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Absence of activating somatic mutations of PI3KCA and AKT1 genes in South Indian women with endometriosis
European Journal of Obstetrics & Gynecology and Reproductive Biology 152 (2010) 78–82
Contents lists available at ScienceDirect
European Journal of Obstetrics & Gynecology and Reproductive Biology journal homepage: www.elsevier.com/locate/ejogrb
Absence of activating somatic mutations of PI3KCA and AKT1 genes in South Indian women with endometriosis Priyanka Rai a, Mamata Deenadayal b, Sisinthy Shivaji a,* a b
Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, Andhra Pradesh, India Infertility Institute and Research Centre, Hyderabad, Andhra Pradesh, India
A R T I C L E I N F O
A B S T R A C T
Article history: Received 7 January 2010 Received in revised form 2 April 2010 Accepted 25 April 2010
Objective: To investigate whether the PI3KCA and AKT1 gene influences the risk of developing endometriosis in South Indian women. Study design: Mutations in exon 9 and 20 of PI3KCA gene and E17K mutation in exon 4 of AKT1 gene were tested for association in a case–control study between eutopic and ectopic endometrium tissue from 30 endometriosis cases and eutopic endometrium tissue from 30 controls. The genotype frequencies of these mutations were compared using polymerase chain reaction and direct sequencing analysis of tissue DNA. Results: The analysis did not reveal any activating somatic mutations in either PI3KCA or AKT1 gene in the cases. Conclusion: In the present study we could not observe any mutation in PI3KCA and AKT1 gene, indicating that these mutations are rarely associated with endometriosis in South Indian women. ß 2010 Elsevier Ireland Ltd. All rights reserved.
Keywords: Endometriosis PI3KCA AKT1 Mutation
1. Introduction Endometriosis is a common gynecological disorder in human females. It is defined as the presence of endometrial glands and stroma within extrauterine sites, most commonly the ovaries and peritoneum, and affects approximately 5–10% of women of reproductive age [1]. The main clinical manifestations are dysmenorrhea, deep dyspareunia, chronic pelvic pain and infertility. The exact etiology and pathogenesis of the disease are still unclear. Among the various theories proposed for its pathogenesis, Sampson’s theory of transplantation of endometrial tissue onto the pelvic peritoneum via retrograde menstruation is most widely accepted [2]. However, the cause is multifactorial involving environmental, immunological, endocrine and genetic processes [3]. An increasing number of gene polymorphisms and mutations have also been associated with endometriosis, but their precise role remains to be determined [4]. Endometriosis is pathohistologically a benign disease; however it is characterized by growth, proliferation, infiltration and adherence of endometrial tissue to the surrounding tissue, suggesting that endometriosis could be viewed as a neoplastic process [5]. This is further supported by evidence that the susceptibility to develop breast cancer, ovarian cancer, ovarian
* Corresponding author. Tel.: +91 40 27192504; fax: +91 40 27160311. E-mail address: [email protected] (S. Shivaji). 0301-2115/$ – see front matter ß 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ejogrb.2010.04.022
clear cell and endometroid cancer increases in the presence of endometriosis [6]. The phosphatidylinositol 3-kinase (PI3K)/Akt (vakt murine thymoma viral oncogene homologue) kinase signaling pathway plays an important role in a wide range of cellular processes associated with malignant behavior including inhibition of apoptosis, cellular growth, proliferation, cell motility, adherence, transformation, angiogenesis and survival [7,8]. PI3K is a family of lipid kinases that exists as heterodimer consisting of catalytic subunits (p110a, p110b or p110d) and regulatory subunits (p85a, p85b or p85g). It is well established that PI3K is activated either by autocrine or paracrine stimulation of receptor tyrosine kinases. Active PI3K phosphorylates phosphotidylinositol 4,5-biphosphate (PIP2) at the 30 -position of inositol ring, converting it to phosphotidylinositol 3,4,5-triphosphate (PIP3). PIP3 recruits its downstream effector, Ser/ Thr kinase Akt and phosphoinositide dependent kinase (PDK) at the plasma membrane. Once at the membrane, Akt is phosphorylated and activated by PDK and 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 PI3KCA, the p110a catalytic subunit of PI3K, resulting in over-activation of Akt signaling [9,10]. Recently, activating somatic mutations of PI3KCA gene were identified in various human cancers particularly in breast, endometroid and clear cell ovarian cancer [11,12] and also in certain benign diseases [13]. More than 75% of these mutations are clustered in the helical domain encoded by exon 9 and the kinase domain encoded by exon 20. In addition, three hot spot mutations in
P. Rai et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 152 (2010) 78–82
these exons (E542K, E545K and H1047R) have been shown to activate the PI3K-Akt pathway via phosphorylation of Akt and results in in vitro transformation [9,14]. Recently, activating mutations in AKT1 have also been characterized [15]. Interestingly, AKT1 mutations have been identified in breast, ovarian and colorectal carcinoma that result in a glutamic acid to lysine substitution at amino acid 17 (E17K) in the plekstrin homology (PH) domain, rather than in the kinase domain [15–17]. Nevertheless, these AKT1 mutations are activating; they transform cells and induce leukemia in nude mice [15]. Recent studies have shown the up-regulation of several genes involved in PI3K/Akt signaling pathway in endometriosis, suggesting that this pathway could be involved in growth and progression of this disease [18,19]. The mechanism for the over-activation of PI3K/Akt signaling pathway in endometriosis has not been defined. Given the frequent mutation of the PI3KCA and AKT1 genes in cancers in which the PI3K/Akt signaling pathway is activated, the present study was conducted to investigate whether such mutations are responsible for the pathogenesis of endometriosis. 