Impact of oviductal versus ovarian epithelial cell of origin on ovarian endometrioid carcinoma phenotype in the mouse

Endometrioid carcinoma (EC) is a relatively indolent ovarian carcinoma subtype that is nonetheless deadly if detected late. Existing genetically engineered mouse models (GEMMs) of the disease, based on transformation of the ovarian surface epithelium (OSE), take advantage of known ovarian EC driver gene lesions, but do not fully recapitulate the disease features seen in patients. An EC model in which the Apc and Pten tumour suppressor genes are conditionally deleted in murine OSE yields tumours that are biologically more aggressive and significantly less differentiated than human ECs. Importantly, OSE is not currently thought to be the tissue of origin of most ovarian cancers, including ECs, suggesting that tumour initiation in Müllerian epithelium may produce tumours that more closely resemble their human tumour counterparts. We have developed Ovgp1‐iCreER^T2 mice in which the Ovgp1 promoter controls expression of tamoxifen (TAM)‐regulated Cre recombinase in oviductal epithelium – the murine equivalent of human Fallopian tube epithelium. Ovgp1‐iCreER^T2;Apc^fl/fl;Pten^fl/fl mice treated with TAM or injected with adenovirus expressing Cre into the ovarian bursa uniformly develop oviductal or ovarian ECs, respectively. On the basis of their morphology and global gene expression profiles, the oviduct‐derived tumours more closely resemble human ovarian ECs than do OSE‐derived tumours. Furthermore, mice with oviductal tumours survive much longer than their counterparts with ovarian tumours. The slow progression and late metastasis of oviductal tumours resembles the relatively indolent behaviour characteristic of so‐called Type I ovarian carcinomas in humans, for which EC is a prototype. Our studies demonstrate the utility of Ovgp1‐iCreER^T2 mice for manipulating genes of interest specifically in the oviductal epithelium, and establish that the cell of origin is an important consideration in mouse ovarian cancer GEMMs. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Alterations in Wnt–β‐catenin and Pten signalling play distinct roles in endometrial cancer initiation and progression

Endometrioid endometrial cancer arises through a gradual series of histological changes, each accompanied by specific alterations in gene expression and activity. Activation of the Wnt–β‐catenin pathway and loss of PTEN activity are frequently observed in endometrial cancers. However, the specific roles played by alterations in these pathways in the initiation and progression of endometrial cancer are currently unclear. Here, we investigated the effects of loss of Pten and Apc gene function in the mouse endometrium by employing tissue‐specific and inducible mutant alleles, followed by immunohistochemical (IHC) and loss of heterozygosity (LOH) analysis of their corresponding cancerous lesions. Loss of the Apc function in the endometrium leads to cytoplasmic and nuclear β‐catenin accumulation in association with uterine hyperplasia and squamous cell metaplasia, but without malignant transformation. Loss of Pten function also resulted in squamous metaplasia but, in contrast to loss of Apc function, it initiates endometrial cancer. On the other hand, loss of Apc function in the endometrium accelerates Pten‐driven endometrial tumourigenesis. Analysis of compound heterozygous mice confirmed that somatic loss of the wild‐type Pten allele represents the rate‐limiting initiation step in endometrial cancer. Simultaneous loss of Pten and Apc resulted in endometrial cancer characterized by earlier onset and a more aggressive malignant behaviour. These observations are indicative of the synergistic action between the Wnt–β‐catenin and Pten signalling pathways in endometrial cancer onset and progression. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Three‐dimensional nuclear telomere architecture changes during endometrial carcinoma development

Endometrioid or type‐I endometrial carcinoma (EC) develops from hyperproliferative glandular pathologies. Inactivation of the tumor suppressor gene PTEN is frequently associated with type‐I EC. Using a previously characterized Pten heterozygous (Pten+/‐) mouse model, this study investigates the three‐dimensional (3D) telomere profiles during progression from hyperplastic lesions to EC to test the hypothesis that altered 3D telomere profiles can be detected prior to Pten loss in early hyperproliferative lesions. We used immunohistochemistry and 3D‐telomere fluorescent in‐situ hybridization to investigate Pten expression, telomere length and signal distribution, average number and spatial distribution of telomeres and formation of telomere aggregates in uterine glandular epithelial cells from wildtype and Pten+/‐ mice. Pten showed nuclear and cytoplasmic localization in WT, predominantly cytoplasmic staining in simple hyperplasia (SH) and was markedly reduced in atypical hyperplasia (AH). Telomere length in glandular epithelial cells does not shorten with age. The average number of telomeres per nucleus was not different in WT and Pten+/‐ mice indicating the lack of substantial numeric chromosome aberrations during EC development. We observed telomere aggregates in lesions of AH and EC. SH lesions in Pten+/‐ mice differed from normal glandular epithelium by an increased relative number of shorter telomeres and by a telomere signal distribution indicative of a heterogeneous cell population. Our study revealed that alterations in the nuclear 3D telomere architecture are present in early proliferative lesions of mouse uterine tissues indicative of EC development. The changes in telomere length distribution and nuclear signal distribution precede the loss of Pten. © 2013 Wiley Periodicals, Inc.     

