Co-existing TP53 and ARID1A mutations promote aggressive endometrial tumorigenesis.

TP53 and ARID1A are frequently mutated across cancer but rarely in the same primary tumor. Endometrial cancer has the highest TP53-ARID1A mutual exclusivity rate. However, the functional relationship between TP53 and ARID1A mutations in the endometrium has not been elucidated. We used genetically engineered mice and in vivo genomic approaches to discern both unique and overlapping roles of TP53 and ARID1A in the endometrium. TP53 loss with oncogenic PIK3CAH1047R in the endometrial epithelium results in features of endometrial hyperplasia, adenocarcinoma, and intraepithelial carcinoma. Mutant endometrial epithelial cells were transcriptome profiled and compared to control cells and ARID1A/PIK3CA mutant endometrium. In the context of either TP53 or ARID1A loss, PIK3CA mutant endometrium exhibited inflammatory pathway activation, but other gene expression programs differed based on TP53 or ARID1A status, such as epithelial-to-mesenchymal transition. Gene expression patterns observed in the genetic mouse models are reflective of human tumors with each respective genetic alteration. Consistent with TP53-ARID1A mutual exclusivity, the p53 pathway is activated following ARID1A loss in the endometrial epithelium, where ARID1A normally directly represses p53 pathway genes in vivo, including the stress-inducible transcription factor, ATF3. However, co-existing TP53-ARID1A mutations led to invasive adenocarcinoma associated with mutant ARID1A-driven ATF3 induction, reduced apoptosis, TP63+ squamous differentiation and invasion. These data suggest TP53 and ARID1A mutations drive shared and distinct tumorigenic programs in the endometrium and promote invasive endometrial cancer when existing simultaneously. Hence, TP53 and ARID1A mutations may co-occur in a subset of aggressive or metastatic endometrial cancers, with ARID1A loss promoting squamous differentiation and the acquisition of invasive properties.

Metronomic dosing of ovarian cancer cells with the ATR inhibitor AZD6738 leads to loss of CDC25A expression and resistance to ATRi treatment.

OBJECTIVE: ATR kinase inhibitors promote cell killing by inducing replication stress and through potentiation of genotoxic agents in gynecologic cancer cells. To explore mechanisms of acquired resistance to ATRi in ovarian cancer, we characterized ATRi-resistant ovarian cancer cells generated by metronomic dosing with the clinical ATR inhibitor AZD6738. METHODS: ATRi-resistant ovarian cancer cells (OVCAR3 and OV90) were generated by dosing with AZD6738 and assessed for sensitivity to Chk1i (LY2603618), PARPi (Olaparib) and combination with cisplatin or a CDK4/6 inhibitor (Palbociclib). Models were characterized by diverse methods including silencing CDC25A in OV90 cells and assessing impact on ATRi response. Serum proteomic analysis of ATRi-resistant OV90 xenografts was performed to identify circulating biomarker candidates of ATRi-resistance. RESULTS: AZD6738-resistant cell lines are refractory to LY2603618, but not to Olaparib or combinations with cisplatin. Cell cycle analyses showed ATRi-resistant cells exhibit G1/S arrest following AZD6738 treatment. Accordingly, combination with Palbociclib confers resistance to AZD6738. AZD6738-resistant cells exhibit altered abundances of G1/S phase regulatory proteins, including loss of CDC25A in AZD6738-resistant OV90 cells. Silencing of CDC25A in OV90 cells confers resistance to AZD6738. Serum proteomics from AZD6738-resistant OV90 xenografts identified Vitamin D-Binding Protein (GC), Apolipoprotein E (APOE) and A1 (APOA1) as significantly elevated in AZD6738-resistant backgrounds. CONCLUSIONS: We show that metronomic dosing of ovarian cancer cells with AZD6738 results in resistance to ATR/ Chk1 inhibitors, that loss of CDC25A expression represents a mechanism of resistance to ATRi treatment in ovarian cancer cells and identify several circulating biomarker candidates of CDC25A low, AZD6738-resistant ovarian cancer cells.

MIG-6 Is Critical for Progesterone Responsiveness in Human Complex Atypical Hyperplasia and Early-Stage Endometrial Cancer.

