HER2 expression patterns in paired primary and metastatic endometrial cancer lesions.

BACKGROUND: Despite successful implementation of drugs targeting the human epidermal growth factor receptor 2 (HER2) receptor in breast and gastric cancers, the potential of HER2 as a therapeutic target in other cancers has been less studied, including endometrial cancer. We investigated expression levels of HER2 (ERBB2) in a large cohort of endometrial cancer lesions, also including complex atypical hyperplasia and metastatic lesions. METHODS: 67 precursor lesions, 790 primary endometrial cancers and 383 metastatic lesions were investigated for HER2 expression in relation to clinicopathologic features and outcome. Protein levels were assessed by immunohistochemistry (using the HercepTest and staining index (SI) criteria), mRNA levels by microarrays and amplification status by chromogenic in situ hybridisation. RESULTS: High HER2 protein levels were significantly associated with features of aggressive disease and increased mRNA ERBB2 levels. HER2 expression defined by the SI proved to be a better predictor of survival compared with the HercepTest. A discordant HER2 expression pattern between paired primary and metastatic lesions was detected, revealing substantial reduction in HER2 expression from primary to metastatic disease. CONCLUSIONS: Loss of HER2 expression is common in metastatic endometrial cancer lesions and assessment of HER2 levels in the metastatic lesions may be important to define the potential benefit of anti-HER2 treatments in endometrial cancer patients.

Suppression of heat shock protein 27 induces long-term dormancy in human breast cancer.

The mechanisms underlying tumor dormancy have been elusive and not well characterized. We recently published an experimental model for the study of human tumor dormancy and the role of angiogenesis, and reported that the angiogenic switch was preceded by a local increase in VEGF-A and basic fibroblast growth factor. In this breast cancer xenograft model (MDA-MB-436 cells), analysis of differentially expressed genes revealed that heat shock protein 27 (HSP27) was significantly up-regulated in angiogenic cells compared with nonangiogenic cells. The effect of HSP27 down-regulation was further evaluated in cell lines, mouse models, and clinical datasets of human patients with breast cancer and melanoma. Stable down-regulation of HSP27 in angiogenic tumor cells was followed by long-term tumor dormancy in vivo. Strikingly, only 4 of 30 HSP27 knockdown xenograft tumors initiated rapid growth after day 70, in correlation with a regain of HSP27 protein expression. Significantly, no tumors escaped from dormancy without HSP27 expression. Down-regulation of HSP27 was associated with reduced endothelial cell proliferation and decreased secretion of VEGF-A, VEGF-C, and basic fibroblast growth factor. Conversely, overexpression of HSP27 in nonangiogenic cells resulted in expansive tumor growth in vivo. By clinical validation, strong HSP27 protein expression was associated with markers of aggressive tumors and decreased survival in patients with breast cancer and melanoma. An HSP27-associated gene expression signature was related to molecular subgroups and survival in breast cancer. Our findings suggest a role for HSP27 in the balance between tumor dormancy and tumor progression, mediated by tumor-vascular interactions. Targeting HSP27 might offer a useful strategy in cancer treatment.

ARID1A loss is prevalent in endometrial hyperplasia with atypia and low-grade endometrioid carcinomas.

ARID1A (AT-rich interactive domain 1A) has recently been identified as a tumor suppressor gene in various, predominantly gynecological cancers. We wanted to investigate the distribution of ARID1A in endometrial hyperplasia, carcinomas and metastatic lesions to elucidate the timing of expression loss of its protein ARID1A in the course of endometrial cancer carcinogenesis. In addition, we wanted to assess the relationship between the loss of ARID1A and clinicopathological variables in endometrial cancer in general and the endometrioid subtype in particular. We analyzed a prospectively collected series of 535 primary endometrial cancers, 77 metastatic lesions, as well as 38 retrospectively collected endometrial hyperplasias with evaluable immunohistochemical staining for ARID1A. Fresh frozen tissue was available for mRNA microarray analysis in 122 primary tumors in parallel. Loss of ARID1A protein expression was noted in none of the hyperplasias without atypia, 16% of hyperplasias with atypia, 19% of primary endometrioid tumors and 28% of metastatic lesions. Loss of ARID1A in primary tumor was significantly associated with endometrioid grade 1 or 2 and clear-cell histology, diploid tumor cells, younger patient age and deeper myometrial infiltration, but not survival. ARID1A RNA expression was significantly correlated with ARID1A protein loss. Thus, loss of ARID1A appears to be an early event in the carcinogenesis of endometrioid uterine carcinomas and the association with deep myometrial infiltration may suggest an importance for invasiveness.

