cancer development, the increased PIK3CA kinase activity by its activating mutations could be a potential target for cancer therapy. In this respect, the present study may provide the basic information of the tumors for future therapies targeting the PIK3CA mutation.

Abbreviations

PCR:

polymerase chain reaction

SSCP:

single-strand conformation polymorphism

PTK:

protein-tyrosine kinase

PI3K:

phosphatidylinositol 3-kinase

PIP3:

phosphatidylinositol-3,4,5-triphosphate

LGDN:

low-grade dysplastic nodule

HGDN:

high-grade dysplastic nodule

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Acknowledgements

This work was supported by the funds from KOSEF (R01-2004-000-10463-0).

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Correspondence to Sug Hyung Lee.

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Lee, J., Soung, Y., Kim, S. et al. PIK3CA gene is frequently mutated in breast carcinomas and hepatocellular carcinomas. Oncogene 24, 1477–1480 (2005). https://doi.org/10.1038/sj.onc.1208304

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Keywords

  • PIK3CA
  • mutation
  • cancer
  • oncogene

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