Women with complex atypical hyperplasia (CAH) or early-stage endometrioid endometrial cancer (EEC) are candidates for fertility preservation. The most common approach is progesterone (P4) therapy and deferral of hysterectomy until after completion of childbearing. However, P4 therapy response rates vary, and molecular mechanisms behind P4 resistance are poorly understood. One potential molecular cause of P4 resistance is a loss or attenuation of PGR expression. Mitogen-inducible gene 6 (MIG-6) is critical for P4 responsiveness. MIG-6 protein expression in the endometrial epithelial and stromal cells from women with CAH and EEC was significantly lower compared to women without CAH or EEC. The P4-responsive women (10/15) exhibited an increase of MIG-6 expression in epithelial and stromal cells compared to P4-resistant women (5/15). In addition, immunohistochemical analysis for PGR results showed that stromal PGR levels are significantly higher in P4-responsive women compared to P4-resistant women, whereas epithelial PGR expression was not different. A reverse correlation of MIG-6 and pAKT levels was observed in early-stage EEC patients. Studies strongly suggest that loss of MIG-6 and PGR and activation of pAKT lead to P4 resistance in CAH and EEC. These results will help to elucidate the molecular mechanism leading to P4 resistance in CAH and EEC.

MIG-6 suppresses endometrial epithelial cell proliferation by inhibiting phospho-AKT.

BACKGROUND: Aberrant hyperactivation of epithelial proliferation, AKT signaling, and association with unopposed estrogen (E2) exposure is the most common endometrial cancer dysfunction. In the normal uterus, progesterone (P4) inhibits proliferation by coordinating stromal-epithelial cross-talk, which we previously showed is mediated by the function of Mitogen-inducible gene 6 (Mig-6). Despite their attractive characteristics, non-surgical conservative therapies based on progesterone alone have not been universally successful. One barrier to this success has been the lack of understanding of the P4 effect on endometrial cells. METHOD: To further understand the role of Mig-6 and P4 in controlling uterine proliferation, we developed a Sprr2f-cre driven mouse model where Mig-6 is specifically ablated only in the epithelial cells of the uterus (Sprr2f cre+ Mig-6 f/f ). We examined P4 effect and regulation of AKT signaling in the endometrium of mutant mice. RESULTS: Sprr2f cre+ Mig-6 f/f mice developed endometrial hyperplasia. P4 treatment abated the development of endometrial hyperplasia and restored morphological and histological characteristics of the uterus. P4 treatment reduced cell proliferation which was accompanied by decreased AKT signaling and the restoration of stromal PGR and ESR1 expression. Furthermore, our in vitro studies revealed an inhibitory effect of MIG-6 on AKT phosphorylation as well as MIG-6 and AKT protein interactions. CONCLUSIONS: These data suggest that endometrial epithelial cell proliferation is regulated by P4 mediated Mig-6 inhibition of AKT phosphorylation, uncovering new mechanisms of P4 action. This information may help guide more effective non-surgical interventions in the future.

Identification and functional characterization of a novel bipartite nuclear localization sequence in ARID1A.

AT-rich interactive domain-containing protein 1A (ARID1A) is a recently identified nuclear tumor suppressor frequently altered in solid tumor malignancies. We have identified a bipartite-like nuclear localization sequence (NLS) that contributes to nuclear import of ARID1A not previously described. We functionally confirm activity using GFP constructs fused with wild-type or mutant NLS sequences. We further show that cyto-nuclear localized, bipartite NLS mutant ARID1A exhibits greater stability than nuclear-localized, wild-type ARID1A. Identification of this undescribed functional NLS within ARID1A contributes vital insights to rationalize the impact of ARID1A missense mutations observed in patient tumors.

Proteomics of the human endometrial glandular epithelium and stroma from the proliferative and secretory phases of the menstrual cycle.

Despite its importance in reproductive biology and women's health, a detailed molecular-level understanding of the human endometrium is lacking. Indeed, no comprehensive studies have been undertaken to elucidate the important protein expression differences between the endometrial glandular epithelium and surrounding stroma during the proliferative and midsecretory phases of the menstrual cycle. We utilized laser microdissection to harvest epithelial cells and stromal compartments from proliferative and secretory premenopausal endometrial tissue and performed a global, quantitative mass spectrometry-based proteomics analysis. This analysis identified 1224 total proteins from epithelial cells, among which 318 were differentially abundant between the proliferative and secretory phases (q < 0.05), and 1005 proteins from the stromal compartments, 19 of which were differentially abundant between the phases (q < 0.05). Several proteins were chosen for validation by immunohistochemistry in an independent set of uterine tissues, including carboxypeptidase M, tenascin C, neprilysin, and ectonucleotide pyrophosphatase/phosphodiesterase family member 3 (ENPP3). ENPP3, which was elevated in epithelial glandular cells in the secretory phase, was confirmed to be elevated in midsecretory-phase baboon uterine lavage samples and also observed to have an N-linked glycosylated form that was not observed in the proliferative phase. This study provides a detailed view into the global proteomic alterations of the epithelial cells and stromal compartments of the cycling premenopausal endometrium. These proteomic alterations during endometrial remodeling provide a basis for numerous follow-up investigations on the function of these differentially regulated proteins and their role in reproductive biology and endometrial pathologies.

MicroRNAs in endometrial cancers from black and white patients.