2. Materials and methods 2.1. Subjects All the women were non-smokers of South Indian origin, and were recruited at the Infertility Institute and Research Centre, Hyderabad, India. Eutopic and ectopic endometrial tissue samples were collected from 30 women with moderate-severe (I–IV) endometriosis, staged using the revised American Fertility Society (rAFS) classification system [20]. All the women had a transvaginal ultrasound scan (TVS) at screening followed by laparoscopy to confirm the diagnosis (rAFS I–3; II-5; III-3; IV-19). Their mean age was 27.5 4.4 (range 20–35) years. All the patients had different forms of endometriosis such as peritoneal lesions, adhesions and endometriomas. Thirty women with normal menstrual cycle, normal hormonal profiles and no uterine abnormalities constituted the control sample group. Their mean age was 26.7 3.9 (range 22–36) years. Women with ovarian cysts, adenomyosis, ovarian cancer, and fibroids were excluded from the study. All tissue samples were collected during routine surgical procedures. The samples were frozen in liquid nitrogen and preserved at 80 8C until DNA was extracted. Written consent was obtained from all participants for the sampling of eutopic and ectopic endometrial tissue for research purposes. The Institutional Review Board of the Centre for Cellular and Molecular Biology, Hyderabad, approved the study. 2.2. Cell lines Immortalized human endometriotic epithelial cells (12-Z) and stromal cells (22-B) [21] and immortalized human endometrial surface epithelial cells (HES) [22] and stromal cells (Sht 290) [23] were also used for mutational analysis. 12-Z and 22-B cells were a generous gift from Dr. A. Starzinski-Powitz, Institut fu¨r Zellbiologie und Neurowissenschaft, Johann Wolfgang Goethe-Universita¨t, Frankfurt, Germany. HES cells were a generous gift from Dr. Asgerally T. Fazleabas, University of Illinois at Chicago, Illinois, USA. Sht 290 cells were a generous gift from David G. Kaufman, University of North Carolina at Chapel Hill, North Carolina, USA. The endometriotic epithelial and stromal cells were cultured in DMEM (Invitrogen, Carlsbad, California, USA) containing 10% FBS (Invitrogen) and normal endometrial epithelial and stromal cells were maintained in phenol red free DMEM (Invitrogen) containing 10% and 2% charcoal treated FBS (Invitrogen), respectively. The media also contained penicillin (100 U/ml) and streptomycin (100 g/ml) (Invitrogen). Cells were maintained in a humidified atmosphere of 5% CO2 and 95% air at 37 8C.
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2.3. DNA extraction Genomic DNA was extracted from eutopic and ectopic endometrium and from cell lines by using DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA, USA). 2.4. Primers and polymerase chain reaction Screening of mutations in exon 9 and 20 of PI3KCA gene was done by PCR and sequencing analysis. Exons 9 and 20 of PIK3CA were individually amplified from genomic DNA with primers complementary to surrounding intronic sequences. PCRs were carried out in a total volume of 10 ml, containing 10 ng genomic DNA, 0.5 pmol of each primer, IX Taq polymerase buffer (1.5 mM MgCl2) and 0.1 U of Amplitaq DNA polymerase (Perkin-Elmer, Foster City, USA). The primers for exon 9 and 20 were 50 -TGAAAATGTATTTGCTTTTTCTGT-30 (forward), and 50 TGTAAATTCTGCTTTATTTATTCC-30 (reverse), and 50 -CATTTGCTCCAAACTGACCA-30 (forward) and 50 -GGTCTTTGCCTGCTGAGAGT-30 (reverse), respectively, using the method described by Campbell et al. [11]. PCR amplification was performed in a programmable thermal cycler gradient PCR system (Eppendorf AG, Hamburg, Germany). The PCR amplification for exon 9 was carried out for 30 cycles (initial denaturation at 95 8C for 10 min, denaturation at 95 8C for 1 min, annealing for 1 min at 54 8C, extension at 72 8C for 1 min and final extension for 10 min at 72 8C). For exon 20, after an initial denaturation step of 95 8C for 10 min, a ‘‘touch-down’’ program was used consisting of two cycles of amplification at annealing temperatures of 63–59 8C; followed by 30 amplification cycles at an annealing temperature of 58 8C and a final extension cycle of 72 8C for 5 min. The PCR condition and the PCR primer for exon 4 of AKT1 were done as described earlier by Shoji et al. [24]. PCR products were analysed using 1.2% agarose gel electrophoresis followed by ethidium bromide staining and ultraviolet visualization and then sequenced with a Taq-Dye deoxy-terminator cycle sequencing kit (Applied BioSystems, Foster City, USA) using an automated ABI 3770 sequencer (Applied BioSystems, Foster City, USA). 3. Results 3.1. PI3KCA mutation in endometriosis Most of the PI3KCA mutations are present in two small clusters in the helical domain from exon 9 and in the kinase domain from exon 20 [9]. All these mutations are missense resulting in amino acid changes in the protein, thus causing aberrant activation of PI3K and of the downstream kinase, Akt. The most common are the transversion mutations of the nucleotide 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 [9]. To investigate the frequency of PI3KCA mutations in South Indian women having endometriosis, we amplified and sequenced these exons by PCR. Shown in Fig. 1A and C are the sequences of endometriosis samples, which are negative for the reported mutations in exon 9 and 20. However, in the breast cancer cell line, MCF-7, which was used as a positive control, the G1633A mutation in exon 9 is clearly visible (Fig. 1B). 3.2. AKT1 mutations in endometriosis The sequencing analysis for exon 4 of the human AKT1 gene in 30 endometriosis samples revealed the absence of the point mutation G > A at nucleotide 49 (E17K) (Fig. 1D) which was first identified by Carpten et al. [15]. This result suggests that E17K
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Fig. 1. Mutational analysis of exon 9 and 20 of PIK3CA and exon 4 of AKT1 gene following PCR amplification and sequencing. Representative human endometrium tissue sample from women having endometriosis showing wild alleles G, A, G, C and A at positions 1633, 1634, 1635, 1636 and 1637, respectively in exon 9 of the PIK3CA gene (A), C3139, A3140 and G3145 in exon 20 of the PIK3CA gene (C), and G49 in exon 4 of the AKT1 gene (D). MCF-7 cell line which was used as a control shows, G1633A mutation in exon 9 of the PI3KCA gene (B).
