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Uncommon Mutation, but Common Amplifications, of the PIK3CA Gene in Thyroid Tumors

Department of Otolaryngology-Head and Neck Surgery (G.W., E.M., Z.G., B.T., D.S.), and Division of Endocrinology and Metabolism, Department of Medicine (S.H., P.W.L., M.X.), The Johns Hopkins University School of Medicine, Baltimore, Maryland 21287; and Department of Human Genetics (X.H.), Graduate School of Public Health, Oral Cancer Center (S.M.G.), University of Pittsburgh, Pittsburgh, Pennsylvania 15261

Address all correspondence and requests for reprints to: Dr. Mingzhao Xing, Division of Endocrinology and Metabolism, The Johns Hopkins University School of Medicine, 1830 East Monument Street, Suite 333, Baltimore, Maryland 21287. E-mail: mxing1{at}jhmi.edu.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Context: As in many other human cancers, overactivation of the phosphotidylinositol 3-kinase (PI3K)/Akt signaling pathway occurs frequently in thyroid cancer, but the mechanism is not completely clear.

Objective: Because activating mutations and genomic amplification of the PIK3CA gene, which encodes the p110a catalytic subunit of PI3K, are common in many cancers, we sought to investigate this phenomenon in thyroid tumors.

Design: To search for PIK3CA mutations, we isolated genomic DNA from primary thyroid tumors of various types and performed direct sequencing of the exons of PIK3CA gene that carry the most common mutations in other cancers. We used real-time quantitative PCR to investigate genomic amplification of the PIK3CA gene.

Results: We found no PIK3CA gene mutations in 37 benign thyroid adenomas, 52 papillary thyroid cancers, 25 follicular thyroid cancers, 13 anaplastic thyroid cancers, 13 medullary thyroid cancers, and seven thyroid tumor cell lines. We found a C3075T single-nucleotide polymorphism in exon 20 of this gene in two cases. With a copy number of 4 or more defined as amplification, we found PIK3CA gene amplification in four of 34 (12%) benign thyroid adenomas, three of 59 (5%) papillary thyroid cancer, five of 21 (24%) follicular thyroid cancer, none of 14 (0%) medullary thyroid cancer, and five of seven (71%) thyroid tumor cell lines. The PIK3CA gene amplification and consequent Akt activation were confirmed by fluorescence in situ hybridization and Western blotting studies using cell lines, respectively.

Conclusion: These data suggest that mutation of the PIK3CA gene is not common, but its amplification is relatively common and may be a novel mechanism in activating the PI3K/Akt pathway in some thyroid tumors.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
THE PHOSPHOTIDYLINOSITOL 3-KINASE (PI3K)/Akt signaling pathway plays an important role in cell growth and proliferation initiated by activation of receptor tyrosine kinases and in tumor genesis and progression (1, 2). PI3K catalyzes phosphorylation of the 3'-hydroxy group of the inositol ring in inositol phospholipids to produce phosphatidylinositol-3,4,5-trisphosphate [PI(3,4,5)P3] and phosphatidylinositol-3,4,5-bisphosphate [PI(3,4)P2]. Activation of PI3K occurs upon engagement with growth factor-activated receptor tyrosine kinase. Through interaction with PI(3,4,5)P3 and PI(3,4)P2 in the plasma membrane produced by PI3K, the down-stream effector, Ser/Thr kinase Akt, is subsequently translocated to plasma membrane and becomes phosphorylated and activated through phosphoinositide-dependent kinase (PDK). Subsequent signaling occurs through phosphorylation of various protein substrates by Akt. This signaling pathway has been shown to be frequently altered in human cancers, often involving increased activity of PIK3CA, the p110 catalytic subunit of PI3K, associated with overactivation of Akt signaling. Recently, activating somatic mutations of the PIK3CA gene were identified in various human cancers (3), particularly in colorectal cancer, glioblastoma, gastric cancer, and breast cancer, with a prevalence ranging from 25–32% in these cancers (3, 4). These data have established the role of PIK3CA as a protooncogene in human cancers in addition to its role as an oncogene through genomic amplification proposed previously (5). Since the initial demonstration of PIK3CA amplification in ovarian cancer (5), this phenomenon has also been shown in several other human cancers, including cervical cancer (6), nonsmall cell lung cancer (7), squamous cell carcinoma (8), esophageal adenocarcinoma (9), and gastric carcinoma (10). Amplification of PIK3CA is generally associated with increased PIK3CA expression, increased PI3K activity, and phosphorylation and activation of Akt in these studies, supporting the oncogenic role of PIK3CA amplification.