Gene expression patterns related to vascular invasion and aggressive features in endometrial cancer.

The presence of tumor cells entering vascular channels is a prognostic marker for many cancers, including endometrial carcinoma. Vascular invasion is considered to be an early step in the metastatic process and important for the progress of malignant tumors. Here, we investigated the gene expression patterns related to vascular involvement in 57 primary endometrial cancers, using DNA microarray and quantitative PCR techniques. A vascular invasion signature of 18 genes was significantly associated with patient survival and clinicopathological phenotype. Vascular involvement was also related to gene sets for epithelial-mesenchymal transition, wound response, endothelial cells, and vascular endothelial growth factor (VEGF) activity. With immunohistochemical validation, both collagen 8 and matrix metalloproteinase 3 (MMP3) were associated with vascular invasion, whereas ANGPTL4 and IL-8 were associated with patient survival. Our findings indicate that vascular involvement within primary tumors is associated with gene expression profiles related to angiogenesis and epithelial-mesenchymal transition. These data could contribute to an improved understanding of potential targets for metastatic spread and may provide clinically important information for better management of endometrial cancer.

Gene expression in tumor cells and stroma in dsRed 4T1 tumors in eGFP-expressing mice with and without enhanced oxygenation.

BACKGROUND: The tumor microenvironment is pivotal in tumor progression. Thus, we aimed to develop a mammary tumor model to elucidate molecular characteristics in the stroma versus the tumor cell compartment by global gene expression. Secondly, since tumor hypoxia influences several aspects of tumor pathophysiology, we hypothesized that hyperoxia might have an inhibitory effect on tumor growth per se. Finally, we aimed to identify differences in gene expression and key molecular mechanisms, both in the native state and following treatment. METHODS: 4T1 dsRed breast cancer cells were injected into eGFP expressing NOD/SCID mice. Group 1 was exposed to 3 intermittent HBO treatments (Day 1, 4 and 7), Group 2 to 7 daily HBO treatments (both 2.5 bar, 100% O2, à 90 min), whereas the controls were exposed to a normal atmosphere. Tumor growth, histology, vascularisation, cell proliferation, cell death and metastasis were assessed. Fluorescence-activated cell sorting was used to separate tumor cells from stromal cells prior to gene expression analysis. RESULTS: The purity of sorted cells was verified by fluorescence microscopy. Gene expression profiling demonstrated that highly expressed genes in the untreated tumor stroma included constituents of the extracellular matrix and matrix metalloproteinases. Tumor growth was significantly inhibited by HBO, and the MAPK pathway was found to be significantly reduced. Immunohistochemistry indicated a significantly reduced microvessel density after intermittent HBO, whereas daily HBO did not show a similar effect. The anti-angiogenic response was reflected in the expression trends of angiogenic factors. CONCLUSIONS: The present in vivo mammary tumor model enabled us to separate tumor and stromal cells, and demonstrated that the two compartments are characterized by distinct gene expressions, both in the native state and following HBO treatments. Furthermore, hyperoxia induced a significant tumor growth-inhibitory effect, with significant down-regulation of the MAPK pathway. An anti-angiogenic effect after intermittent HBO was observed, and reflected in the gene expression profile.

Lipocalin 2 expression is associated with aggressive features of endometrial cancer.