Table 1 PIK3CA and AKT1 mutations in immortalized human endometrial surface epithelial cells (HES) and stromal cells (Sht 290) and immortalized human endometriotic epithelial cells and stromal cells. Cell type
PI3KCA exon 9 mutation
PI3KCA exon 20 mutation
AKT1 exon 4 mutation
HES cells Sht 290 cells Endometriotic epithelial cells Endometriotic stromal cells
Absent Absent Absent Absent
Absent Absent Absent Absent
Absent Absent Absent Absent
transformation mutation of the AKT1 gene may be a rare event in the development of endometriosis in the South Indian women. 3.3. PI3KCA and AKT1 mutational analysis in human normal and endometriotic cell lines We also analysed mutations in exon 9 and 20 of PI3KCA gene and exon 4 of AKT1 gene in immortalized human endometriotic epithelial cells (12-Z) and stromal cells (22-B) and immortalized human endometrial surface epithelial cells (HES) and stromal cells (Sht 290) by PCR amplification and sequencing analysis. Both PI3KCA and AKT1 mutations were found to be absent in both endometriotic and endometrial cell lines (Table 1). 4. Comment Endometriosis is a polygenically inherited disease with multifactorial pathogenesis. Several case–control studies have been done to assess whether association exists between endometriosis and genes involved in inflammation, steroid hormone production and detoxification [4]. These genes play a critical role in the pathophysiology of the disease. In the present study, mutations in exon 9 and 20 of PI3KCA gene, which codes for the catalytic subunit of PI3K and in exon 4 of AKT1 gene, were investigated to ascertain whether these mutations are associated with endometriosis in South Indian women.
Numerous genetic and functional studies have clearly established the involvement of the PI3K-Akt pathway in the development of neoplasia. The PI3K-Akt pathway is deregulated in human cancers at several levels. The tumour suppressor PTEN, which dephosphorylates PIP3 to PIP2, thus antagonizing PI3K activity, is deleted or mutated in several cancers [25,26] and amplification of the genomic region containing AKT or PI3K genes has also been reported [11,27,28]. PI3KCA, which codes for the catalytic subunit of PI3K and AKT1 are somatically mutated at significant frequencies in many cancers. These studies suggest an oncogenic role for PI3KCA and AKT1. PI3KCA mutation has also been reported in benign or pre-neoplastic conditions such as epidermal nevi and seborrheic keratoses [13], which suggest that in addition to their role in cancer, oncogenic PIK3CA mutations contribute to the pathogenesis of benign diseases. PI3KCA is located on chromosome 3q26.32 and consists of 20 exons. Interestingly, however, more than 75% of the mutations occur in two small clusters in the helical (exon 9) and kinase domain (exon 20). A recent report by Carpten et al. [15] identified a transforming E17K mutation in PH domain of AKT1. Interestingly, the E17K-AKT1 transforming mutation resulted in not only an increased level of Akt phosphorylation but also of PIP2, and the PIP3-independent recruitment of Akt1 to the cell membrane. This recurring mutation in a regulatory domain of AKT1 leads to structural alteration of the ligand-binding site, pathological membrane association, constitutive activation of the kinase, in vitro transformation, soft agar colony formation and
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leukemia in mice. Functional studies on PI3KCA mutants show attenuation of apoptosis and enhancement of growth, invasion and metastasis [29]. These observations reflect the importance of PI3KCA and AKT1 mutations in the progression of cancer. Our study showed that the most common activating PI3KCA mutation (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) and transforming AKT1 (nucleotide G49A) mutations, which are reported to be extremely common in endometriod, ovarian, breast and clear cell ovarian cancer, with 45% harboring one or other alteration, were not present in South Indian women with rAFS stage I–IV endometriosis. We further analysed the human endometriotic epithelial cells and stromal cells for the presence of PI3KCA and AKT1 mutations, which were derived from a European population, to investigate whether these mutations are responsible for the pathogenesis of endometriosis. We observed that these mutations are even absent in the endometriotic cell lines. This result suggests that some other mechanisms are involved in the activation of the PI3K/AKT pathway in endometriosis. PI3KCA and AKT1 mutations were also studied in immortalized human endometrial surface epithelial cells and stromal cells and as anticipated the mutations were absent in these cell lines as they were derived from normal endometrium. Our data are further supported by report of Tanaka et al. [30], where they studied PI3KCA mutation in hepatocellular carcinoma in a Japanese population and found that PI3KCA is not somatically mutated in this tumour lineage. Further reports by Kim et al. [17] and Reiner et al. [31] in Korean and European populations, respectively, showed absence of AKT1 mutation in hepatocellular carcinoma, suggesting that alternative mechanisms might be involved in the activation of the PI3K/Akt pathway in malignant diseases. The PI3k/Akt pathway is activated by estrogen in uterine endometrium and upon activation it regulates many physiological processes such as blastocyst attachment and implantation, regulation of trophoblast invasion, immune surveillance and efficient disposal of blood and desquamated cellular debris with menstruation [32]. This is further supported by the evidence that rapid activation of intracellular signaling cascades ERK1/2 (extracellular signal-regulated kinases) and PI3K/Akt by growth factors and estrogens are involved in the migration of normal endometrial stromal cells [33]. Given that endometriosis is an estrogen-dependent disease [34], the PI3K/Akt pathway appears to have a role in disease development and progression. Recent studies by Matsuzaki et al. [18] and Wu et al. [19], using complementary DNA microarray and laser capture microdissection, revealed an upregulation of PI3K-Akt and several other genes involved in this pathway, in epithelial and stromal cells from