Whole‐exome DNA sequence analysis of Brca2‐ and Trp53‐deficient mouse mammary gland tumours

Germline mutations in the tumour suppressor BRCA2 predispose to breast, ovarian and a number of other human cancers. Brca2‐deficient mouse models are used for preclinical studies but the pattern of genomic alterations in these tumours has not yet been described in detail. We have performed whole‐exome DNA sequencing analysis of mouse mammary tumours from Blg–Cre Brca2^f/f Trp53^f/f animals, a model of BRCA2‐deficient human cancer. We also used the sequencing data to estimate DNA copy number alterations in these tumours and identified a recurrent copy number gain in Met, which has been found amplified in other mouse mammary cancer models. Through a comparative genomic analysis, we identified several mouse Blg–Cre Brca2^f/f Trp53^f/f mammary tumour somatic mutations in genes that are also mutated in human cancer, but few of these genes have been found frequently mutated in human breast cancer. A more detailed analysis of these somatic mutations revealed a set of genes that are mutated in human BRCA2 mutant breast and ovarian tumours and that are also mutated in mouse Brca2‐null, Trp53‐null mammary tumours. Finally, a DNA deletion surrounded by microhomology signature found in human BRCA1/2‐deficient cancers was not common in the genome of these mouse tumours. Although a useful model, there are some differences in the genomic landscape of tumours arising in Blg–Cre Brca2^f/f Trp53^f/f mice compared to human BRCA‐mutated breast cancers. Therefore, this needs to be taken into account in the use of this model. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Methylation profiles of hereditary and sporadic ovarian cancer

Bol G M, Suijkerbuijk K P M, Bart J, Vooijs M, van der Wall E & van Diest P J
(2010) Histopathology 57, 363–370
Methylation profiles of hereditary and sporadic ovarian cancer Aims: Tumour suppressor gene silencing through promoter hypermethylation plays an important role in oncogenesis. Carcinogenesis of hereditary cancers usually differs from that of their sporadic counterparts, but methylation has hardly been studied in hereditary ovarian cancer. The aim of this study was to investigate promoter methylation of a set of common tumour suppressor genes in BRCA1‐related ovarian cancer in comparison with sporadic ovarian cancer. Methods and results: Methylation‐specific multiplex ligation‐dependent probe amplification was used to assess the extent of promoter methylation of 24 different tumour suppressor genes in BRCA1‐associated (n = 25) and matched sporadic ovarian tumours (n = 50). A cumulative methylation index (CMI) was calculated and differences between individual genes were analysed. There was no significant difference in cumulative methylation between BRCA1‐associated and sporadic ovarian carcinomas (median CMI 108; CMI 110; P = 0.86). Also, methylation patterns of individual genes did not show distinct differences after correction for multiple comparisons. CDH13, GSTP1 and RASSF1 were frequently methylated in both sporadic and hereditary ovarian cancers. BRCA1 methylation occurred in 14% of sporadic tumours, but was not detected in BRCA1‐associated tumours. Conclusions: CDH13, GSTP1 and RASSF1 are frequently methylated in both sporadic and BRCA1‐associated ovarian cancers. Interestingly, methylation of BRCA1, while frequent in sporadic ovarian cancer, never occurred in the hereditary group. BRCA1‐associated ovarian cancers mimic their sporadic counterparts in extent and pattern of promoter methylation of several common tumour suppressor genes. This finding could have implications for future chemotherapy regimens based on epigenetic changes.     

