Mutations of the PIK3CA gene in hereditary colorectal cancers



Somatic mutations of the PIK3CA gene have recently been detected in various human cancers, including sporadic colorectal cancer. However, mutations of the PIK3CA gene in hereditary colorectal cancers have not been clarified. To elucidate the mutation status in familial adenomatous polyposis (FAP) and hereditary nonpolyposis colorectal cancer (HNPCC), which are the most common hereditary colorectal cancers, we investigated PIK3CA mutations in 163 colorectal tumors, including adenomas, intramucosal carcinomas and invasive carcinomas. For comparison, we also analyzed mutations of the same gene in 160 sporadic colorectal tumors at various histopathological stages. Analysis at exons 1, 7, 9 and 20 of the PIK3CA gene revealed somatic mutations in 21% (8 of 39) of FAP invasive carcinomas, 21% (7 of 34) of HNPCC invasive carcinomas, 15% (8 of 52) of sporadic invasive carcinomas, and 14% (7 of 50) of sporadic colorectal metastases in the liver. Mutations in FAP and HNPCC carcinomas predominantly occurred in the kinase domain (exon 20), while the majority of mutations in sporadic cases occurred in the helical domain (exon 9). Adenomas and intramucosal carcinomas from all patients exhibited no mutations (0 of 148). Our data suggest that PIK3CA mutations contribute to the invasion step from intramucosal carcinoma to invasive carcinoma in colorectal carcinogenesis in FAP and HNPCC patients at a similar extent to that seen in sporadic patients. © 2007 Wiley-Liss, Inc.

Familial adenomatous polyposis (FAP)1 and hereditary nonpolyposis colorectal cancer (HNPCC)2 are the most common autosomal dominant diseases predisposed to colorectal cancer. FAP is characterized by numerous colorectal adenomas, with nearly all patients developing colorectal carcinomas if left untreated. FAP is caused by a heterozygous germline mutation of the tumor suppressor APC gene,3, 4 and inactivation of the APC gene through mutation or a loss in the normal allele results in the formation of adenomas that develop into carcinomas via the adenoma–carcinoma sequence. In addition to inactivation of the APC gene, this sequence includes inactivation of additional suppressor genes, such as the TP53 and SMAD4 genes, and activation of oncogenes, such as the KRAS2 gene. HNPCC is characterized by the early onset of colorectal cancer, and is caused by heterozygous germline mutations in any one of the DNA mismatch repair genes, including the hMSH2,5hMLH1,6hMSH67 and hPMS28 genes. Inactivation of mismatch repair genes through germline mutation and somatic mutation in the normal allele results in a high frequency of replication errors at microsatellite regions and at repeated sequences in the coding regions of various growth-related genes, such as TGFβRII(A)10, BAX(G)8 and ACVR2(A)8. These gene alterations lead to carcinogenesis in HNPCC patients.

Recently, Samuels et al.9 have identified somatic mutations in the PIK3CA gene in various human cancers. These cancers include cancer of the colon,9, 10, 11 stomach,9, 11, 12 breast,9, 10, 13 brain,9, 14 lung,9, 13 ovary10, 15 and thyroid gland.16 PIK3CA is the catalytic subunit of phosphoinositide 3-kinase (PI3K),17 and activation of PI3K causes the generation of phosphatidylinositol-3,4,5-triphosphate (PIP3) from phosphatidylinositol-4,5-biphosphate (PIP2). Association with PIP3 at the membrane facilitates the phosphorylation of v-akt murine thymoma viral oncogene homologue kinase (AKT) by phosphoinositide-dependent protein kinase-1 (PDK1). This phosphorylation stimulates the AKT signaling activity, affecting cell growth, cell survival and cell movement through the acceleration of various downstream pathways.18 Mutant PIK3CA has been shown to stimulate AKT signaling, and promote the cell growth and invasion of human colon cancer cell lines.19

PIK3CA mutations have been reported to occur in sporadic colorectal cancer at a frequency of 31.6%,9 18.8%10 or 13.6%,11 generally at a later stage of tumorigenesis. However, PIK3CA mutations have not been reported in the case of hereditary tumors. To clarify the status of PIK3CA mutations in hereditary colorectal tumors, we analyzed colorectal tumors at various histopathological stages from FAP and HNPCC patients. We found that PIK3CA mutations apparently contribute to carcinogenesis in both FAP and HNPCC patients.

Material and methods


In the present study, 119 FAP colorectal tumors from 72 patients, 44 HNPCC colorectal tumors from 38 patients and 160 sporadic colorectal tumors from 145 patients were analyzed for somatic PIK3CA mutations. These tumors, including adenomas, intramucosal carcinomas, invasive carcinomas and colorectal metastases in the liver, were obtained from patients who underwent surgical resections of colorectal carcinomas and colorectal metastases in the liver. Normal tissues were obtained from surgically resected tissues which were located at least 5 cm away from the tumors. Microsatellite instability was assayed using BAT25, BAT26, D2S123, D5S346 and D17S250. All FAP and sporadic tumors were microsatellite stable (no alterations at 5 loci), and all HNPCC tumors showed high microsatellite instability (alterations at more than 4 of 5 loci). Tumor samples were obtained after full informed consent, and analysis of these samples was approved by the Komagome Hospital Review Committee.