deep endometriosis compared with their matched eutopic endometrium. This finding further support the importance of the PI3K/Akt pathway in endometriosis. Absence of PI3KCA and AKT1 mutation raises the question as to how the over-activation or up-regulation of the PI3K-Akt signaling pathway occurs in endometriosis. Alternative mechanisms responsible for the activation of the PI3K-Akt signaling pathway presumably exist in this disease. One such mechanism may involve the amplification of PI3KCA or AKT1 gene as reported in several human cancers including ovarian cancer [35]. It is also possible that absence of these mutations in our study may be attributed due to different geographical origin of the patients, variation in risk factors and the methods used for DNA mutational analysis. In conclusion, our result suggests that mutation in PI3KCA and AKT1 genes is absent in South Indian women with endometriosis. Other alternative mechanisms for the activation of the PI3K/Akt
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signaling pathway in endometriosis remain to be further investigated. Conflicts of interest The authors have declared no conflict of interest. Acknowledgments Priyanka Rai is the recipient of CSIR fellowship from Government of India. We thank Dr. Archana B. Siva, Venkatesh Kota and Y. Kameshwari for their valuable suggestions and technical help. References [1] Eskenazi B, Warner ML. Epidemiology of endometriosis. Obstet Gynecol Clin North Am 1997;24:235–58. [2] Sampson JA. Peritoneal endometriosis due to menstrual dissemination of endometrial tissue into peritoneal cavity. Am J Obstet Gynecol 1927;14: 422–69. [3] Bulun SE. Endometriosis. N Engl J Med 2009;360:268–79. [4] Montgomery GW, Nyholt DR, Zhao ZZ, et al. The search for genes contributing to endometriosis risk. Hum Reprod Update 2008;14:447–57. [5] Varma R, Rollason T, Gupta JK, Maher ER. Endometriosis and the neoplastic process. Reproduction 2004;127:293–304. [6] Melin A, Spare´n P, Bergqvist A. The risk of cancer and the role of parity among women with endometriosis. Hum Reprod 2007;22:3021–6. [7] Vivanco I, Sawyers CL. The phosphatidylinositol 3-kinase-AKT pathway in human cancer. Nat Rev Cancer 2002;2:489–501. [8] Brader S, Eccles SA. Phosphoinositide 3-kinase signalling pathways in tumor progression, invasion and angiogenesis. Tumori 2004;90:2–8. [9] Samuels Y, Wang Z, Bardelli A, et al. High frequency of mutations of the PIK3CA gene in human cancers. Science 2004;304:554. [10] Altomare DA, Testa JR. Perturbations of the AKT signaling pathway in human cancer. Oncogene 2005;24:7455–64. [11] Campbell IG, Russell SE, Choong DY, et al. Mutation of the PIK3CA gene in ovarian and breast cancer. Cancer Res 2004;64:7678–81. [12] Velasco A, Bussaglia E, Pallares J, et al. PIK3CA gene mutations in endometrial carcinoma. Correlation with PTEN and K-RAS alterations. Hum Pathol 2006;37:1465–72. [13] Hafner C, Lo´pez-Knowles E, Luis NM, et al. Oncogenic PIK3CA mutations occur in epidermal nevi and seborrheic keratoses with a characteristic mutation pattern. Proc Natl Acad Sci USA 2007;104:13450–4. [14] Kang S, Bader AG, Vogt PK. Phosphatidylinositol 3-kinase mutations identified in human cancer are oncogenic. Proc Natl Acad Sci USA 2005;102:802–7. [15] Carpten JD, Faber AL, Horn C, et al. A transforming mutation in the pleckstrin homology domain of AKT1 in cancer. Nature 2007;448:439–44. [16] Bleeker FE, Felicioni L, Buttitta F, et al. AKT1E17K in human solid tumours. Oncogene 2008;27:5648–50. [17] Kim MS, Jeong EG, Yoo NJ, Lee SH. Mutational analysis of oncogenic AKT E17K mutation in common solid cancers and acute leukaemias. Br J Cancer 2008;98:1533–5. [18] Matsuzaki S, Canis M, Vaurs-Barriere C, Boespflug-Tanguy O, Dastugue B, Mage G. DNA microarray analysis of gene expression in eutopic endometrium from patients with deep endometriosis using laser capture microdissection. Fertil Steril 2005;84:1180–90. [19] Wu Y, Kajdacsy-Balla A, Strawn E, et al. Transcriptional characterizations of differences between eutopic and ectopic endometrium. Endocrinology 2006;147:232–46. [20] American Fertility Society. Classification of endometriosis. Fertil Steril 1979;32:633–45. [21] Zeitvogel A, Baumann R, Starzinski-Powitz A. Identification of an invasive, Ncadherin-expressing epithelial cell type in endometriosis using a new cell culture model. Am J Pathol 2001;159:1839–52. [22] Desai NN, Kennard EA, Kniss DA, Friedman CI. Novel human endometrial cell line promotes blastocyst development. Fertil Steril 1994;61:760–6. [23] Barbier CS, Becker KA, Troester MA, Kaufman DG. Expression of exogenous human telomerase in cultures of endometrial stromal cells does not alter their hormone responsiveness. Biol Reprod 2005;73:106–14. [24] Shoji K, Oda K, Nakagawa S, et al. The oncogenic mutation in the pleckstrin homology domain of AKT1 in endometrial carcinomas. Br J Cancer 2009;101:145–8. [25] Li J, Yen C, Liaw D, et al. PTEN, a putative protein tyrosine phosphatase gene mutated in human brain, breast and prostate cancer. Science 1997;275: 1943–7. [26] Sansal I, Sellers WR. The biology and clinical relevance of the PTEN tumor suppressor pathway. J Clin Oncol 2004;22:2954–63. [27] Bellacosa A, de Feo D, Godwin AK, et al. Molecular alterations of the AKT2 oncogene in ovarian and breast carcinomas. Int J Cancer 1995;64:280–5. [28] Wu G, Mambo E, Guo Z, et al. Uncommon mutation, but common amplifications, of the PIK3CA gene in thyroid tumors. J Clin Endocrinol Metab 2005;90:4688–93. [29] Samuels Y, Diaz Jr LA, Schmidt-Kittler O, et al. Mutant PIK3CA promotes cell growth and invasion of human cancer cells. Cancer Cell 2005;7:561–73.
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[30] Tanaka Y, Kanai F, Tada M, et al. Absence of PIK3CA hotspot mutations in hepatocellular carcinoma in Japanese patients. Oncogene 2006;25:2950–2. [31] Riener MO, Bawohl M, Clavien PA, Jochum W. Analysis of oncogenic AKT1 p.E17K mutation in carcinomas of the biliary tract and liver. Br J Cancer 2008;99:836. [32] Toyofuku A, Hara T, Taguchi T, Katsura Y, Ohama K, Kudo Y. Cyclic and characteristic expression of phosphorylated Akt in human endometrium and decidual cells in vivo and in vitro. Hum Reprod 2006;21:1122–8.