High incidence of mammary intraepithelial neoplasia development in Men1‐disrupted murine mammary glands

Mutations of the MEN1 tumour suppressor gene predispose patients to the development of multiple endocrine neoplasia type 1 (MEN1) syndrome, which is characterized by multiple endocrine tumours, including prolactinomas. The recent findings of the interaction between menin, encoded by the MEN1 gene, and the oestrogen receptor, as well as the observation of rare cases of mammary carcinomas in our heterozygous Men1 mutant mice, led us to investigate a putative tumour suppressor function of the Men1 gene in mouse mammary cells by disrupting the gene in luminal epithelial cells. A significantly higher incidence of mammary intraepithelial neoplasia (MIN) was observed in mutant WapCre‐Men1^F/F mice (51.5%) than in WapCre‐Men1^+/+ (0%) or Men1^F/F (7.1%) control mice. The majority of MIN observed in the mutant mice displayed complete menin inactivation. Because of the leakage of WapCre transgene expression, prolactinomas were observed in 83.3% of mutant mice, leading to premature death. As there was no correlation between MIN development and elevated serum prolactin levels, and phospho‐STAT5 expression was decreased in mammary lesions, the increased incidence of MIN lesions was most likely due to Men1 disruption rather than to prolactinoma development. Interestingly, in MIN lesions, we found a decrease in membrane‐associated E‐cadherin and beta‐catenin expression, the latter of which is a menin partner. Finally, reduced menin expression was found in a large proportion of two independent cohorts of patients with breast carcinomas. Taken together, the current work indicates a role of Men1 inactivation in the development of mammary pre‐cancerous lesions in mice and a potential role in human mammary cancer.     

A tissue reconstitution model to study cancer cell‐intrinsic and ‐extrinsic factors in mammary tumourigenesis

The contribution of cancer cell‐intrinsic and ‐extrinsic factors to metastatic breast cancer is still poorly understood, hampering development of novel therapeutic strategies that decrease breast cancer mortality. Cre/loxP‐based conditional mouse models of breast cancer present unique opportunities to study sporadic tumour formation and progression in a controlled setting. Unfortunately, the generation of mouse strains carrying multiple mutant alleles needed for such studies is very time‐consuming. Moreover, conditional mouse tumour models do not permit independent manipulation of tumour cell‐intrinsic and ‐extrinsic factors. Although the latter can be achieved by cleared fat‐pad transplantation of mouse mammary epithelial cells (MMECs) from tumour suppressor gene (TSG) knockouts into wild‐type or mutant recipients, this procedure is not possible for mutations that cause embryonic lethality or preclude mammary gland development. Here we show that cleared fat‐pad transplantations with MMECs isolated from K14cre;Cdh1^F/F; Trp53^F/F mice expressing Cre recombinase under control of the cytokeratin‐14 promoter and carrying conditional null alleles for p53 and E‐cadherin (Cdh1) first resulted in the formation of phenotypically normal mammary glands, followed by the development of invasive metastatic mammary tumours. Tumour formation in the recipients mimicked tumour latency, spectrum, morphology, immunophenotype, and metastatic characteristics of the original mammary tumour model. This transplantation system, which can be expanded to other conditional TSG knockouts, permits independent genetic analysis of stromal factors and testing of additional cancer cell‐intrinsic mutations that would otherwise be embryonic lethal or require intensive breeding. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

RNF43 is a tumour suppressor gene mutated in mucinous tumours of the ovary

Mucinous carcinomas represent a distinct morphological subtype which can arise from several organ sites, including the ovary, and their genetic characteristics are largely under‐described. Exome sequencing of 12 primary mucinous ovarian tumours identified RNF43 as the most frequently somatically mutated novel gene, secondary to KRAS and mutated at a frequency equal to that of TP53 and BRAF. Further screening of RNF43 in a larger cohort of ovarian tumours identified additional mutations, with a total frequency of 2/22 (9%) in mucinous ovarian borderline tumours and 6/29 (21%) in mucinous ovarian carcinomas. Seven mutations were predicted to truncate the protein and one missense mutation was predicted to be deleterious by in silico analysis. Six tumours had allelic imbalance at the RNF43 locus, with loss of the wild‐type allele. The mutation spectrum strongly suggests that RNF43 is an important tumour suppressor gene in mucinous ovarian tumours, similar to its reported role in mucinous pancreatic precancerous cysts. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Prophylactic window therapy with the clinical poly(ADP‐ribose) polymerase inhibitor olaparib delays BRCA1‐deficient mammary tumour formation in mice