BACKGROUND: Increased expression of lipocalin 2 (LCN2) has been observed in several cancers. The aim of the present study was to investigate LCN2 in endometrial cancer in relation to clinico-pathologic phenotype, angiogenesis, markers of epithelial-mesenchymal transition (EMT), and patient survival. METHODS: Immunohistochemical staining was performed using a human LCN2 antibody on a population-based series of endometrial cancer patients collected in Hordaland County (Norway) during 1981-1990 (n = 256). Patients were followed from the time of primary surgery until death or last follow-up in 2007. The median follow-up time for survivors was 17 years. Gene expression data from a prospectively collected endometrial cancer series (n = 76) and a publicly available endometrial cancer series (n = 111) was used for gene correlation studies. RESULTS: Expression of LCN2 protein, found in 49% of the cases, was associated with non-endometrioid histologic type (p = 0.001), nuclear grade 3 (p = 0.001), >50% solid tumor growth (p = 0.001), ER and PR negativity (p = 0.028 and 0.006), and positive EZH2 expression (p < 0.001). LCN2 expression was significantly associated with expression of VEGF-A (p = 0.021), although not with other angiogenesis markers examined (vascular proliferation index, glomeruloid microvascular proliferation, VEGF-C, VEGF-D or bFGF2 expression). Further, LCN2 was not associated with several EMT-related markers (E-cadherin, N-cadherin, P-cadherin, ß-catenin), nor with vascular invasion (tumor cells invading lymphatic or blood vessels). Notably, LCN2 was significantly associated with distant tumor recurrences, as well as with the S100A family of metastasis related genes. Patients with tumors showing no LCN2 expression had the best outcome with 81% 5-year survival, compared to 73% for intermediate and 38% for the small subgroup with strong LCN2 staining (p = 0.007). In multivariate analysis, LCN2 expression was an independent prognostic factor in addition to histologic grade and FIGO stage. CONCLUSION: Increased LCN2 expression is associated with aggressive features and poor prognosis in endometrial cancer.

Primary human acute myelogenous leukemia cells release matrix metalloproteases and their inhibitors: release profile and pharmacological modulation.

OBJECTIVES: Angiogenesis seems important for both leukemogenesis and chemosensitivity in acute myelogenous leukemia (AML). Angiogenesis is regulated by the balance between pro- and antiangiogenic cytokines, which also indicates an important role of matrix metalloproteases (MMPs) and their natural inhibitors, tissue inhibitors of metalloproteases (TIMPs). We investigated the constitutive release of MMPs and TIMPs for a large group of consecutive AML patients. METHODS: AML cells were cultured in vitro either alone or together with microvascular endothelial cells, and levels of MMPs and TIMPs were determined in culture supernatants. RESULTS: AML cells showed constitutive release of several MMPs and TIMPs. For all patients, detectable MMP-10 release was observed, and most patients showed detectable release of at least one additional MMP, usually MMP-9 or MMP-2. A significant correlation was found between MMP-9 and TIMP-1 release and the release of several CCL and CXCL chemokines. MMP-9 release was higher for AML cells with monocytic differentiation corresponding to the FAB-subtype M4/M5 AML; it was mainly released in its inactive form, but endogenously active MMP-9 could be detected even in the presence of the constitutively released TIMP-1/2. Endothelial cells released relatively high levels of MMP-10, and these levels were further increased by coculture with AML cells. Patients achieving complete hematological remission after only one induction cycle showed relatively low constitutive MMP-2 release. CONCLUSION: We conclude that primary human AML cells show constitutive release of both MMPs and TIMPs, and this release may be important for leukemogenesis and possibly also for chemosensitivity.

Primary human acute myeloid leukaemia cells increase the proliferation of microvascular endothelial cells through the release of soluble mediators.