[33] Gentilini D, Busacca M, Di Francesco S, Vignali M, Vigano` P, Di Blasio AM. PI3K/ Akt and ERK1/2 signalling pathways are involved in endometrial cell migration induced by 17beta-estradiol and growth factors. Mol Hum Reprod 2007;13:317–22. [34] Gurates B, Bulun SE. Endometriosis: the ultimate hormonal disease. Semin Reprod Med 2003;21:125–34. [35] Shayesteh L, Lu Y, Kuo WL, et al. PIK3CA is implicated as an oncogene in ovarian cancer. Nat Genet 1999;21:99–102.
Contents lists available at ScienceDirect
European Journal of Obstetrics & Gynecology and Reproductive Biology journal homepage: www.elsevier.com/locate/ejogrb
Absence of activating somatic mutations of PI3KCA and AKT1 genes in South Indian women with endometriosis Priyanka Rai a, Mamata Deenadayal b, Sisinthy Shivaji a,* a b
Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, Andhra Pradesh, India Infertility Institute and Research Centre, Hyderabad, Andhra Pradesh, India
A R T I C L E I N F O
A B S T R A C T
Article history: Received 7 January 2010 Received in revised form 2 April 2010 Accepted 25 April 2010
Objective: To investigate whether the PI3KCA and AKT1 gene influences the risk of developing endometriosis in South Indian women. Study design: Mutations in exon 9 and 20 of PI3KCA gene and E17K mutation in exon 4 of AKT1 gene were tested for association in a case–control study between eutopic and ectopic endometrium tissue from 30 endometriosis cases and eutopic endometrium tissue from 30 controls. The genotype frequencies of these mutations were compared using polymerase chain reaction and direct sequencing analysis of tissue DNA. Results: The analysis did not reveal any activating somatic mutations in either PI3KCA or AKT1 gene in the cases. Conclusion: In the present study we could not observe any mutation in PI3KCA and AKT1 gene, indicating that these mutations are rarely associated with endometriosis in South Indian women. ß 2010 Elsevier Ireland Ltd. All rights reserved.
Keywords: Endometriosis PI3KCA AKT1 Mutation
1. Introduction Endometriosis is a common gynecological disorder in human females. It is defined as the presence of endometrial glands and stroma within extrauterine sites, most commonly the ovaries and peritoneum, and affects approximately 5–10% of women of reproductive age [1]. The main clinical manifestations are dysmenorrhea, deep dyspareunia, chronic pelvic pain and infertility. The exact etiology and pathogenesis of the disease are still unclear. Among the various theories proposed for its pathogenesis, Sampson’s theory of transplantation of endometrial tissue onto the pelvic peritoneum via retrograde menstruation is most widely accepted [2]. However, the cause is multifactorial involving environmental, immunological, endocrine and genetic processes [3]. An increasing number of gene polymorphisms and mutations have also been associated with endometriosis, but their precise role remains to be determined [4]. Endometriosis is pathohistologically a benign disease; however it is characterized by growth, proliferation, infiltration and adherence of endometrial tissue to the surrounding tissue, suggesting that endometriosis could be viewed as a neoplastic process [5]. This is further supported by evidence that the susceptibility to develop breast cancer, ovarian cancer, ovarian
* Corresponding author. Tel.: +91 40 27192504; fax: +91 40 27160311. E-mail address: [email protected] (S. Shivaji). 0301-2115/$ – see front matter ß 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ejogrb.2010.04.022
clear cell and endometroid cancer increases in the presence of endometriosis [6]. The phosphatidylinositol 3-kinase (PI3K)/Akt (vakt murine thymoma viral oncogene homologue) kinase signaling pathway plays an important role in a wide range of cellular processes associated with malignant behavior including inhibition of apoptosis, cellular growth, proliferation, cell motility, adherence, transformation, angiogenesis and survival [7,8]. PI3K is a family of lipid kinases that exists as heterodimer consisting of catalytic subunits (p110a, p110b or p110d) and regulatory subunits (p85a, p85b or p85g). It is well established that PI3K is activated either by autocrine or paracrine stimulation of receptor tyrosine kinases. Active PI3K phosphorylates phosphotidylinositol 4,5-biphosphate (PIP2) at the 30 -position of inositol ring, converting it to phosphotidylinositol 3,4,5-triphosphate (PIP3). PIP3 recruits its downstream effector, Ser/ Thr kinase Akt and phosphoinositide dependent kinase (PDK) at the plasma membrane. Once at the membrane, Akt is phosphorylated and activated by PDK and 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 PI3KCA, the p110a catalytic subunit of PI3K, resulting in over-activation of Akt signaling [9,10]. Recently, activating somatic mutations of PI3KCA gene were identified in various human cancers particularly in breast, endometroid and clear cell ovarian cancer [11,12] and also in certain benign diseases [13]. More than 75% of these mutations are clustered in the helical domain encoded by exon 9 and the kinase domain encoded by exon 20. In addition, three hot spot mutations in
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these exons (E542K, E545K and H1047R) have been shown to activate the PI3K-Akt pathway via phosphorylation of Akt and results in in vitro transformation [9,14]. Recently, activating mutations in AKT1 have also been characterized [15]. Interestingly, AKT1 mutations have been identified in breast, ovarian and colorectal carcinoma that result in a glutamic acid to lysine substitution at amino acid 17 (E17K) in the plekstrin homology (PH) domain, rather than in the kinase domain [15–17]. Nevertheless, these AKT1 mutations are activating; they transform cells and induce leukemia in nude mice [15]. Recent studies have shown the up-regulation of several genes involved in PI3K/Akt signaling pathway in endometriosis, suggesting that this pathway could be involved in growth and progression of this disease [18,19]. The mechanism for the over-activation of PI3K/Akt signaling pathway in endometriosis has not been defined. Given the frequent mutation of the PI3KCA and AKT1 genes in cancers in which the PI3K/Akt signaling pathway is activated, the present study was conducted to investigate whether such mutations are responsible for the pathogenesis of endometriosis. 