Women with heterozygous germline mutations in the BRCA1 tumour suppressor gene are strongly predisposed to developing early‐onset breast cancer through loss of the remaining wild‐type BRCA1 allele and inactivation of TP53. Although tumour prevention strategies in BRCA1‐mutation carriers are still limited to prophylactic surgery, several therapeutic strategies have been developed to target the DNA repair defects (also known as ‘BRCAness’) of BRCA1‐deficient tumours. In particular, DNA‐damaging agents such as platinum drugs and poly(ADP‐ribose) polymerase (PARP) inhibitors show strong activity against BRCA1‐mutated tumours. However, it is unclear whether drugs that target BRCAness can also be used to prevent tumour formation in BRCA1‐mutation carriers, especially as loss of wild‐type BRCA1 may not be the first event in BRCA1‐associated tumourigenesis. We performed prophylactic treatments in a genetically engineered mouse model in which de novo development of BRCA1‐deficient mammary tumours is induced by stochastic loss of BRCA1 and p53. We found that prophylactic window therapy with nimustine, cisplatin or olaparib reduced the amount and size of mammary gland lesions, and significantly increased the median tumour latency. Similar results were obtained with intermittent prophylactic treatment with olaparib. Importantly, prophylactic window therapy with nimustine and cisplatin resulted in an increased fraction of BRCA1‐proficient mammary tumours, suggesting selective survival and malignant transformation of BRCA1‐proficient lesions upon prophylactic treatment with DNA‐damaging agents. Prophylactic therapy with olaparib significantly prolonged mammary tumour‐free survival without any significant increase in the fraction of BRCA1‐proficient tumours, warranting the evaluation of this PARP inhibitor in prophylactic trials in BRCA1‐mutation carriers. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Type I to Type II Ovarian Carcinoma Progression: Mutant Trp53 or Pik3ca Confers a More Aggressive Tumor Phenotype in a Mouse Model of Ovarian Cancer