Mutation analysis

DNA was extracted from fresh frozen tissues using SDS-Proteinase K and phenol-chloroform. Mutation analysis was performed by PCR-SSCP and sequencing of exon 1 (including the p85 domain), exon 7 (core domain), exon 9 (helical domain) and exon 20 (kinase domain) of the PIK3CA gene. The PCR reaction mixture (6 μl) contained 300 ng of genomic DNA, each 0.2 μM primer, 25 μM each of 4 dNTPs, 1× PCR buffer, Taq polymerase and [α-32P]dCTP. Primers for exons 1, 7, 9 and 20 of the PIK3CA gene were the same as those previously reported.9 PCR was performed under the following conditions: 5 min at 97°C once; 1 min at 94°C, 1 min at 58°C and 1 min at 72°C for 35 cycles; and 10 min at 72°C once. The products were diluted fivefold with formamide dye solution, followed by heating for 5 min at 80°C. For SSCP analysis, 2 μl of each diluted sample was applied to 5% polyacrylamide gel containing 5% glycerol, electrophoresed and gel was exposed to X-ray film. Aberrant single-strand DNA fragments were extracted with water from the corresponding band on SSCP gel. The extracted DNA fragments were amplified through the asymmetrical PCR in 100 μl mixture under the same conditions as those for PCR-SSCP analysis, with the exception that ratio of primers was 100/1 or 1/100 for sense and antisense primers. The amplified DNA was purified using Purification Kit (QIAGEN), and then sequenced with the dideoxy chain-termination reaction using T7 Sequenase V2.0 (USB). Primers used for sequencing were the same as those in PCR-SSCP. These primers were prelabeled with [γ-32P]ATP. The reaction mixture was applied to denaturing electrophoresis in 6% polyacrylamide containing 7 M urea, and the gel was exposed to X-ray film. DNA samples exhibiting mutant bands were subjected to PCR-SSCP at least twice, and only reproducible cases were sequenced at least twice.

Results and discussion

Somatic mutations detected in colorectal tumors from FAP, HNPCC and sporadic patients are listed in Table I. Examples of mutations are shown in Figure 1. In the present analysis 30 somatic mutations were detected in 125 invasive carcinomas from FAP, HNPCC and sporadic patients and 50 sporadic colorectal metastases in the liver. None of these mutations were detected in normal DNA. With respect to exon 9, PIK3CA mutation of E545K was detected in 8 carcinomas, E542K was in 5 carcinomas and Q546K was in 2 carcinomas. In exon 20, H1047R was detected in 8 carcinomas, H1047Y was in 2 carcinomas, Y1021C was in 2 carcinomas, A1035V was in 1 carcinoma and M1055V was in 1 carcinoma. In exon 1, R88Q was detected in 1 carcinoma. In exon 7, no mutations were detected. An HNPCC patient had 2 invasive carcinomas, PLK416Ca1 and PLK416Ca2, the former being produced at transverse colon and the latter at descending colon. Both carcinomas had the same PIK3CA mutations, H1047R. One polymorphic site was detected at codon T1025T (C3075T) in exon 20, the allele frequency of which was 1% in sporadic cases.

Figure 1.

Examples of somatic mutations of PIK3CA gene in colorectal carcinomas from FAP, HNPCC and sporadic patients.

Table I. PIK3CA Mutations Detected in Colorectal Carcinomas from FAP, HNPCC and Sporadic Patients
TumorDiseaseDiagnosisExonNucleotideAmino acidFunctional domain
  1. IVC, invasive carcinoma; LM, colorectal metastasis in the liver; FAP, familial adenomatous polyposis; HNPCC, hereditary nonpolyposis colorectal cancer.


PIK3CA amino acid substitutions at codons 542, 545 and 1047 were the most common hot spot mutations in the various cancers. In a previous study of sporadic colorectal cancer, there were 3 mutational hot spots, E542K, E545K and H1047R, which consisted of 12, 28 and 20%, respectively, of mutations detected.9 These 3 mutations have been confirmed to be functionally active, showing cell growth and invasion19 and transforming activity.20, 21, 22 In the present study, these mutations consisted of 17, 27 and 27%, respectively, of mutations detected. Accordingly, the mutation pattern regarding all colorectal carcinomas, including FAP, HNPCC and sporadic carcinomas, was similar to that of the forementioned study by Samuels et al.9 However, the distribution of mutations in PIK3CA gene was found to be different between hereditary and sporadic carcinomas. Mutations in FAP and HNPCC carcinomas predominantly occurred in the kinase domain (exon 20) at 63 and 86%, respectively, of mutations detected, whereas the majority (80%) of mutations in sporadic carcinomas occurred in the helical domain (exon 9). Samuels et al.9 also reported that mutations in sporadic colorectal cancers were more frequent in the helical domain than those in the kinase domain. From in vivo assays of mutant E542K, E545K and H1047R, Bader et al.22 suggested that H1047R mutation in the catalytic (kinase) domain is more potent than the E542K and E545K mutations in the helical domain. Such data is of interest in the comparison of the different mutation patterns between hereditary carcinomas and sporadic carcinomas.