Several studies have shown that aberrant PI3K/Akt signaling may also play a role in thyroid tumor genesis or progression (11, 12, 13, 14). Thyroid tumors consist of several histologically distinct types, including the follicular epithelial cell-derived benign adenoma, papillary thyroid cancer (PTC), follicular thyroid cancer (FTC), and anaplastic thyroid cancer, and parafollicular C cell-derived medullary thyroid cancer (MTC). Akt was shown to be activated in thyroid cancer associated with Cowden’s syndrome in which the PI3K signaling pathway was overactivated due to inactivating mutations of PTEN, the protein product of which is a phosphatase that normally dephosphorylates the 3'-hydroxy position of inositol in phospholipids, thus antagonizing the action of PI3K (15). Increased phosphorylation and activation of Akt were also reported in sporadic FTC (11) and PTC (12, 13). However, mutation of PTEN was extremely rare in sporadic thyroid cancers and is therefore unlikely to be the mechanism for the activated Akt signaling pathway in these cancers. Thus, the mechanism for the overactivation of the PI3K/Akt signaling pathway in sporadic thyroid tumors has not been clearly defined. Given the frequent mutations and amplifications of the PIK3CA gene in other tumors in which the PI3K/Akt signaling pathway is activated, the present study was conducted to investigate whether similar genetic alterations of this gene occur in thyroid tumors.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Human thyroid tissues and DNA isolation

The study was conducted based on related institutional review board-approved protocols with appropriate patient consenting where required. The tumors were prepared and microdissected, and DNA was isolated from paraffin-embedded samples as previously described (16). Briefly, paraffin-embedded tissues were first treated for 8 h at room temperature with xylene, followed by digestion with 1% sodium dodecyl sulfate and 0.5 mg/ml proteinase K at 48 C for 48 h, with a midinterval addition of a spiking aliquot of concentrated sodium dodecyl sulfate-proteinase K to facilitate the digestion. DNA was subsequently isolated by standard phenol-chloroform extraction and ethanol precipitation procedures. Thyroid tumor cell line DNA was similarly isolated. The human thyroid tumor cell lines used in this study were provided by the following researchers: the KAK-1, KAT-5, KAT-7, and KAT-10 cells were from Dr. K. B. Ain (University of Kentucky Medical Center, Lexington, KY); the DRO-90-1 and ARO-81-1 cells were from Dr. G. J. F. Juillard (University of California School of Medicine, Los Angeles, CA); and the C643 cell was from Dr. N. E. Heldin (University of Uppsala, Uppsala, Sweden).

Mutation analysis of the PIK3CA gene

Because the vast majority of PIK3CA gene mutations were found in exon 9 (for the regulatory helical domain) and exon 20 (for kinase domain), and occasionally in exon 1 (3), we focused our mutation analysis on these exons in thyroid tumors. Genomic DNA was amplified by PCR using the amplifying and sequencing primers for these exons of PIK3CA gene as described previously (3). Step-down PCR was performed as follows. After a 3-min denaturing at 95 C, the PCR was run with each temperature for 1 min at six step-down steps, for two cycles each. The denaturing temperature was 95 C, and extension temperature was 72 C for each step, with the annealing temperature of 66, 64, 62, 60, 58, and 56 C from the first to the last step. The PCR was finally run at 95, 54, and 72 C each for 1 min for 35 cycles, followed by an elongation at 72 C for 5 min. In a final volume of 30 µl, the PCR mixture contained about 60 ng genomic DNA, 16.6 mM ammonium sulfate, 67 mM Tris (pH 8.8), 5% dimethylsulfoxide, 6.7 mM MgCl₂, 10 mM 2-mercaptoethanol, 1.5 mM of each deoxynucleotide triphosphate, 1.67 µM of each primer (forward and reverse), and 0.5 U platinum DNA Taq polymerase (Invitrogen Life Technologies, Inc., Gaithersburg, MD). The efficiency and quality of the amplification PCR were confirmed by running the PCR products on a 1.5% agarose gel. The PCR products were subsequently subjected to direct sequencing PCR with BigDye terminator V 3.0 cycle sequencing reagents (Applied Biosystems, Foster City, CA) with the following cycles: 95 C for 30 sec for one cycle, and 95 C for 15 sec, 50 C for 15 sec, and 60 C for 4 min for 35 cycles. The samples were finally analyzed on an ABI PRISM 3700 DNA Analyzer (Applied Biosystems) at The Johns Hopkins biosynthesis and sequencing facility for mutation identification.