A dualistic pathway model of ovarian carcinoma (OvCA) pathogenesis has been proposed: type I OvCAs are low grade, genetically stable, and relatively more indolent than type II OvCAs, most of which are high-grade serous carcinomas. Endometrioid OvCA (EOC) is a prototypical type I tumor, often harboring mutations that affect the Wnt and phosphatidylinositol 3-kinase/AKT/mammalian target of rapamycin signaling pathways. Molecular and histopathologic analyses indicate type I and II OvCAs share overlapping features, and a subset of EOCs may undergo type I→type II progression accompanied by acquisition of somatic TP53 or PIK3CA mutations. We used a murine model of EOC initiated by conditional inactivation of the Apc and Pten tumor suppressor genes to investigate mutant Trp53 or Pik3ca alleles as key drivers of type I→type II OvCA progression. In the mouse EOC model, the presence of somatic Trp53 or Pik3ca mutations resulted in shortened survival and more widespread metastasis. Activation of mutant Pik3ca alone had no demonstrable effect on the ovarian surface epithelium but resulted in papillary hyperplasia when coupled with Pten inactivation. Our findings indicate that the adverse prognosis associated with TP53 and PIK3CA mutations in human cancers can be functionally replicated in mouse models of type I→type II OvCA progression. Moreover, the models should represent a robust platform for assessment of the contributions of Trp53 or Pik3ca defects in the response of EOCs to conventional and targeted drugs.Surgical pathologists continue to use traditional morphology-based schemes for classifying ovarian carcinomas (OvCAs). The schemes are based largely on the degree of resemblance of the OvCAs to non-neoplastic epithelia in the female genital tract. However, mounting clinicopathologic and molecular data have led Kurman and Shih¹ to propose a “dualistic” model of OvCA pathogenesis in which OvCAs are divided into two main categories, type I and type II.^1–3 Type I OvCAs are suggested to be low-grade, relatively indolent and genetically stable tumors that typically arise from recognizable precursor lesions such as endometriosis or so-called borderline (low malignant potential) tumors. Type I OvCAs frequently harbor somatic mutations (eg, KRAS, BRAF, CTNNB1, PTEN) that dysregulate specific cell signaling pathway factors or certain chromatin remodeling complexes (eg, ARID1A). Type I OvCAs include most endometrioid, clear cell, and mucinous carcinomas and low-grade serous carcinomas. In contrast, type II OvCAs are proposed to be high-grade, biologically aggressive tumors from their outset, with a propensity for metastasis from small-volume primary lesions. Most type II OvCAs are high-grade serous carcinomas, virtually all of which harbor mutant TP53 alleles.⁴ These tumors have a high level of chromosomal instability.⁵ Although varied gene defects besides TP53 mutation have been identified in type II tumors, with the exception of frequent genetic/epigenetic inactivation of BRCA1/2 and NF1, RB1, and CDK12 mutations, each of the somatic gene defects is found in a small fraction of tumors.^6–8 Notably, recent data suggest that many (and perhaps most) high-grade serous carcinomas arise from epithelium in the fallopian tube, rather than the ovarian surface epithelium (OSE).^9–11 The identification of precursor lesions with p53 protein overexpression and clonal TP53 mutations in the fallopian tube epithelium (including “p53 signature lesions” and tubal intraepithelial carcinomas) suggests TP53 mutation is an early event in the pathogenesis of most type II OvCAs.¹²The dualistic pathway model represents an advance in conceptualizing OvCA pathogenesis, but the model is likely an oversimplified view of a complex group of cancers. For example, there are uncertainties about classification of clear cell carcinomas as type I versus type II, because their molecular features are more in keeping with type I tumors, but cytologic grade and clinical behavior are often more like type II tumors.³ As another example, there is significant overlap in the morphologic and molecular features of high-grade serous and high-grade endometrioid OvCAs (hereafter referred to as EOCs) such that some pathologists now default the majority of gland-forming or near-solid cytologically high-grade OvCAs to the serous category and consider “true” high-grade EOCs to be rare or nonexistent.¹³ In this scenario, all low-grade EOCs would be classified as type I, and the category of high-grade EOCs would be eliminated.In a prior analysis of a substantial number of primary human EOCs, we found that mutations predicted to activate the canonical Wnt and phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling pathways frequently co-occur in low-grade EOC, a prototypical type I OvCA.¹⁴ Activation of Wnt signaling typically occurs as a consequence of oncogenic CTNNB1 mutations, or rarely bi-allelic APC inactivation, whereas PI3K/AKT/mTOR signaling is usually activated because of PTEN inactivation and/or oncogenic PIK3CA mutations. Notably, we identified 4 of 21 tumors with gene expression and/or mutational profiles like type I EOCs, but the tumors had also acquired TP53 mutations, suggesting type I→type II progression may occur in a sizeable subset (nearly 20%) of EOCs.¹⁴ Similarly, we identified four EOCs with mutations in both PTEN and PIK3CA, rather than a single mutation dysregulating PI3K/AKT/mTOR signaling. Importantly, TP53 mutation has been associated with adverse outcome in women with endometrioid carcinomas of the endometrium and ovary,^15–18 and PIK3CA mutation has been linked to poor outcome in patients with several types of cancer, including carcinomas of the endometrium, breast, and colon.^19–22 The effect of PIK3CA mutations on the prognosis of patients with OvCA is unclear, because some studies have shown an adverse effect on outcome, whereas others showed no or even a favorable effect.^23–25 The notion that type II tumors can progress from type I tumors is not unique to EOCs. Indeed, type I→type II progression in serous carcinomas has also been described and is often associated with acquisition of TP53 mutation.^26–29We previously described a murine model for type I human EOC in which concurrent dysregulation of Wnt and PI3K/AKT/mTOR signaling is achieved through conditional inactivation of the Apc and Pten tumor suppressor genes in the OSE.¹⁴ In this model, ovarian bursal injection of recombinant adenovirus expressing Cre recombinase (AdCre) in Apc^flox/flox;Pten^floxf/flox mice results in the development of EOC-like tumors with complete penetrance. The purpose of the present study was to determine the effect of mutant Trp53 and Pik3ca on the Apc^−/−;Pten^−/− murine EOC tumor phenotype as models of type I→type II progression.     

Lgr5‐expressing stem cells are not the cells of origin of pyloric neuroendocrine carcinomas in mice

In intestinal and pyloric epithelia, leucine‐rich repeat‐containing G protein‐coupled receptor 5 (Lgr5)‐expressing cells represent long‐lived adult stem cells that give rise to all epithelial cell types, including endocrine cells. Ablation of the Apc gene in Lgr5‐expressing cells leads to intestinal and pyloric adenomas. To assess whether all epithelial tumours of the gastrointestinal tract are derived from LGR5‐positive stem cells, we crossed Lgr5–EGFP–IRES–creER^T2 mice, which express EGFP and Cre recombinase driven by the Lgr5 promoter, with CEA424–SV40–TAg mice, which develop pyloric neuroendocrine carcinomas of epithelial origin. In 19 day‐old mice, single SV40 T antigen (TAg)‐positive cells were identified preferentially at the the bases of pyloric glands, close to the stem cell compartment. However, contrary to previous publications describing subpopulations of LGR5‐positive cells in gastrointestinal neoplasia, we could not detect Lgr5–EGFP‐positive tumour cells in malignant lesions. The lack of expression of the Wnt target gene Lgr5 is probably not caused by suppression of Wnt signalling by TAg, since β‐catenin‐mediated Wnt signalling, as measured by the TOPflash assay, was not inhibited. To determine the cellular origin of CEA424–SV40–TAg tumours, we performed tracing experiments using Lgr5–EGFP–IRES–creERT2:CEA424–SV40–TAg:ROSA26–tdRFP mice. Following tamoxifen induction, it was possible to efficiently trace the progeny of Lgr5‐expressing cells in gastrointestinal tissue via red fluorescent protein (RFP) expression. No RFP‐positive tumour cells were detected, even when RFP gene activation occurred in 7 day‐old mice well before the appearance of TAg‐positive tumour cells. Hence, we conclude that Lgr5‐expressing stem cells probably do not constitute the cells of origin in CEA424–SV40–TAg mice. Consequently, not all epithelial tumours in the pyloric region are initiated by transformation of LGR5‐positive stem cells. Thus, additional long‐lived LGR5‐negative stem cells or progenitor cells with a low turnover rate might exist in the pyloric region, which could give rise to tumours. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Conditional abrogation of transforming growth factor‐β receptor 1 in PTEN‐inactivated endometrium promotes endometrial cancer progression in mice