2. Materials and methods 2.1. Subjects All the women were non-smokers of South Indian origin, and were recruited at the Infertility Institute and Research Centre, Hyderabad, India. Eutopic and ectopic endometrial tissue samples were collected from 30 women with moderate-severe (I–IV) endometriosis, staged using the revised American Fertility Society (rAFS) classification system [20]. All the women had a transvaginal ultrasound scan (TVS) at screening followed by laparoscopy to confirm the diagnosis (rAFS I–3; II-5; III-3; IV-19). Their mean age was 27.5 4.4 (range 20–35) years. All the patients had different forms of endometriosis such as peritoneal lesions, adhesions and endometriomas. Thirty women with normal menstrual cycle, normal hormonal profiles and no uterine abnormalities constituted the control sample group. Their mean age was 26.7 3.9 (range 22–36) years. Women with ovarian cysts, adenomyosis, ovarian cancer, and fibroids were excluded from the study. All tissue samples were collected during routine surgical procedures. The samples were frozen in liquid nitrogen and preserved at 80 8C until DNA was extracted. Written consent was obtained from all participants for the sampling of eutopic and ectopic endometrial tissue for research purposes. The Institutional Review Board of the Centre for Cellular and Molecular Biology, Hyderabad, approved the study. 2.2. Cell lines Immortalized human endometriotic epithelial cells (12-Z) and stromal cells (22-B) [21] and immortalized human endometrial surface epithelial cells (HES) [22] and stromal cells (Sht 290) [23] were also used for mutational analysis. 12-Z and 22-B cells were a generous gift from Dr. A. Starzinski-Powitz, Institut fu¨r Zellbiologie und Neurowissenschaft, Johann Wolfgang Goethe-Universita¨t, Frankfurt, Germany. HES cells were a generous gift from Dr. Asgerally T. Fazleabas, University of Illinois at Chicago, Illinois, USA. Sht 290 cells were a generous gift from David G. Kaufman, University of North Carolina at Chapel Hill, North Carolina, USA. The endometriotic epithelial and stromal cells were cultured in DMEM (Invitrogen, Carlsbad, California, USA) containing 10% FBS (Invitrogen) and normal endometrial epithelial and stromal cells were maintained in phenol red free DMEM (Invitrogen) containing 10% and 2% charcoal treated FBS (Invitrogen), respectively. The media also contained penicillin (100 U/ml) and streptomycin (100 g/ml) (Invitrogen). Cells were maintained in a humidified atmosphere of 5% CO2 and 95% air at 37 8C.
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2.3. DNA extraction Genomic DNA was extracted from eutopic and ectopic endometrium and from cell lines by using DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA, USA). 2.4. Primers and polymerase chain reaction Screening of mutations in exon 9 and 20 of PI3KCA gene was done by PCR and sequencing analysis. Exons 9 and 20 of PIK3CA were individually amplified from genomic DNA with primers complementary to surrounding intronic sequences. PCRs were carried out in a total volume of 10 ml, containing 10 ng genomic DNA, 0.5 pmol of each primer, IX Taq polymerase buffer (1.5 mM MgCl2) and 0.1 U of Amplitaq DNA polymerase (Perkin-Elmer, Foster City, USA). The primers for exon 9 and 20 were 50 -TGAAAATGTATTTGCTTTTTCTGT-30 (forward), and 50 TGTAAATTCTGCTTTATTTATTCC-30 (reverse), and 50 -CATTTGCTCCAAACTGACCA-30 (forward) and 50 -GGTCTTTGCCTGCTGAGAGT-30 (reverse), respectively, using the method described by Campbell et al. [11]. PCR amplification was performed in a programmable thermal cycler gradient PCR system (Eppendorf AG, Hamburg, Germany). The PCR amplification for exon 9 was carried out for 30 cycles (initial denaturation at 95 8C for 10 min, denaturation at 95 8C for 1 min, annealing for 1 min at 54 8C, extension at 72 8C for 1 min and final extension for 10 min at 72 8C). For exon 20, after an initial denaturation step of 95 8C for 10 min, a ‘‘touch-down’’ program was used consisting of two cycles of amplification at annealing temperatures of 63–59 8C; followed by 30 amplification cycles at an annealing temperature of 58 8C and a final extension cycle of 72 8C for 5 min. The PCR condition and the PCR primer for exon 4 of AKT1 were done as described earlier by Shoji et al. [24]. PCR products were analysed using 1.2% agarose gel electrophoresis followed by ethidium bromide staining and ultraviolet visualization and then sequenced with a Taq-Dye deoxy-terminator cycle sequencing kit (Applied BioSystems, Foster City, USA) using an automated ABI 3770 sequencer (Applied BioSystems, Foster City, USA). 3. Results 3.1. PI3KCA mutation in endometriosis Most of the PI3KCA mutations are present in two small clusters in the helical domain from exon 9 and in the kinase domain from exon 20 [9]. All these mutations are missense resulting in amino acid changes in the protein, thus causing aberrant activation of PI3K and of the downstream kinase, Akt. The most common are the transversion mutations of the nucleotide 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 [9]. To investigate the frequency of PI3KCA mutations in South Indian women having endometriosis, we amplified and sequenced these exons by PCR. Shown in Fig. 1A and C are the sequences of endometriosis samples, which are negative for the reported mutations in exon 9 and 20. However, in the breast cancer cell line, MCF-7, which was used as a positive control, the G1633A mutation in exon 9 is clearly visible (Fig. 1B). 3.2. AKT1 mutations in endometriosis The sequencing analysis for exon 4 of the human AKT1 gene in 30 endometriosis samples revealed the absence of the point mutation G > A at nucleotide 49 (E17K) (Fig. 1D) which was first identified by Carpten et al. [15]. This result suggests that E17K
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Fig. 1. Mutational analysis of exon 9 and 20 of PIK3CA and exon 4 of AKT1 gene following PCR amplification and sequencing. Representative human endometrium tissue sample from women having endometriosis showing wild alleles G, A, G, C and A at positions 1633, 1634, 1635, 1636 and 1637, respectively in exon 9 of the PIK3CA gene (A), C3139, A3140 and G3145 in exon 20 of the PIK3CA gene (C), and G49 in exon 4 of the AKT1 gene (D). MCF-7 cell line which was used as a control shows, G1633A mutation in exon 9 of the PI3KCA gene (B).
