A recent study revealed that the p110α (PIK3CA), catalytic subunit of phosphatidylinositol 3-kinase (PI3K), is somatically mutated in many types of cancer. For example, PIK3CA is mutated in an estimated 35.6% of hepatocellular carcinoma (HCC) cases. To measure the frequency of PIK3CA hotspot mutations in Japanese HCC patients, exons 9 and 20 of the PIK3CA gene were sequenced in 47 clinical HCC samples. Contrary to expectations, no hotspot mutations were found any of the HCC samples. In addition, we found abnormally migrating waves near the end of exon 9 in the PCR chromatograms from 13 of the 47 samples. PCR amplification and subsequent cloning and sequencing revealed that these chromatograms contained two distinct sequences, the wild-type p110α sequence and a different sequence found on human chromosome 22q11.2, the Cat Eye Syndrome region, which contains a putative pseudogene of PIK3CA. These abnormally migrating waves were also found in noncancerous liver tissue, indicating that this was not a result of HCC-associated mutations. Therefore, it is likely that the percentage of hotspot mutations in the PIK3CA gene of Japanese HCC patients is lower than was previously reported.
Phosphatidylinositol 3-kinase (PI3K) is a family of lipid kinases that regulate the signaling pathways involved in cell survival and proliferation (Cantley, 2002). The aberrant activation of this pathway has been reported in many types of cancers, including colorectal carcinoma, ovarian carcinoma, and breast carcinoma (Vivanco and Sawyers, 2002). PI3Ks are expressed as heterodimers of catalytic and regulatory subunits. The catalytic subunits are encoded by three genes (α, β, γ), of which the p110α subunit (PIK3CA) was reported to be overexpressed in ovarian carcinomas and was thus implicated as an oncogene (Shayesteh et al., 1999). Recently, Samuels et al. (2004) sequenced the coding exons of PIK3CA and showed that this gene was somatically mutated in a high frequency of colorectal carcinomas. They reported that the PIK3CA gene was mutated in 74 of 234 (32%) colorectal carcinomas, but in only two of 76 (3%) premalignant colorectal tumors, suggesting that these mutations arise late in tumorigenesis. Moreover, they found PIK3CA mutations in 27% of glioblastomas, 25% of gastric carcinomas, 8% of breast carcinomas, and 4% of lung carcinomas, indicating that these mutations are prevalent in many types of cancer. This observation was confirmed by several other groups (Campbell et al., 2004; Levine et al., 2005; Li et al., 2005; Velho et al., 2005). The PIK3CA mutations were located mostly at hotspots within the helical domain (encoded by exon 9) and the kinase domain (encoded by exon 20), and they resulted in gain of function mutations that were implicated in the initiation of oncogenicity (Ikenoue et al., 2005; Kang et al., 2005; Samuels et al., 2005).
The PIK3CA gene was also reported to be highly mutated in approximately 35.6% of hepatocellular carcinoma (HCC) cases (Lee et al., 2005). The most frequent PIK3CA mutations in HCC include a substitution mutation in the helical domain (A1634C) and an insertion mutation in the kinase domain (3204_3205 insA). The substitution mutation results in an amino acid change at the same residue (aa 545) in the p110α protein as that affected by a hotspot mutation reported in colorectal carcinomas. In HCC, the A1634C mutation results in a Glu (E) to Ala (A) change, thus replacing a charged (acidic) residue with a neutral residue, whereas, in colorectal carcinoma, the G1633C mutation changes a Glu (E) to Lys (K), thus replacing the charged (acidic) residue with a basic residue. A novel insertion mutation, first reported by them, changes the C-terminal Asn (N) of the p110α protein to Lys (K) and adds three amino acids, resulting in a more basic sequence, Lys-Leu-Lys-Arg (KLKR). Although functional analyses have yet to be performed, these mutations might cause the aberrant activation of PI3K and of the downstream kinase Akt, as seen in HCC.
To investigate the frequency of PIK3CA mutations in Japanese patients with HCC, we sequenced the two hotspot-containing exons (9 and 20) in 47 HCC samples. The male to female ratio of the patients was 38:9, and their ages ranged from 28 to 81 years, with an average age of 62 years. In all, 10 patients had HCC associated with hepatitis B virus infection, 30 patients had HCC associated with hepatitis C virus infection, and seven patients had HCC not associated with hepatitis virus infection. The tumor sizes ranged from 1.2 to 10 cm (average 4.7 cm). This study was performed with appropriate Institutional Review Board approval.