Although a putative role for transforming growth factor‐β (TGFB) signalling in the pathogenesis of human endometrial cancer has long been proposed, the precise function of TGFB signalling in the development and progression of endometrial cancer remains elusive. Depletion of phosphatase and tensin homologue (PTEN) in the mouse uterus causes endometrial cancer. To identify the potential role of TGFB signalling in endometrial cancer, we simultaneously deleted TGFB receptor 1 (Tgfbr1) and Pten in the mouse uterus by using Cre‐recombinase driven by the progesterone receptor (termed Pten^d/d;Tgfbr1^d/d). We found that Pten^d/d;Tgfbr1^d/d mice developed severe endometrial lesions that progressed more rapidly than those resulting from conditional deletion of Pten alone, suggesting that TGFB signalling synergizes with PTEN to suppress endometrial cancer progression. Remarkably, Pten^d/d;Tgfbr1^d/d mice developed distant pulmonary metastases, leading to a significantly reduced lifespan. The development of metastasis and accelerated tumour progression in Pten^d/d;Tgfbr1^d/d mice are associated with increased production of proinflammatory chemokines, enhanced cancer cell motility, as shown by myometrial invasion and disruption, and an altered tumour microenvironment characterized by recruitment of tumour‐associated macrophages. Thus, conditional deletion of Tgfbr1 in PTEN‐inactivated endometrium leads to a disease that recapitulates invasive and lethal human endometrial cancer. This mouse model may be valuable for preclinical testing of new cancer therapies, particularly those targeting metastasis, one of the hallmarks of cancer and a major cause of death in endometrial cancer patients. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Polarized Ovaries of the Long‐tongued Bat,Glossophaga soricina: A Novel Model for Studying Ovarian Development,Folliculogenesis, and Ovulation

Glossophaga soricina is a spontaneously ovulating, monovular, polyestrous bat with a simplex uterus, exhibiting true menstruation. Studies conducted on reproductively active, captive‐maintained animals established that G. soricina also has polarized ovaries, with the ovarian surface epithelium (OSE) restricted to the medial side of the ovary, and primordial follicles limited to an immediately adjacent zone. Follicles selected for further development are recruited from the medullary side of this zone, and ovulation is restricted to the portion of the ovary covered by the OSE. To further develop G. soricina as a model for studying ovarian development and physiology, ovaries were collected from fetal, neonatal, and adult females and processed for morphological and immunohistochemical analyses. The latter included staining for factor VIII‐related antigen (von Willebrand factor) to assess regional differences in ovarian vascularity. The ovarian structure in fetal and neonatal animals was very similar to that in other species that do not have polarized ovaries at comparable stages of development. This indicated that polarization of the ovary does not occur during fetal development, but rather sometime between the neonatal period and adulthood. Vascular elements were abundant in areas of the ovary surrounding early growing follicles, but sparse in the zone of the ovary containing primordial follicles. The polarized nature of the ovaries in G. soricina suggests that this species might be used as a model to investigate the formation, long‐term maintenance, and activation of primordial follicles, and the role of the OSE in ovulation and folliculogenesis. Anat Rec, 290:1439–1448, 2007. © 2007 Wiley‐Liss, Inc.     

