Early aberrant insulin-like growth factor signaling in the progression to endometrial carcinoma is augmented by tamoxifen Pamela L. Strissel 1 , Stephan Ellmann 1 , Elke Loprich 1 , Falk Thiel 1 , Peter A. Fasching 1 , Elisabeth Stiegler 1 , Arndt Hartmann 2 , Matthias W. Beckmann 1 and Reiner Strick 1 * 1 Department of Gynaecology and Obstetrics, Laboratory for Molecular Medicine, University-Clinic Erlangen, Erlangen, Germany 2 Institute for Pathology, University-Clinic Erlangen, Krankenhausstr. 12, 91054 Erlangen, Germany Tamoxifen is an important selective estrogen receptor (ER) mod- ulator for treatment of steroid hormone positive breast cancer. In addition to the beneficial effect, tamoxifen is one risk factor for endometrial carcinoma (EnCa) development. We hypothe- sized that, (1) dysregulation of gene expression and protein phos- phorylation of the insulin-like growth factor (IGF) and steroid hormone receptor-signaling occur early in benign endometrial tissues and (2) signaling differences would be detected between patients with or without tamoxifen treatment. Seventy-eight tis- sues, including 2 benign cohorts from patients treated with (n 5 24) or without tamoxifen (n 5 28) (hyperproliferative endome- trium, hyperplasia, polyps), EnCa (n 5 12) with endometrium controls (n 5 14) were analyzed for expression of 15 genes from the IGF and steroid hormone receptor-signaling, including the target genes Syncytin-1, PAX2 and c-myc. Total and phosphoryl- ated protein expression were examined for ERa, PTEN, AKT, mTOR and Syncytin-1. Compared to controls similar significant deregulation of IGF and steroid hormone receptor-signaling, Syncytin-1 and PAX2 occurred in both benign cohorts, irrelevant of tamoxifen treatment. Comparing both benign cohorts with and without tamoxifen significant expression differences were noted. Increased total protein and phosphorylation of pERa- Ser118, pPTEN-Thr380, pAKT-Thr308, pAKT-Ser473, pmTOR- Ser2448 and Syncytin-1 were noted in early benign tissue stages associating with tamoxifen, especially polyps. Functional kinetic studies following tamoxifen treatment of the PTEN mutated RL95-2 EnCa cell line, demonstrated a doubling of phosphoryla- tion of pERa-Ser118 and a 4.2-fold induction of pAKT-Thr308 along with Syncytin-1 induction. This study supports that dysre- gulated IGF and steroid hormone receptor signaling is promi- nent in endometrial benign stages and these alterations could represent clinical indicators for the risk of EnCa and also help in development of new therapies. ' 2008 Wiley-Liss, Inc. Key words: endometrial carcinoma; tamoxifen; IGF; Syncytin-1; PAX2 Endometrial carcinoma (EnCa) primarily occurs in postmeno- pausal women and represents the 7th most common malignant dis- order. 1 The estimated incidence and mortality rate for EnCa was recently reported at 33.5 and 9.3 per 100,000 in Europe, respec- tively. 2 The mean age-adjusted 5-year survival for EnCa in Europe was also calculated at 78%, 1 percent below the female breast can- cer rate at 79%. 3 According to the International Federation of Gynaecology and Obstetrics, EnCa is staged according to tumor location (Ia is endometrial to IVb with distant metastasis) and graded according to the growth pattern (G1–3). More than 85% of all EnCa cases are histologically classified as endometrioid (type I), mainly expressing steroid hormone receptors. The rare, but more aggressive nonendometrioid EnCa (type II) often lacks ER and PR expression and may develop directly from transformed en- dometrial surface epithelium. 4 Estrogens are considered to act as tumor promoters in the development of endometrioid EnCa by inducing endometrial progression from a hyperproliferative state to hyperplasia and then progressing to EnCa. 5 Two percent of complex hyperplasias, but 52% of atypical hyperplasias can pro- gress into EnCa. 6,7 A study analyzing 513 endometrial polyps determined that 66 (13%) were malignant and approximately 3- fold higher in the age group older than 65. 