Bone marrow angiogenesis is suggested to play a role in the pathogenesis of acute myeloid leukaemia (AML) and endothelial cells may mediate chemosensitivity. This study investigated in vitro endothelial effects of coculture of microvascular endothelial cells (MVEC) with AML cells derived from 33 consecutive AML patients. A proliferation assay showed that (i) AML cells from the majority of patients examined increased endothelial cell proliferation, while cytokine neutralizing experiments had divergent effects on proliferation and (ii) the angiopoietin/Tie2 system was important for growth of AML cells, and angiopoietin-1 induced phosphorylation of signal transducers and activators of transcription (STAT) proteins in AML cells. Finally, gene expression profiling of MVEC cocultured with AML cells was conducted in non-contact cultures. Microarray analysis revealed that the majority of significantly expressed genes could be categorized into gene ontology terms involved in transcription, cellular organization and intracellular signalling. Our study indicates a role for the leukaemic-endothelium crosstalk in leukaemogenesis with enhancement of endothelial cell growth and increased AML cell proliferation possibly mediated by angiopoietin-1 and the STAT signalling pathway.

Dual androgen receptor (AR) and STAT3 inhibition by a compound targeting the AR amino-terminal domain.

Prostate cancer (PCa) often recurs as incurable castration-resistant prostate cancer (CRPC) after the failure of androgen deprivation therapy. CRPC development relies on androgen receptor (AR) signaling. The IL6/STAT3 pathway is also a key driver of CRPC. The crosstalk between IL6/STAT3 and the AR pathways provides opportunities to explore next-generation agents to treat PCa. Through screening of around 600 natural compounds in our newly established prostate tumorigenesis model, potential STAT3 signaling inhibitors were found and additionally examined for effects on AR signaling. The small molecular compound 154 exhibited dual effects on IL6/STAT3 and AR pathways. We show here that compound 154 inhibits AR and STAT3 transcriptional activity, reduces the expression of phosphorylation of STAT3 (Y705) and downregulates the mRNA levels of AR target genes. Compound 154 also inhibits protein expression of AR and AR splice variants (ARv567es and AR-V7) without altering AR mRNA levels. Compound 154 binds to AR directly, but not to STAT3 and is identified as an antagonist of the AR amino-terminal domain (NTD) by disrupting protein-protein interactions between STAT3 and the AR NTD. Moreover, compound 154 does not reduce AR nuclear translocation. Compound 154 possesses the potential to become a leading compound in novel therapies against CRPC.

Identification of highly connected and differentially expressed gene subnetworks in metastasizing endometrial cancer.

We have identified nine highly connected and differentially expressed gene subnetworks between aggressive primary tumors and metastatic lesions in endometrial carcinomas. We implemented a novel pipeline combining gene set and network approaches, which here allows integration of protein-protein interactions and gene expression data. The resulting subnetworks are significantly associated with disease progression across tumor stages from complex atypical hyperplasia, primary tumors to metastatic lesions. The nine subnetworks include genes related to metastasizing features such as epithelial-mesenchymal transition (EMT), hypoxia and cell proliferation. TCF4 and TWIST2 were found as central genes in the subnetwork related to EMT. Two of the identified subnetworks display statistically significant association to patient survival, which were further supported by an independent validation in the data from The Cancer Genome Atlas data collection. The first subnetwork contains genes related to cell proliferation and cell cycle, while the second contains genes involved in hypoxia such as HIF1A and EGLN3. Our findings provide a promising context to elucidate the biological mechanisms of metastasis, suggest potential prognostic markers and further identify therapeutic targets. The pipeline R source code is freely available, including permutation tests to assess statistical significance of the identified subnetworks.

PIK3CA exon9 mutations associate with reduced survival, and are highly concordant between matching primary tumors and metastases in endometrial cancer.