Identification of cellular and genetic drivers of breast cancer heterogeneity in genetically engineered mouse tumour models

The heterogeneous nature of mammary tumours may arise from different initiating genetic lesions occurring in distinct cells of origin. Here, we generated mice in which Brca2, Pten and p53 were depleted in either basal mammary epithelial cells or luminal oestrogen receptor (ER)‐negative cells. Basal cell‐origin tumours displayed similar histological phenotypes, regardless of the depleted gene. In contrast, luminal ER‐negative cells gave rise to diverse phenotypes, depending on the initiating lesions, including both ER‐negative and, strikingly, ER‐positive invasive ductal carcinomas. Molecular profiling demonstrated that luminal ER‐negative cell‐origin tumours resembled a range of the molecular subtypes of human breast cancer, including basal‐like, luminal B and ‘normal‐like’. Furthermore, a subset of these tumours resembled the ‘claudin‐low’ tumour subtype. These findings demonstrate that not only do mammary tumour phenotypes depend on the interactions between cell of origin and driver genetic aberrations, but also multiple mammary tumour subtypes, including both ER‐positive and ‐negative disease, can originate from a single epithelial cell type. This is a fundamental advance in our understanding of tumour aetiology. © 2014 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

Conditional Pten knock‐out mice: a model for metastatic phaeochromocytoma

Phaeochromocytomas (PCCs) are neuro‐endocrine tumours of the adrenal medulla that are usually benign, but approximately 10% of patients develop metastases. Malignant PCCs can only be diagnosed with certainty if metastases are present. Here we describe adrenal tumours generated in a Pten conditional knock‐out (KO) mouse model. We characterized the molecular alterations in these tumours and compared them with human PCC. Thirty‐two of 41 (78%) male Psa‐Cre;Pten‐loxP/loxP mice presented adrenal tumours that were shown to be PCC by histology and by immunohistochemical staining for enzymes in the catecholamine biosynthetic pathway. In 6 of 17 investigated mice, histological and immunohistochemical evidence was obtained for the presence of PCC lung metastases. Array comparative genomic hybridization (CGH) analysis of the primary tumours showed loss of chromosomes 6 and 19, which are syntenic to human 3p and 11q. Another frequent alteration found was gain of chromosome 15, which is syntenic to human chromosome 5. The molecular aberrations in the mouse model corresponded to the alterations found in a subtype of human PCC, suggesting that the PCC of the Pten KO mice might be representative of human PCC. The mouse model should allow further studies into the pathogenesis of human malignant PCCs and into therapeutic strategies for these tumours. Copyright © 2008 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

PTEN loss and KRAS activation leads to the formation of serrated adenomas and metastatic carcinoma in the mouse intestine

Mutation or loss of the genes PTEN and KRAS have been implicated in human colorectal cancer (CRC), and have been shown to co‐occur despite both playing a role in the PI3' kinase (PI3'K) pathway. We investigated the role of these genes in intestinal tumour progression in vivo, using genetically engineered mouse models, with the aim of generating more representative models of human CRC. Intestinal‐specific deletion of Pten and activation of an oncogenic allele of Kras was induced in wild‐type (WT) mice and mice with a predisposition to adenoma development (Apc^fl/+). The animals were euthanized when they became symptomatic of a high tumour burden. Histopathological examination of the tissues was carried out, and immunohistochemistry used to characterize signalling pathway activation. Mutation of Pten and Kras resulted in a significant life‐span reduction of mice predisposed to adenomas. Invasive adenocarcinoma was observed in these animals, with evidence of activation of the PI3'K pathway but no metastasis. However, mutation of Pten and Kras in WT animals not predisposed to adenomas led to perturbed homeostasis of the intestinal epithelium and the development of hyperplastic polyps, dysplastic sessile serrated adenomas and metastasizing adenocarcinomas with serrated features. These studies demonstrate synergism between Pten and Kras mutations in intestinal tumour progression, in an autochthonous and immunocompetent murine model, with potential application to preclinical drug testing. In particular, they show that Pten and Kras mutations alone predispose mice to the spectrum of serrated lesions that reflect the serrated pathway of CRC progression in humans. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk     

Combined deletion of Vhl,Trp53 and Kif3a causes cystic and neoplastic renal lesions

