Oncogene (2015) 34, 2471–2482 © 2015 Macmillan Publishers Limited All rights reserved 0950-9232/15 www.nature.com/onc ORIGINAL ARTICLE Loss of Cdh1 and Trp53 in the uterus induces chronic inflammation with modification of tumor microenvironment GR Stodden1,5, ME Lindberg1,5, ML King1, M Paquet2, JA MacLean1, JL Mann3, FJ DeMayo4, JP Lydon4 and K Hayashi1 Type II endometrial carcinomas (ECs) are estrogen independent, poorly differentiated tumors that behave in an aggressive manner. As TP53 mutation and CDH1 inactivation occur in 80% of human endometrial type II carcinomas, we hypothesized that mouse uteri lacking both Trp53 and Cdh1 would exhibit a phenotype indicative of neoplastic transformation. Mice with conditional ablation of Cdh1 and Trp53 (Cdh1d/dTrp53d/d) clearly demonstrate architectural features characteristic of type II ECs, including focal areas of papillary differentiation, protruding cytoplasm into the lumen (hobnailing) and severe nuclear atypia at 6 months of age. Further, Cdh1d/dTrp53d/d tumors in 12-month-old mice were highly aggressive, and metastasized to nearby and distant organs within the peritoneal cavity, such as abdominal lymph nodes, mesentery and peri-intestinal adipose tissues, demonstrating that tumorigenesis in this model proceeds through the universally recognized morphological intermediates associated with type II endometrial neoplasia. We also observed abundant cell proliferation and complex angiogenesis in the uteri of Cdh1d/dTrp53d/d mice. Our microarray analysis found that most of the genes differentially regulated in the uteri of Cdh1d/dTrp53d/d mice were involved in inflammatory responses. CD163 and Arg1, markers for tumor-associated macrophages, were also detected and increased in the uteri of Cdh1d/dTrp53d/d mice, suggesting that an inflammatory tumor microenvironment with immune cell recruitment is augmenting tumor development in Cdh1d/dTrp53d/d uteri. Further, inflammatory mediators secreted from CDH1-negative, TP53 mutant endometrial cancer cells induced normal macrophages to express inflammatory-related genes through activation of nuclear factor-κB signaling. These results indicate that absence of CDH1 and TP53 in endometrial cells initiates chronic inflammation, promotes tumor microenvironment development following the recruitment of macrophages and promotes aggressive ECs. Oncogene (2015) 34, 2471–2482; doi:10.1038/onc.2014.193; published online 7 July 2014 INTRODUCTION and harmful cellular phenotypes that are characteristic of cancer Endometrial cancer is the most common malignancy of the female cells, such as resistance to apoptosis, neoangiogenesis and genital tract, affecting 452000 women and leading to approxi- enhanced proliferative and invasive potential. Approximately mately 8500 deaths in the United States each year.1 Two types of 80% of T2ECs harbor TP53 mutations. Although TP53 mutations endometrial carcinomas (ECs) have been distinguished, type I are less common in T1ECs, those reported have been largely 8 (T1EC) and type II (T2EC).2 T1ECs, which account for 80% of all ECs, confined to high-grade tumors (grades 3 and 4). In addition to are associated with hyperestrogenism, have a good prognosis, TP53 mutation, inactivation of CDH1 is also a common molecular exhibit atypical hyperplasia and usually develop in premenopausal feature in T2ECs.4,10 CDH1 is critical in the establishment of cell and peri-menopausal women.3,4 T2ECs are high-grade, estrogen- polarity and maintenance of the epithelial phenotype.13 CDH1 is – independent and diagnosed mainly in postmenopausal women. often downregulated or lost during tumor progression,14 17 – Although only 15–20% of ECs belong to T2ECs, these tumors leading to increased tumor invasiveness and metastasis.4,18 22 appear to be very aggressive, and 450% of patients present with Mice with either Trp53 heterozygous or homozygous deletion recurrent disease shortly after primary treatment.5–7 Further, develop a variety of cancers, with most homozygous mice dying o30% of the patients with T2ECs survive 5 years after initial by 6 months due to development of widespread lymphoma, but – diagnosis.5 Therefore, a clear understanding of the mechanisms Trp53-null mice rarely form ECs.23 25 PgrCre/+ mice have been involved in tumor development of T2ECs is necessary for early recognized as an excellent model to target genes in the uterus stage diagnosis as well as rational design of therapies. after birth.26 Although conditional uterine ablation of Pten driven T2ECs are poorly differentiated, aggressive tumors.8–10 The by Pgr-Cre results in the development of T1ECs in mice,27,28 the most common histological tumor type is serous carcinoma uteri of mice lacking Trp53 alone do not exhibit any abnormal comprised of atypical cells