Mutations of the phosphoinositide-3-kinase (PI3K) catalytic subunit alpha gene (PIK3CA) are frequent in endometrial cancer. We sequenced exon9 and exon20 of PIK3CA in 280 primary endometrial cancers to assess the relationship with clinicopathologic variables, patient survival and associations with PIK3CA mRNA and phospho-AKT1 by gene expression and protein data, respectively. While PIK3CA mutations generally had no impact on survival, and were not associated with clinicopathological variables, patients with exon9 charge-changing mutations, providing a positive charge at the substituted amino acid residue, were associated with poor survival (p = 0.018). Furthermore, we characterized PIK3CA mutations in the metastatic setting, including 32 patients with matched primary tumors and metastases, and found a high level of concordance (85.7%; 6 out of 7 patients), suggesting limited heterogeneity. PIK3CA mRNA levels were increased in metastases compared to the primary tumors (p = 0.031), independent of PIK3CA mutation status, which rather associated with reduced PIK3CA mRNA expression. PIK3CA mutated tumors expressed higher p-AKT/AKT protein levels, both within primary (p < 0.001) and metastatic lesion (p = 0.010). Our results support the notion that the PI3K signaling pathway might be activated, both dependent- and independently of PIK3CA mutations, an aspect that should be considered when designing PIK3 pathway targeting strategies in endometrial cancer.

Androgen receptor as potential therapeutic target in metastatic endometrial cancer.

PURPOSE: The expression and involvement of estrogen (ER) and progesterone receptor (PR) is extensively studied in endometrial cancer. Androgen receptor (AR) is a hormone receptor less studied in female cancers, and we here aim to investigate the expression level of AR in endometrial cancer precursor lesions, primary tumors and metastases, and its potential as therapeutic target. RESULTS: Expression of AR was observed in 93% of hyperplasias, but only in 41% of non-endometrioid tumors. Compared to estrogen and progesterone receptor AR is more commonly expressed in metastatic lesions, and AR status is discordant in primary and metastatic lesions in a large proportion of cases. AR protein level was significantly associated with survival (P < 0.001), and a calculated AR to ERα ratio identified a subgroup of patients with particular poor outcome. The anti-androgen enzalutamide may have a growth inhibitory effect in endometrial cancer cells based on experiments with primary endometrial tumor cells. MATERIALS AND METHODS: 718 primary endometrial cancers and 298 metastatic lesions (from 142 patients) were investigated for expression of AR in relation to survival, clinical and histopathological data. Protein levels were investigated by immunohistochemistry and reverse phase protein array; mRNA levels by DNA oligonucleotide microarray. The effect of androgen stimulation and inhibition was tested on primary endometrial tumor cells. CONCLUSIONS: A large proportion of metastatic endometrial cancer lesions express AR, which may be a potential target in these patients. Treatment targeting AR may be of particular benefit in patients with high AR levels compared to ERα levels.

Nucleosome binding by the bromodomain and PHD finger of the transcriptional cofactor p300.

The PHD finger and the bromodomain are small protein domains that occur in many proteins associated with phenomena related to chromatin. The bromodomain has been shown to bind acetylated lysine residues on histone tails. Lysine acetylation is one of several histone modifications that have been proposed to form the basis for a mechanism for recording epigenetically stable marks in chromatin, known as the histone code. The bromodomain is therefore thought to read a part of the histone code. Since PHD fingers often occur in proteins next to bromodomains, we have tested the hypothesis that the PHD finger can also interact with nucleosomes. Using two different in vitro assays, we found that the bromodomain/PHD finger region of the transcriptional cofactor p300 can bind to nucleosomes that have a high degree of histone acetylation. In a nucleosome retention assay, both domains were required for binding. Replacement of the p300 PHD finger with other PHD fingers resulted in loss of nucleosome binding. In an electrophoretic mobility shift assay, each domain alone showed, however, nucleosome-binding activity. The binding of the isolated PHD finger to nucleosomes was independent of the histone acetylation levels. Our data are consistent with a model where the two domains cooperate in nucleosome binding. In this model, both the bromodomain and the PHD finger contact the nucleosome while simultaneously interacting with each other.

Tumor necrosis is an important hallmark of aggressive endometrial cancer and associates with hypoxia, angiogenesis and inflammation responses.