Subsequently, the frequencies of PIK3CA mutations were compared with histopathological stages of colorectal tumors from FAP, HNPCC and sporadic patients (Table II). In the case of invasive carcinomas, PIK3CA mutations were detected in 21% (8 of 39) of FAP, 21% (7 of 34) of HNPCC and 15% (8 of 52) of sporadic patients. However, adenomas and intramucosal carcinomas from FAP, HNPCC and sporadic patients exhibited no mutations. These data suggested that PIK3CA mutations contribute to the invasion step from intramucosal carcinoma to invasive carcinoma in FAP and HNPCC patients, as well as in sporadic patients. Since colorectal metastases in the liver exhibited a similar mutation frequency (14%, 7 of 50) to that seen in invasive carcinomas (15%), PIK3CA mutations are assumed to not contribute to metastatic changes of colorectal carcinomas. The present data confirmed that somatic mutations of the PIK3CA gene occur in FAP and HNPCC colorectal carcinomas at similar frequencies to that seen in sporadic colorectal carcinomas.

Table II. Frequencies of PIK3CA Mutations in Colorectal Tumors from FAP, HNPCC and Sporadic Patients
TumorNumber of mutation (%)
  1. ND, not done; FAP, familial adenomatous polyposis; HNPCC, hereditary nonpolyposis colorectal cancer. p-value of mutation frequency for FAP intramucosal carcinoma versus FAP invasive carcinoma was 0.002, and that for sporadic intramucosal carcinoma versus sporadic invasive carcinoma was 0.03. (Fisher's exact probability).

Adenoma0/40 (0)0/5 (0)0/31 (0)
Intramucosal carcinoma0/40 (0)0/5 (0)0/27 (0)
Invasive carcinoma8/39 (21)7/34 (21)8/52 (15)
Colorectal metastasis in the liverNDND7/50 (14)

Although genetic alterations in HNPCC tumors usually occur at repeated sequences, mutations at genes without repeated sequences, such as PIK3CA, were also found to occur frequently in this study. This suggests that defect of the mismatch repair function causes replication errors not only at repeated sequences but also causes single-base substitutions at nonrepeated sequences. Previously, spectra of spontaneous mutations have been determined by mutation at the hprt locus in colorectal cell lines deficient in mismatch repair and exhibiting high microsatellite instability.23, 24 Mutation frequency in these cells was 2 orders higher than that in wild-type repair-proficient cells: both single-base substitutions (57 and 66%) and frameshifts (43 and 28%) occurred in hMLH1- and an hPMS2-deficient cell lines, respectively, while single-base substitution (92%) was preferentially observed in an hMSH6-deficient cell line. These data indicate that single-base substitution of PIK3CA detected in HNPCC carcinomas may be due to high microsatellite instability. Such mutations have also been observed in APC and β-catenin genes in HNPCC colorectal carcinomas.25

In the present study 77% of PIK3CA mutations occurred in 3 codons (codons 542, 545 and 1047) in the helical (exon 9) and kinase (exon 20) domains in FAP, HNPCC and sporadic carcinomas. Heterozygous somatic mutations at these hot spots have been shown to increase the kinase activity of PIK3CA, which activated downstream target kinase AKT in the PI3K pathway and caused cell transformation.20, 21, 22 This suggests that these PIK3CA mutations are oncogenic. Heterozygous KRAS2 mutations are also oncogenic activating RTK pathway26 and resulting in neoplastic transformation. KRAS2 mutations are frequently detected during colorectal carcinogenesis, similar to PIK3CA mutations. However, these two oncogenic mutations play different roles in colorectal carcinogenesis: KRAS2 mutations are involved in an earlier stage such as adenoma,27 whereas PIK3CA mutations are involved in a later stage such as invasive carcinoma (this study).

In conclusion, the PI3K signaling pathway contributes to colorectal carcinogenesis, especially at the invasion step from intramucosal carcinoma to invasive carcinoma, in FAP and HNPCC patients at a similar extent to that seen in sporadic patients. The present findings will be valuable for understanding the more detailed mechanism of carcinogenesis in hereditary colorectal cancers. This pathway could be one of the potential targets for therapy involving these hereditary cancers.