8 The most important risk factors in the development of EnCa include the following: (i) a body mass index above 30 kg/m 2 , which triples the risk for EnCa 1 ;(ii) unopposed estrogen replace- ment therapy, which increases the EnCa risk 2.3-fold for post- menopausal women 9 and (iii) selective estrogen receptor modula- tors (SERMs), like tamoxifen used for adjuvant therapy of breast cancer patients, where the EnCa risk was increased 2.0- and 6.9- fold with treatments of 2.5 and 5 years, respectively. 10–15 In addi- tion, conflicting reports have also been observed for a possible association between tamoxifen and higher staging and poor prog- nosis of EnCa and the development of rare malignant mixed meso- dermal tumors and sarcomas of the endometrium and gastrointesti- nal and colorectal cancers. 10–16 Interestingly, tamoxifen therapy also correlated with hyperproliferative endometrium characterized with increased endometrial thickness and occurrence of endome- trial polyps. 17,18 In a study involving 700 breast cancer patients treated with tamoxifen, benign endometrial changes were observed in 38.85%, including 23.14% polyps, 8% hyperplasia, 3% metaplasia and 4.71% EnCa. Of these polyps 10% demon- strated a carcinoma in situ. 19 Common DNA alterations in endometrioid EnCa include increased microsatellite instability, due to defects in mismatch repair genes and gene mutations in the tumor suppressor and lipid phosphatase PTEN, whereas non-endometrioid EnCa demon- strated chromosome aneuploidy and p53 mutations. 20 Other DNA alterations detected in EnCa included gene mutations in b-catenin 1, K-ras, and amplifications of c-myc and INT2. Many of the above DNA mutations were also identified in EnCa from patients treated with tamoxifen. 21 Ferguson et al. 22 determined that there were no differences in RNA expression between EnCa from tamoxifen and non-tamoxifen treated patients using gene chip analysis. Impor- tantly, specific pathways have been implicated with the develop- ment of EnCa, like IGF, insulin and steroid hormone signaling. 1 IGF signaling is a protein phosphorylation cascade regulating growth. 23 IGF1 and 2 share 62% homology to each other and 40% homology to proinsulin in amino acid sequence. The cellular actions of IGF1 & 2 can be modulated by interaction with a family of 6 IGF-binding proteins, IGFBP1 to IGFBP6. More than 90% of circulating IGF1 is complexed to IGFBP3. 24 The IGF1 receptor (IGF1R) belongs to the family of receptor-tyrosine-kinases where upon ligand binding of IGF1, IGF2 or insulin the receptor kinase Abbreviations: EnCa, endometrial carcinoma; ER, estrogen receptor; IGF, insulin-like growth factor; IGF1R, insulin-like growth factor 1 recep- tor; IGFBP, insulin-like growth factor binding protein; mTOR, mammalian target of Rapamycin; PAX2, paired box gene 2; PR, progesterone receptor; PTEN, phosphatase and tensin homolog. The first two authors contributed equally to this paper. Grant sponsor: Interdisciplinary Centre for Clinical Research (IZKF), University of Erlangen-Nuremberg. *Correspondence to: Department of Gynaecology and Obstetrics, Lab- oratory for Molecular Medicine, University-Clinic Erlangen, Universi- taetsstr 21-23, D-91054 Erlangen, Germany. Fax: +49-9131-85-36670. E-mail: reiner.strick@uk-erlangen.de Received 29 January 2008; Accepted after revision 16 July 2008 DOI 10.1002/ijc.23900 Published online 23 September 2008 in Wiley InterScience (www.interscience. wiley.com). Int. J. Cancer: 123, 2871–2879 (2008) ' 2008 Wiley-Liss, Inc. Publication of the International Union Against Cancer