AIMS: Tumor necrosis is associated with aggressive features of endometrial cancer and poor prognosis. Here, we investigated gene expression patterns and potential treatment targets related to presence of tumor necrosis in primary endometrial cancer lesions. METHODS AND RESULTS: By DNA microarray analysis, expression of genes related to tumor necrosis reflected multiple tumor-microenvironment interactions like tissue hypoxia, angiogenesis and inflammation pathways. A tumor necrosis signature of 38 genes and a related patient cluster (Cluster I, 67% of the cases) were associated with features of aggressive tumors such as type II cancers, estrogen receptor negative tumors and vascular invasion. Further, the tumor necrosis signature was increased in tumor cells grown in hypoxic conditions in vitro. Multiple genes with increased expression are known to be activated by HIF1A and NF-kB. CONCLUSIONS: Our findings indicate that the presence of tumor necrosis within primary tumors is associated with hypoxia, angiogenesis and inflammation responses. HIF1A, NF-kB and PI3K/mTOR might be potential treatment targets in aggressive endometrial cancers with presence of tumor necrosis.

Increased angiogenesis is associated with a 32-gene expression signature and 6p21 amplification in aggressive endometrial cancer.

BACKGROUND: Angiogenesis is a hallmark of cancer. The aim of this study was to explore whether microvessel proliferation is associated with gene expression profiles or copy number alterations in endometrial cancer. METHODS: A prospective series of endometrial carcinomas was studied for angiogenesis markers, gene expression profiles, and gene copy number data. For validation, an independent series of endometrial carcinomas as well as an external cohort of endometrial cancer patients were examined by gene expression microarrays. RESULTS: Increased microvessel proliferation (MVP) was associated with aggressive tumor features and reduced survival, and a 32-gene expression signature was found to separate tumors with high versus low MVP. An increased 32-gene signature score was confirmed to associate with high-grade tumor features and reduced survival by independent cohorts. Copy number studies revealed that amplification of the 6p21 region was significantly associated with MVP, a high 32-gene score, as well as reduced survival. CONCLUSION: Increased MVP was significantly associated with aggressive endometrial cancer and reduced survival. Integrated analyses demonstrated significant associations between increased vascular proliferation, amplification of the 6p21 region, VEGF-A mRNA expression, and the 32-gene angiogenesis signature. Our findings indicate amplification of 6p21 as a possible driver of tumor vascular proliferation in endometrial cancer.

ATAD2 overexpression links to enrichment of B-MYB-translational signatures and development of aggressive endometrial carcinoma.

We have explored the potential for clinical implementation of ATAD2 as a biomarker for aggressive endometrial cancer by investigating to what extent immunohistochemical (IHC) staining for ATAD2 is feasible, reflects clinical phenotype and molecular subgroups of endometrial carcinomas. Increased expression of the ATAD2 gene has been implicated in cancer development and progression in a number of tissues, but few studies have investigated ATAD2 expression using IHC. Here we show that high ATAD2 protein expression is significantly associated with established clinical-pathological variables for aggressive endometrial cancer, also in the subset of estrogen receptor α (ERα) positive tumors. Protein and mRNA expression of ATAD2 were highly correlated (P < 0.001), suggesting that IHC staining may represent a more clinically applicable measure of ATAD2 level in routinely collected formalin fixed paraffin embedded specimens. Gene expression alterations in samples with high ATAD2 expression revealed upregulation of several cancer-related genes (B-MYB, CDCs, E2Fs) and gene sets that previously have been linked to aggressive disease and potential for new targeting therapies. Our results support that IHC staining for ATAD2 may be a clinically applicable biomarker reflecting clinical phenotype and targetable alterations in endometrial carcinomas to be further explored in controlled clinical trials.

Molecular profiling of endometrial carcinoma precursor, primary and metastatic lesions suggests different targets for treatment in obese compared to non-obese patients.