Copy number analysis of PIK3CA with real-time quantitative PCR

Real-time PCR for the PIK3CA amplification study was performed using a PE Applied Biosystem ABI 7900 TaqMan sequence detector (Foster City, CA) following the manufacturer’s instructions. Specific primers and probes were designed with the Applied Biosystems software to amplify both the PIK3CA and control ß-actin genes. For the PIK3CA gene, the probe used was 5'-6-carboxyfluorescein-CACTGCACTGTTAATAACTCTCAGCAGGCAAA-tetramethylrhodamine-3', and the primers were 5'-AAATGAAAGCTCACTCTGGATTCC-3' (forward) and 5'-TGTGCAATTCCTATGCAATCG-3' (reverse). For the ß-actin gene, the probe was 5'-6-carboxyfluorescein-ATGCCCTCCCCCATGCCATCC-tetramethylrhodamine-3', and the primers were TCACCCACACTGTGCCCATCTACGA-3' (forward) and 5'-TCGGTGAGGATCTTCATGAGGTA-3' (reverse). Using these primers and probes and the protocol described previously (17), samples were run in triplicate, and primers and probes to ß-actin were run in parallel to standardize the input DNA. Standard curves were established using serial dilutions of DNA extracted from MCF12A cells with 0.01–20 ng DNA.

Fluorescence in situ hybridization (FISH)

To validate the real-time PCR technique in detecting the PIK3CA gene amplification, we conducted FISH studies on cell lines with various numbers of PIK3CA gene copies.

The bacterial artificial chromosome (BAC) clone RP11-466H15 for PIK3CA was obtained from Research Genetics (Invitrogen Life Technologies, Inc., Carlsbad, CA). The chromosome 3 -satellite plasmid and BAC DNA were labeled directly with SpectrumOrange-dUTP and SpectrumGreen-dUTP (Vysis, Inc., Downers Grove, IL), respectively, using a nick translation kit (Vysis) following the manufacturer’s instructions. Dual-color FISH was performed using a standard protocol. Slides were counterstained with 4',6-diamido-2-phenylindole hydrochloride (Sigma-Aldrich Corp., St. Louis, MO), mounted with antifade (Vysis), and stored at –20 C. Analysis was carried out using an Olympus (New Hyde Park, NY) BHS fluorescence microscope, and images were captured using a CytoVision Ultra (Applied Imaging, Santa Clara, CA).

Western blotting of phosphorylated Akt

To evaluate Akt phosphorylation status, we performed Western blotting analysis on C643 and KAT-10 thyroid tumor cells. The C643 cell harbored no PIK3CA amplifications, whereas the KAT-10 cell did. Cell lysates were collected in sodium dodecyl sulfate lysis buffer (Cell Signaling Technology, Beverly, MA), and protein concentrations were determined with a protein assay kit (Bio-Rad Laboratories, Hercules, CA). Approximately 50 µg total protein from each sample was denatured in loading buffer for 10 min, electrophoresed on 10% polyacrylamide gels, and electroblotted to nitrocellulose membranes (Hybond-C extra, Amersham Biosciences, Arlington Heights, IL). The membrane was incubated overnight with primary antibody Akt Ser⁴⁷³ (antirabbit; Cell Signaling Technology), Akt (antirabbit; Cell Signaling Technology), or ß-actin (antimouse antibody; Sigma-Aldrich Corp.) at 4 C. The membrane was washed three times in PBS with 0.1% Tween 20 at room temperature and incubated with horseradish peroxidase-labeled secondary antibody (goat antirabbit IgG or goat antimouse IgG; Sigma-Aldrich Corp.) or 1 h at room temperature. Signal detection was performed by horseradish peroxidase chemiluminescent reaction (ECL, Amersham Biosciences).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
The vast majority of PIK3CA mutations occur in other cancers in two small clusters in the helical domain from exon 9, in the kinase domain from exon 20, and occasionally in exon 1 of the gene. These mutations are all missense mutations causing amino acid changes in the protein with expected functional consequences. The most common are the transversion mutations of nucleotides C1616G, G1624A, A1625G, A1625T, G1633A, A1634G, G1635T, C1636A, and A1637C in exon 9 and of nucleotides T3022C, A3073G, C3074A, G3129T, C3139T, A3140G, A3140T, and G3145A in exon 20 (3). We therefore amplified and sequenced these exons by PCR to search for the mutations in thyroid tumors. Shown in Fig. 1 are examples of wild-type alleles in thyroid tumors negative for the reported mutations in exons 9 and 20 (Fig. 1, A and C, respectively). As positive controls, the G1633A mutation in exon 9 in the breast cancer MCF7 cell line (Fig. 1B) and the A3140G mutation in exon 20 in the breast cancer BT20 cell line (Fig. 1D) were used. We found none of the reported common mutations in exons 9 and 20 in 37 benign thyroid adenomas, 52 PTC, 25 FTC, 13 anaplastic thyroid cancer, 13 MTC, and seven thyroid tumor cell lines. Instead, we found a C3075T nucleotide change in exon 20 in two cases, one in an adenoma and the other in an FTC tumor. This C3075T change also occurred in matched normal tissues and resulted in no change in the amino acid sequence of the protein product. It therefore represents a single nucleotide polymorphism. Because PIK3CA gene mutations were occasionally seen also in exon 1 in other cancers (3), we sequenced exon 1 of the PIK3CA gene in 10 thyroid cancer samples. Again, no mutations were found in this exon in thyroid cancers.