The von Hippel–Lindau (VHL) tumour suppressor gene is bi‐allelically inactivated in the majority of cases of clear cell renal cell carcinoma (ccRCC); however, Vhl knockout mouse models do not recapitulate human ccRCC, implying that additional mutations are required for tumour formation. Mutational inactivation of VHL sensitises renal epithelial cells to lose the primary cilium in response to other mutations or extracellular stimuli. Loss of cilia is believed to represent a second hit in VHL mutant cells that causes the development of cystic lesions that, in some cases, can progress to ccRCC. Supporting this idea, genetic ablation of the primary cilium by deletion of the kinesin family member 3A (Kif3a) gene cooperates with loss of Vhl to accelerate cyst formation in mouse kidneys. Additionally, aged Vhl/Trp53 double‐mutant mice develop renal cysts and tumours at a relatively low incidence, suggesting that there is a genetic cooperation between VHL and TP53 mutation in the development of ccRCC. Here we generated renal epithelium‐specific Kif3a/Trp53 and Vhl/Kif3a/Trp53 mutant mice to investigate whether primary cilium deletion would accelerate the development of cystic precursor lesions or cause their progression to ccRCC. Longitudinal microcomputed tomography (μCT) imaging and histopathological analyses revealed an increased rate of cyst formation, increased proportion of cysts with proliferating cells, higher frequency of atypical cysts as well as the development of neoplasms in Vhl/Kif3a/Trp53 mutant kidneys compared to Kif3a/Trp53 or Vhl/Kif3a mutant kidneys. These findings demonstrate that primary cilium loss, in addition to Vhl and Trp53 losses, promotes the transition towards malignancy and provide further evidence that the primary cilium functions as a tumour suppressor organelle in the kidney. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Palbociclib has antitumour effects on Pten‐deficient endometrial neoplasias

PTEN is one of the most frequently mutated genes in human cancers. The frequency of PTEN alterations is particularly high in endometrial carcinomas. Loss of PTEN leads to dysregulation of cell division, and promotes the accumulation of cell cycle complexes such as cyclin D1–CDK4/6, which is an important feature of the tumour phenotype. Cell cycle proteins have been presented as key targets in the treatment of the pathogenesis of cancer, and several CDK inhibitors have been developed as a strategy to generate new anticancer drugs. Palbociclib (PD‐332991) specifically inhibits CDK4/6, and it has been approved for use in metastatic breast cancer in combination with letrazole. Here, we used a tamoxifen‐inducible Pten knockout mouse model to assess the antitumour effects of cyclin D1 knockout and CDK4/6 inhibition by palbociclib on endometrial tumours. Interestingly, both cyclin D1 deficiency and palbociclib treatment triggered shrinkage of endometrial neoplasias. In addition, palbociclib treatment significantly increased the survival of Pten‐deficient mice, and, as expected, had a general effect in reducing tumour cell proliferation. To further analyse the effects of palbociclib on endometrial carcinoma, we established subcutaneous tumours with human endometrial cancer cell lines and primary endometrial cancer xenografts, which allowed us to provide more translational and predictive data. To date, this is the first preclinical study evaluating the response to CDK4/6 inhibition in endometrial malignancies driven by PTEN deficiency, and it reveals an important role of cyclin D–CDK4/6 activity in their development. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Comprehensive genetic and functional characterization of IPH‐926: a novel CDH1‐null tumour cell line from human lobular breast cancer

Infiltrating lobular breast cancer (ILBC) is a clinically and biologically distinct tumour entity defined by a characteristic linear cord invasion pattern and inactivation of the CDH1 tumour suppressor gene encoding for E‐cadherin. ILBCs also lack β‐catenin expression and show aberrant cytoplasmic localization of the E‐cadherin binding protein p120‐catenin. The lack of a well‐characterized ILBC cell line has hampered the functional characterization of ILBC cells in vitro. We report the establishment of a permanent ILBC cell line, named IPH‐926, which was derived from a patient with metastatic ILBC. The DNA fingerprint of IPH‐926 verified genetic identity with the patient and had no match among the human cell line collections of several international biological resource banks. IPH‐926 expressed various epithelial cell markers but lacked expression of E‐cadherin due to a previously unreported, homozygous CDH1 241ins4 frameshift mutation. Detection of the same CDH1 241ins4 mutation in archival tumour tissue of the corresponding primary ILBC proved the clonal origin of IPH‐926 from this particular tumour. IPH‐926 also lacked β‐catenin expression and showed aberrant cytoplasmic localization of p120‐catenin. Array‐CGH analysis of IPH‐926 revealed a profile of genomic imbalances that included many distinct alterations previously observed in primary ILBCs. Spectral karyotyping of IPH‐926 showed a hyperdiploid chromosome complement and numerous clonal, structural aberrations. IPH‐926 cells were anti‐cancer drug‐resistant, clonogenic in soft agar, and tumourigenic in SCID mice. In xenograft tumours, IPH‐926 cells recapitulated the linear cord invasion pattern that defines ILBCs. In summary, IPH‐926 significantly extends the biological spectrum of the established breast cancer cell lines and will facilitate functional analyses of genuine human ILBC cells in vitro and in vivo. Copyright © 2008 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.