Genomic DNA was extracted from tumors and normal tissues obtained from frozen surgical or biopsy specimens. The primers used for PCR amplification of PIK3CA exons and for sequencing were described previously (Samuels et al., 2004). PCR amplification was performed using a high-fidelity PCR master mix (Roche, Mannheim, Germany), and reaction conditions were 30 cycles of 96°C for 1 min, 55°C for 1 min, and 72°C for 1 min. Direct sequencing was performed using the ABI 3730xl DNA sequencer (Applied Biosystems, Foster City, CA, USA).
In these experiments, no mutations were detected in exon 9 or 20 in any of the HCC samples. However, in 13 of 47 clinical samples, we noted that the chromatograms resulting from the exon 9 PCR reaction contained abnormally migrating waves near the end of exon, as shown in Figure 1a. PCR products from noncancerous liver tissue also had these abnormally migrating waves, indicating that this was not a result of HCC-associated mutations. PCR amplification and subsequent cloning and sequencing of the PCR products revealed that the sequence chromatograms contained two distinct sequences (Figure 1b and c), the wild-type p110α sequence (IndexTermGAGCAGGAGAAAGATTTTCTATGGAGTCACAG) and a similar but distinct sequence (IndexTermGCGCAGGAGAAAGATTTTCTATGGACCACAG; base changes are underlined). A BLAST search showed that the latter sequence is found on human chromosome 22q11.2, the Cat Eye Syndrome (CES) region. CES is a hereditary disease characterized by ocular colobomata, anal atresia, congenital heart defects, and mental retardation. Patients with CES have a partial tetrasomy of the region that spans the p-arm and part of 22q11 (McDermid and Morrow, 2002). The 22q11 region contains partial sequences that are highly homologous to exons 9, 10, 11, 12, and 13 of the PIK3CA gene (98, 74, 98, 100 and 98%, respectively), thus this region is considered to be a putative pseudogene that arose as a result of gene duplication. This pseudogene does not seem to be transcribed, as an NCBI BLAST search revealed no expresses sequence tags (ESTs) with the same sequence. The primer for exon 9 anneals to both the PIK3CA and the pseudogene sequences, indicating that these primers were not specific for PIK3CA. The CES region sequence resembles a previously reported mutation in exon 9, but we did not consider this as a mutation because it reflects the sequence of the putative pseudogene rather than a mutated wild-type sequence. Similar findings have been reported by other groups (Or et al., 2005; Saal et al., 2005).
To confirm the hypothesis that the A1634C sequence change was a result of the amplification of the pseudogene and not a mutation, we designed a new reverse primer set specific for the amino-terminal region of exon 10 of the PIK3CA gene (forward primer, 5′-IndexTermGATTGGTTCTTTCCTGTCTCTG-3′; reverse primer; 5′-IndexTermGTAGAATTTCGGGGATAGTT-3′) and performed PCR and sequence analysis. This primer yielded an amplicon of approximately 1100 base pairs, the sequence of which matched that of the wild-type PIK3CA gene (Figure 1b) but not that of the pseudogene (Figure 1c).
Although our results do not suggest that mutations in PIK3CA do not occur, the percentage of mutations is likely to be lower than previously reported (Lee et al., 2005). Whether the PI3K-Akt pathway is activated in HCC remains controversial. A recent study indicated that the phosphorylation of the downstream kinases mTOR and P70 S6 was observed in 15 and 45% of HCC cases, respectively, suggesting that the PI3K-AKT pathway is indeed activated in a subset of HCC cases (Sahin et al., 2004). Our findings do not address whether PIK3CA mutations directly cause the activation of the PI3K-Akt pathway in HCC. The relationship between HCC and the PI3K-Akt pathway remains to be determined.
In conclusion, our results suggest that the percentage of hotspot mutations in the PIK3CA gene is lower among Japanese patients with HCC than was previously reported.
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This work was supported in part by the Health Science Research Grants for Medical Frontier Strategy Research from the Ministry of Health, Labor, and Welfare of Japan, and the Grants-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.
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Tanaka, Y., Kanai, F., Tada, M. et al. Absence of PIK3CA hotspot mutations in hepatocellular carcinoma in Japanese patients. Oncogene 25, 2950–2952 (2006). https://doi.org/10.1038/sj.onc.1209311
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