View larger version (33K): [in this window] [in a new window]   FIG. 1. Mutational analysis of PIK3CA gene by sequencing. A, Representative thyroid tumor sample harboring wild alleles of exon 9 of the PIK3CA gene. B, G1633A mutation in exon 9 of the gene in MCF-7 cell line. C, Representative thyroid tumor sample harboring wild alleles of exon 20 of the gene. D, A3140G mutation in exon 20 in BT20 cell line.

View larger version (36K): [in this window] [in a new window]   FIG. 2. Real-time quantitative PCR amplification curves. A, A standard curve was generated using H1299 cell line DNA and shows a linear range from 0.01–20 ng. B, Real-time amplification curves for the ß-actin gene from two representative tumor samples showing complete overlap. C, Real-time amplification curves for the PIK3 gene from the same two samples in B, showing a one-cycle shift to the right in one of the tumor samples, representing four (left curve) and two (right curve) copies of the gene, respectively. DRn = (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 is initiated. Each experiment was performed in triplicate and is shown by overlapping amplification curves. More details are given in Materials and Methods.

View larger version (19K): [in this window] [in a new window]   FIG. 3. FISH study of the PIK3CA gene amplification. A, Normal lymphocyte cell in metaphase, showing normal two copies of the PIK3CA gene (green) matching the corresponding two centromeres (pink) on the same chromosome. B and C, KAT-10 cell (B) and KAT-5 cell (C), both showing a much higher number of copies of the PIK3CA gene, demonstrating the amplification of this gene. Pink, Centromere probe; green, BAC 466H15 probe (specific for the PIK3CA gene). For the PIK3CA gene, two copies in normal cells, at least 14 copies in KAT-10 cells, and at least five copies in KAT-5 cells are revealed.

View larger version (17K): [in this window] [in a new window]   FIG. 4. PIK3CA gene amplification in thyroid tumors detected by real-time quantitative PCR. A, Shown is the copy number of the PIK3CA gene in each individual case of thyroid tumor or cell line. B, Shown is the average copy number of the PIK3CA gene of each group of thyroid tumors, with mean ± SD of 2.63 ± 0.83 for FTC, 2.38 ± 0.70 for PTC, 2.42 ± 0.78 for adenoma, 1.80 ± 0.40 for MTC, and 3.84 ± 1.50 for cell lines. The total number of cases of each type of tumor is indicated in Table 1.