Table 1 PIK3CA and AKT1 mutations in immortalized human endometrial surface epithelial cells (HES) and stromal cells (Sht 290) and immortalized human endometriotic epithelial cells and stromal cells. Cell type
PI3KCA exon 9 mutation
PI3KCA exon 20 mutation
AKT1 exon 4 mutation
HES cells Sht 290 cells Endometriotic epithelial cells Endometriotic stromal cells
Absent Absent Absent Absent
Absent Absent Absent Absent
Absent Absent Absent Absent
transformation mutation of the AKT1 gene may be a rare event in the development of endometriosis in the South Indian women. 3.3. PI3KCA and AKT1 mutational analysis in human normal and endometriotic cell lines We also analysed mutations in exon 9 and 20 of PI3KCA gene and exon 4 of AKT1 gene in immortalized human endometriotic epithelial cells (12-Z) and stromal cells (22-B) and immortalized human endometrial surface epithelial cells (HES) and stromal cells (Sht 290) by PCR amplification and sequencing analysis. Both PI3KCA and AKT1 mutations were found to be absent in both endometriotic and endometrial cell lines (Table 1). 4. Comment Endometriosis is a polygenically inherited disease with multifactorial pathogenesis. Several case–control studies have been done to assess whether association exists between endometriosis and genes involved in inflammation, steroid hormone production and detoxification [4]. These genes play a critical role in the pathophysiology of the disease. In the present study, mutations in exon 9 and 20 of PI3KCA gene, which codes for the catalytic subunit of PI3K and in exon 4 of AKT1 gene, were investigated to ascertain whether these mutations are associated with endometriosis in South Indian women.
Numerous genetic and functional studies have clearly established the involvement of the PI3K-Akt pathway in the development of neoplasia. The PI3K-Akt pathway is deregulated in human cancers at several levels. The tumour suppressor PTEN, which dephosphorylates PIP3 to PIP2, thus antagonizing PI3K activity, is deleted or mutated in several cancers [25,26] and amplification of the genomic region containing AKT or PI3K genes has also been reported [11,27,28]. PI3KCA, which codes for the catalytic subunit of PI3K and AKT1 are somatically mutated at significant frequencies in many cancers. These studies suggest an oncogenic role for PI3KCA and AKT1. PI3KCA mutation has also been reported in benign or pre-neoplastic conditions such as epidermal nevi and seborrheic keratoses [13], which suggest that in addition to their role in cancer, oncogenic PIK3CA mutations contribute to the pathogenesis of benign diseases. PI3KCA is located on chromosome 3q26.32 and consists of 20 exons. Interestingly, however, more than 75% of the mutations occur in two small clusters in the helical (exon 9) and kinase domain (exon 20). A recent report by Carpten et al. [15] identified a transforming E17K mutation in PH domain of AKT1. Interestingly, the E17K-AKT1 transforming mutation resulted in not only an increased level of Akt phosphorylation but also of PIP2, and the PIP3-independent recruitment of Akt1 to the cell membrane. This recurring mutation in a regulatory domain of AKT1 leads to structural alteration of the ligand-binding site, pathological membrane association, constitutive activation of the kinase, in vitro transformation, soft agar colony formation and
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leukemia in mice. Functional studies on PI3KCA mutants show attenuation of apoptosis and enhancement of growth, invasion and metastasis [29]. These observations reflect the importance of PI3KCA and AKT1 mutations in the progression of cancer. Our study showed that the most common activating PI3KCA mutation (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) and transforming AKT1 (nucleotide G49A) mutations, which are reported to be extremely common in endometriod, ovarian, breast and clear cell ovarian cancer, with 45% harboring one or other alteration, were not present in South Indian women with rAFS stage I–IV endometriosis. We further analysed the human endometriotic epithelial cells and stromal cells for the presence of PI3KCA and AKT1 mutations, which were derived from a European population, to investigate whether these mutations are responsible for the pathogenesis of endometriosis. We observed that these mutations are even absent in the endometriotic cell lines. This result suggests that some other mechanisms are involved in the activation of the PI3K/AKT pathway in endometriosis. PI3KCA and AKT1 mutations were also studied in immortalized human endometrial surface epithelial cells and stromal cells and as anticipated the mutations were absent in these cell lines as they were derived from normal endometrium. Our data are further supported by report of Tanaka et al. [30], where they studied PI3KCA mutation in hepatocellular carcinoma in a Japanese population and found that PI3KCA is not somatically mutated in this tumour lineage. Further reports by Kim et al. [17] and Reiner et al. [31] in Korean and European populations, respectively, showed absence of AKT1 mutation in hepatocellular carcinoma, suggesting that alternative mechanisms might be involved in the activation of the PI3K/Akt pathway in malignant diseases. The PI3k/Akt pathway is activated by estrogen in uterine endometrium and upon activation it regulates many physiological processes such as blastocyst attachment and implantation, regulation of trophoblast invasion, immune surveillance and efficient disposal of blood and desquamated cellular debris with menstruation [32]. This is further supported by the evidence that rapid activation of intracellular signaling cascades ERK1/2 (extracellular signal-regulated kinases) and PI3K/Akt by growth factors and estrogens are involved in the migration of normal endometrial stromal cells [33]. Given that endometriosis is an estrogen-dependent disease [34], the PI3K/Akt pathway appears to have a role in disease development and