Obesity is linked to increased incidence of endometrioid endometrial cancer (EEC) and complex atypical hyperplasia (CAH). We here explore pattern and sequence of molecular alterations characterizing endometrial carcinogenesis in general and related to body mass index (BMI), to improve diagnostic stratification and treatment strategies. We performed molecular characterization of 729 prospectively collected EEC and CAH. Candidate biomarkers were identified in frozen samples by whole-exome and Sanger sequencing, oligonucleotide gene expression and Reverse Phase Protein Arrays (investigation cohort) and further explored in formalin fixed tissues by immunohistochemistry and Fluorescent in Situ Hybridization (validation cohort). We here demonstrate that PIK3CA mutations, PTEN loss, PI3K and KRAS activation are early events in endometrial carcinogenesis. Molecular changes related to KRAS activation and inflammation are more common in obese CAH patients, suggesting different prevention and systemic treatment strategies in obese and non-obese patients. We also found that oncoprotein Stathmin might improve preoperative diagnostic distinction between premalignant and malignant endometrial lesions.

Switch in FOXA1 status associates with endometrial cancer progression.

BACKGROUND: The transcription factor Forkhead box A1 (FOXA1) is suggested to be important in hormone dependent cancers, although with little data for endometrial cancer. We investigated expression levels of FOXA1 in primary and metastatic endometrial cancer in relation to clinical phenotype, and transcriptional alterations related to FOXA1 status. METHODS: Protein expression of FOXA1 was explored by immunohistochemistry in 529 primary and 199 metastatic endometrial carcinoma lesions. mRNA levels from corresponding 158 fresh frozen primary and 42 metastatic lesions were analyzed using Agilent Microarrays (44k) in parallel. RESULTS: Low FOXA1 protein expression in primary tumors significantly correlated with low FOXA1 mRNA, high age, non-endometrioid histology, high grade, loss of ERα and PR and poor survival (all p-values <0.05). Through a Connectivity Map search, HDAC inhibitors were suggested as potential treatment for patients with low FOXA1 expression. An increase in FOXA1 expression was observed from primary to metastatic lesions and it correlated with CDKN2A expression in metastases. CONCLUSION: Low FOXA1 is associated with poor survival and suggests a potential for HDAC inhibitors in endometrial carcinoma. A switch in FOXA1 expression from primary to metastatic lesions is observed and gene expression indicates a link between FOXA1 and CDKN2A in metastatic lesions.

Hypomethylation of the CTCFL/BORIS promoter and aberrant expression during endometrial cancer progression suggests a role as an Epi-driver gene.

Cancers arise through accumulating genetic and epigenetic alterations, considered relevant for phenotype and approaches to targeting new therapies. We investigated a unique collection of endometrial cancer precursor samples and clinically annotated primary and metastatic lesions for two evolutionary and functionally related transcription factors, CCCTC-binding factor (zinc finger protein) (CTCF) and its paralogue CTCF-like factor, also denoted Brother of the Regulator of Imprinted Sites (CTCFL/BORIS). CTCF, a chromatin modeling- and transcription factor, is normally expressed in a ubiquitous fashion, while CTCFL/BORIS is restricted to the testis. In cancer, CTCF is thought to be a tumor suppressor, while CTCFL/BORIS has been suggested as an oncogene. CTCF mutations were identified in 13%, with CTCF hotspot frameshift mutations at p.T204, all observed solely in the endometrioid subtype, but with no association with outcome. Interestingly, CTCFL/BORIS was amongst the top ranked genes differentially expressed between endometrioid and non-endometrioid tumors, and increasing mRNA level of CTCFL/BORIS was highly significantly associated with poor survival. As aberrant CTCFL/BORIS expression might relate to loss of methylation, we explored methylation status in clinical samples from complex atypical hyperplasia, through primary tumors to metastatic lesions, demonstrating a pattern of DNA methylation loss during disease development and progression in line with the increase in CTCFL/BORIS mRNA expression observed. Thus, CTCF and CTCFL/BORIS are found to diverge in the different subtypes of endometrial cancer, with CTCFL/BORIS activation through demethylation from precursors to metastatic lesions. We thus propose, CTCFL/BORIS as an Epi-driver gene in endometrial cancer, suggesting a potential for future vaccine development.