View larger version (65K): [in this window] [in a new window]   FIG. 5. Western blotting analysis of Akt phosphorylation in thyroid tumor cell lines. Cellular proteins were prepared and blocked as described in Materials and Methods. Antiphosphorylated Akt (P-Akt) was used as primary antibody to detect Akt phosphorylation, and antiwhole Akt (Akt) was used to demonstrate the comparable levels of Akt protein in the two cellular protein preparations. ß-Actin levels were examined to ensure the integrity of the protein preparations. It should be noted that C643 thyroid tumor cell lines harbor two copies of the PIK3CA gene, and KAT-10 cells harbor five copies of the PIK3CA gene in the real-time PCR study (14 copies in the FISH study; Fig. 3).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Nevertheless, the PIK3CA amplification is less common than other thyroid tumor-associated oncogenic genetic alterations, such as the Ras mutation (18), the BRAF mutation (16, 19, 20), and Ret/PTC rearrangements (21) in thyroid tumors. This, together with the lack of PIK3CA mutation, raises the question of how the commonly seen overactivation of the PIK3/Akt signaling pathway occurs in thyroid cancers (11, 12, 13, 14). Alternative mechanisms responsible for activation of the PI3K/Akt signaling pathway presumably exist in these tumors. One such mechanism may involve the well-established classical oncogenic pathways in thyroid tumors. For example, a recent study demonstrated that the Akt-phosphorylating and -activating PDK could be directly phosphorylated and activated by Ret/PTC tyrosine kinase in a phosphoinositide-independent manner (22). PDK is an important component of the PI3K/Akt pathway in which the former is activated by phosphoinositides produced by PI3K and subsequently phosphorylates and activates Akt (1, 2). A more recent study showed that Ret/PTC3 could also activate the Akt signaling pathway partially through activation of PI3K, because the PI3K-specific inhibitor LY294002 could reduce the activation level of Akt in Ret/PTC-transfected thyroid cells (23). Therefore, Ret/PTC seems to be able to activate Akt signaling pathway in a PDK-dependent, but PI3K-independent, pathway and in a PI3K-dependent pathway as well. This may be a mechanism particularly in PTC which, among various types of thyroid tumors, most commonly harbors Ret/PTC rearrangements (21). One can also speculate that Akt signaling pathway could be activated through a similar mechanism involving Ret tyrosine kinase in MTC, in which the activating Ret mutation is a major genetic driving force for tumorigenesis. The oncogenic Ras mutation may represent another alternative mechanism for PI3K/Akt activation in thyroid tumors. In our preliminary studies (data not shown), in which we examined the relationship between Ras mutations and PIK3CA amplification, we found Ras mutation in none of 10 samples that harbored PIK3CA amplification, but found Ras mutation in five of 23 samples that did not harbor PIK3CA amplification. Although the data did not reach statistical significance (P = 0.65, by Fisher’s exact t test with two tails), it suggested a possible mutual exclusivity between the Ras mutation and PIK3CA amplification. This is consistent with the idea that, like PI3K, Ras may independently activate the PI3K/Akt pathway. The PIK3CA possesses a Ras binding domain, and PI3K is a well-characterized immediate downstream effector of Ras (24). One study showed that activation of PI3K occurred in thyroid cells upon expression of mutated Ras protein, with a consequent increased dependence of the cell on PI3K for survival (25). Another study demonstrated that PI3K was an essential antiapoptotic effector in the proliferative response of human thyroid cells to mutant Ras (26). Activation of the PI3K/Akt pathway by Ras mutants may particularly occur in FTC, which, among various types of thyroid tumors, most commonly harbor activating Ras mutations (18). Thus, Ret/PTC rearrangements, Ras mutations, and Ret mutations may conceivably play an important role in activating the PI3K/Akt signaling pathway in PTC, FTC, and MTC, respectively. Consequently, PIK3CA mutation and amplification may not be frequent genetic events in thyroid tumors, because they would not be (redundantly) selected during thyroid tumorigenesis. These and other alternative mechanisms for activation of the PI3K/Akt signaling pathway in thyroid tumors remain to be investigated.

	Acknowledgments

 
We thank Drs. Vasily Vasko and Giovanni Tallini for kindly providing us thyroid tumor tissues that were previously used in our published studies and partially used in the present study.

	Footnotes

 
This work was supported in part by National Institutes of Health Grants UO1-CA-98-028 and RO1-DE13561-01. M.X. is the recipient of a Flight Attendant Medical Research Institute Grant and a Johns Hopkins Clinician Scientist Award that partially supported this project.

First Published Online May 31, 2005

¹ G.W. and E.M. contributed equally to this work.

Abbreviations: BAC, Bacterial artificial chromosome; FISH, fluorescence in situ hybridization; FTC, follicular thyroid cancer; MTC, medullary thyroid cancer; PDK, phosphoinositide-dependent kinase; PI3K, phosphotidylinositol 3-kinase; PI(3,4)P2, phosphatidylinositol-3,4,5-bisphosphate; PI(3,4,5)P3, phosphatidylinositol-3,4,5-trisphosphate; PTC, papillary thyroid cancer.

Received November 22, 2004.

Accepted May 25, 2005.

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