that grow into papillary, glandular or morphology by 5 months.27 We have recently reported that solid tumors.8,9,11 A number of studies have reported that TP53 conditional ablation of Cdh1 in the mouse uterus results in a mutations are associated with poor prognosis.4,8,12 Inactivation of disorganized cellular structure of the epithelium and ablation of TP53 renders cells non-responsive to signals that challenge endometrial glands, leading to implantation defects.29 However, genomic integrity, thereby promoting the acquisition of novel loss of Cdh1 alone in the uterus does not predispose 1Department of Physiology, Southern Illinois University School of Medicine, Carbondale, IL, USA; 2Departement de Pathologie et de Microbiologie, Université de Montreal, St-Hyacinthe, Quebec, Canada; 3Department of Pathology, Southern Illinois University School of Medicine, Springfield, IL, USA and 4Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX, USA. Correspondence: Dr K Hayashi, Department of Physiology, Southern Illinois University School of Medicine, 1135 Lincoln Dr, Carbondale, IL 62901, USA. E-mail: [email protected] 5These authors contributed equally to this work. Received 20 November 2013; revised 5 May 2014; accepted 28 May 2014; published online 7 July 2014 The impact of Cdh1 and Trp53 ablation in the mouse uterus GR Stodden et al 2472 mice to tumors. Conditional ablation of Cdh1 does not induce Cdh1d/dTrp53d/d mice was significantly reduced (Po0.0001, – tumors in mammary glands30 32 or stomach,33 whereas loss Figure 1k). Loss of both Cdh1 and Trp53 affected survival as early of Cdh1 and Trp53 induces invasive lobular carcinoma in as 5.5 months, and half of Cdh1d/dTrp53d/d mice died or reached mammary glands with massive angiogenesis.31,32 Thus, these tumor burden euthanasia criteria by 12 months. Although the results indicate that single gene ablation in the uterus is not ovariectomized model is the most appropriate to study T2EC, we sufficient to understand the etiology of heterogeneous aggressive also examined mice with intact ovaries (Supplementary Figure S3). types of ECs. We observed massive fluid accumulation in the uterine lumen of In the present study, we generated a mouse model in Cdh1d/d, Cdh1d/dTrp53d/+ and Cdh1d/dTrp53d/d mice at 6 months. which Cdh1 and Trp53 were conditionally ablated in the uterus. This phenotype did not affect survival time until 6 months but Ablation of Trp53 and Cdh1 accelerated endometrial neoplastic caused thin uterine layers without any carcinogenesis. Although transformation and induced cell invasion and dissemination. estrogen induces engorging of the uterus at estrus, the uterus Further, the results of the present study suggest that ablation of metabolizes substances/fluids and recovers to normal during the Cdh1 and Trp53 in the mouse uterus initiates chronic inflammation estrous cycle. Our previous study demonstrated that loss of Cdh1 with tumor microenvironment modification, which promotes causes abnormal uterine development and infertility.29 Thus loss aggressive ECs. of Cdh1 in the uterine epithelium causes abnormal endometrial function and accumulation of fluids in the uterine lumen. Because RESULTS the intact model did not result in any carcinogenesis, we excluded it from further analysis. Generation of mice with Cdh1 and Trp53 ablation in the mouse Histological analysis demonstrated that single ablation of either uterus Trp53 or Cdh1 in the uterus at 12 months did not result in any Because TP53 mutation and CDH1 inactivation are the two most abnormalities (Figures 2a–c). In contrast, Cdh1d/dTrp53d/d uteri common found molecular features in human T2ECs,3,4 our showed abnormal histology starting at 2 months, including objective was to study the combined effect of dysfunctional disorganized epithelium with cellular budding like papillae uterine TRP53 and CDH1. As Cdh1-null mice show early embryonic (Figure 2d) and frequent invasion into myometrium (Figure 2e). lethality,34,35 we conditionally deleted both Cdh1 and Trp53 in the Our subsequent studies examining 6- and 12-month Cdh1d/d uterus using PgrCre/+ mice. PgrCre/+ mice were crossed with Cdh1f/f Trp53d/d mice revealed poorly differentiated high-grade nuclear and/or Trp53f/fmice to provide a tissue-specific knockout of Cdh1 features with atypia (Figures 2f–h), which are characteristics of and/or Trp53 in Pgr-expressing cells: Pgr+/+Cdh1f/fTrp53f/f = control, T2ECs. Further, tumors exhibited architectural features of PgrCre/+Cdh1f/fTrp53+/+ = Cdh1d/d, PgrCre/+Cdh1+/+Trp53f/f = Trp53d/d, T2ECs, including focal areas of papillary differentiation Cre/+ f/f f/+ d/d d/+ Cre/+ f/+ f/f Pgr Cdh1 Trp53 = Cdh1 Trp53 , Pgr Cdh1 Trp53 = (Figure 2i), protruding cytoplasm into the lumen (hobnailing) d/+ d/d Cre/+ f/f