progression. Recent studies by Matsuzaki et al. [18] and Wu et al. [19], using complementary DNA microarray and laser capture microdissection, revealed an upregulation of PI3K-Akt and several other genes involved in this pathway, in epithelial and stromal cells from deep endometriosis compared with their matched eutopic endometrium. This finding further support the importance of the PI3K/Akt pathway in endometriosis. Absence of PI3KCA and AKT1 mutation raises the question as to how the over-activation or up-regulation of the PI3K-Akt signaling pathway occurs in endometriosis. Alternative mechanisms responsible for the activation of the PI3K-Akt signaling pathway presumably exist in this disease. One such mechanism may involve the amplification of PI3KCA or AKT1 gene as reported in several human cancers including ovarian cancer [35]. It is also possible that absence of these mutations in our study may be attributed due to different geographical origin of the patients, variation in risk factors and the methods used for DNA mutational analysis. In conclusion, our result suggests that mutation in PI3KCA and AKT1 genes is absent in South Indian women with endometriosis. Other alternative mechanisms for the activation of the PI3K/Akt
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signaling pathway in endometriosis remain to be further investigated. Conflicts of interest The authors have declared no conflict of interest. Acknowledgments Priyanka Rai is the recipient of CSIR fellowship from Government of India. We thank Dr. Archana B. Siva, Venkatesh Kota and Y. Kameshwari for their valuable suggestions and technical help. References [1] Eskenazi B, Warner ML. Epidemiology of endometriosis. Obstet Gynecol Clin North Am 1997;24:235–58. [2] Sampson JA. Peritoneal endometriosis due to menstrual dissemination of endometrial tissue into peritoneal cavity. Am J Obstet Gynecol 1927;14: 422–69. [3] Bulun SE. Endometriosis. N Engl J Med 2009;360:268–79. [4] Montgomery GW, Nyholt DR, Zhao ZZ, et al. The search for genes contributing to endometriosis risk. Hum Reprod Update 2008;14:447–57. [5] Varma R, Rollason T, Gupta JK, Maher ER. Endometriosis and the neoplastic process. Reproduction 2004;127:293–304. [6] Melin A, Spare´n P, Bergqvist A. The risk of cancer and the role of parity among women with endometriosis. Hum Reprod 2007;22:3021–6. [7] Vivanco I, Sawyers CL. The phosphatidylinositol 3-kinase-AKT pathway in human cancer. Nat Rev Cancer 2002;2:489–501. [8] Brader S, Eccles SA. Phosphoinositide 3-kinase signalling pathways in tumor progression, invasion and angiogenesis. Tumori 2004;90:2–8. [9] Samuels Y, Wang Z, Bardelli A, et al. High frequency of mutations of the PIK3CA gene in human cancers. Science 2004;304:554. [10] Altomare DA, Testa JR. Perturbations of the AKT signaling pathway in human cancer. Oncogene 2005;24:7455–64. [11] Campbell IG, Russell SE, Choong DY, et al. Mutation of the PIK3CA gene in ovarian and breast cancer. Cancer Res 2004;64:7678–81. [12] Velasco A, Bussaglia E, Pallares J, et al. PIK3CA gene mutations in endometrial carcinoma. Correlation with PTEN and K-RAS alterations. Hum Pathol 2006;37:1465–72. [13] Hafner C, Lo´pez-Knowles E, Luis NM, et al. Oncogenic PIK3CA mutations occur in epidermal nevi and seborrheic keratoses with a characteristic mutation pattern. Proc Natl Acad Sci USA 2007;104:13450–4. [14] Kang S, Bader AG, Vogt PK. Phosphatidylinositol 3-kinase mutations identified in human cancer are oncogenic. Proc Natl Acad Sci USA 2005;102:802–7. [15] Carpten JD, Faber AL, Horn C, et al. A transforming mutation in the pleckstrin homology domain of AKT1 in cancer. Nature 2007;448:439–44. [16] Bleeker FE, Felicioni L, Buttitta F, et al. AKT1E17K in human solid tumours. Oncogene 2008;27:5648–50. [17] Kim MS, Jeong EG, Yoo NJ, Lee SH. Mutational analysis of oncogenic AKT E17K mutation in common solid cancers and acute leukaemias. Br J Cancer 2008;98:1533–5. [18] Matsuzaki S, Canis M, Vaurs-Barriere C, Boespflug-Tanguy O, Dastugue B, Mage G. DNA microarray analysis of gene expression in eutopic endometrium from patients with deep endometriosis using laser capture microdissection. Fertil Steril 2005;84:1180–90. [19] Wu Y, Kajdacsy-Balla A, Strawn E, et al. Transcriptional characterizations of differences between eutopic and ectopic endometrium. Endocrinology 2006;147:232–46. [20] American Fertility Society. Classification of endometriosis. Fertil Steril 1979;32:633–45. [21] Zeitvogel A, Baumann R, Starzinski-Powitz A. Identification of an invasive, Ncadherin-expressing epithelial cell type in endometriosis using a new cell culture model. Am J Pathol 2001;159:1839–52. [22] Desai NN, Kennard EA, Kniss DA, Friedman CI. Novel human endometrial cell line promotes blastocyst development. Fertil Steril 1994;61:760–6. [23] Barbier CS, Becker KA, Troester MA, Kaufman DG. Expression of exogenous human telomerase in cultures of endometrial stromal cells does not alter their hormone responsiveness. Biol Reprod 2005;73:106–14. [24] Shoji K, Oda K, Nakagawa S, et al. The oncogenic mutation in the pleckstrin homology domain of AKT1 in endometrial carcinomas. Br J Cancer 2009;101:145–8. [25] Li J, Yen C, Liaw D, et al. PTEN, a putative protein tyrosine phosphatase gene mutated in human brain, breast and prostate cancer. Science 1997;275: 1943–7. [26] Sansal I, Sellers WR. The biology and clinical relevance of the PTEN tumor suppressor pathway. J Clin Oncol 2004;22:2954–63. [27] Bellacosa A, de Feo D, Godwin AK, et al. Molecular alterations of the AKT2 oncogene in ovarian and breast carcinomas. Int J Cancer 1995;64:280–5. [28] Wu G, Mambo E, Guo Z, et al. Uncommon mutation, but common amplifications, of the PIK3CA gene in thyroid tumors. J Clin Endocrinol Metab 2005;90:4688–93. [29] Samuels Y, Diaz Jr LA, Schmidt-Kittler O, et al. Mutant PIK3CA promotes cell growth and invasion of human cancer cells. Cancer Cell 2005;7:561–73.
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