OBJECTIVE: Previous studies have identified differences in gene mutations among endometrial cancers from whites and blacks suggesting that differences in tumor biology may explain racial disparities in patient outcome. Micro RNAs (miRNAs) have emerged as regulators of transcript expression and their aberrant expression has been discovered in many diseases, including endometrial cancer. We performed quantitative polymerase chain reaction-based analysis in a set of endometrial cancers to identify whether there are racial differences in miRNA expression. STUDY DESIGN: Tumor cells from 50 stage-I endometrioid endometrial cancer specimens from 41 white and 9 black patients were prepared by laser microdissection and miRNA extracts were analyzed using TaqMan (Life Technologies, Carlsbad, CA) low-density arrays. Statistically significant, differentially expressed miRNAs between blacks and whites were identified using multidimensional scaling, Wilcoxon testing, and analysis of variance. RESULTS: There were no global differences in miRNA expression between endometrial cancers from 41 white and 9 black patients. To minimize potential bias introduced by unbalanced sample size, we performed a subset analysis with stage- and histology-matched specimens from 9 whites and 9 blacks that identified 18 differentially abundant miRNAs (>2-fold at P < .005). Quantitative polymerase chain reaction validated miRNA-337-3p in an independent set of endometrial cancer specimens from 23 white and 24 black women. There were no racial differences in hsa-miR-337-3p expression in normal endometrium. CONCLUSION: These data indicate that hsa-mir-337-3p is more frequently down-regulated in endometrial cancers from whites compared to blacks. Future studies are focused on determining the phenotypic impact of miR-337-3p and whether its differential expression is associated with clinical outcome.

Human ovarian cancer stroma contains luteinized theca cells harboring tumor suppressor gene GT198 mutations.

Ovarian cancer is a highly lethal gynecological cancer, and its causes remain to be understood. Using a recently identified tumor suppressor gene, GT198 (PSMC3IP), as a unique marker, we searched for the identity of GT198 mutant cells in ovarian cancer. GT198 has germ line mutations in familial and early onset breast and ovarian cancers and recurrent somatic mutations in sporadic fallopian tube cancers. GT198 protein has been shown as a steroid hormone receptor coregulator and also as a crucial factor in DNA repair. In this study, using GT198 as a marker for microdissection, we find that ovarian tumor stromal cells harboring GT198 mutations are present in various types of ovarian cancer including high and low grade serous, endometrioid, mucinous, clear cell, and granulosa cell carcinomas and in precursor lesions such as inclusion cysts. The mutant stromal cells consist of a luteinized theca cell lineage at various differentiation stages including CD133(+), CD44(+), and CD34(+) cells, although the vast majority of them are differentiated overexpressing steroidogenic enzyme CYP17, a theca cell-specific marker. In addition, wild type GT198 suppresses whereas mutant GT198 protein stimulates CYP17 expression. The chromatin-bound GT198 on the human CYP17 promoter is decreased by overexpressing mutant GT198 protein, implicating the loss of wild type suppression in mutant cells. Together, our results suggest that GT198 mutant luteinized theca cells overexpressing CYP17 are common in ovarian cancer stroma. Because first hit cancer gene mutations would specifically mark cancer-inducing cells, the identification of mutant luteinized theca cells may add crucial evidence in understanding the cause of human ovarian cancer.

Normal viability of Kai1/Cd82 deficient mice.

The KAI1/CD82 tetraspanin is a widely expressed cell surface molecule thought to organize diverse cellular signaling processes. KAI1/CD82 suppresses metastasis but not tumorigenicity, establishing it as one of a class of metastasis suppressor genes. In order to further assess its functions, we have characterized the phenotypic properties of Kai1/Cd82 deleted mice, including viability, fertility, lymphocyte composition, blood chemistry and tissue histopathology, and of their wild-type and heterozygote littermates. Interestingly, Kai1/Cd82(-/-) showed no obvious genotype associated defects in any of these processes and displayed no genotype associated histopathologic abnormalities after 12 or 18 months of life. Expression profiles of non-immortal, wild-type and Kai1/Cd82(-/-) mouse embryo fibroblast (MEFs) indicated distinct sex-specific and genotype-specific profiles. These data identify 191 and 1,271 differentially expressed transcripts (by twofold at P < 0.01) based on Kai1/CD82 genotype status in female and male MEFs, respectively. Differentially expressed genes in male MEFs were surprisingly enriched for cell division related processes, suggesting that Kai1/Cd82 may functionally affect these processes. This suggests that Kai/Cd82 has an unappreciated role in the early establishment of proliferation and division when challenged with a new environment that might play a role in adaptability to new metastatic sites.

Transcript expression in endometrial cancers from Black and White patients.

OBJECTIVE: Previous studies suggest that differences in molecular features of endometrial cancers between racial groups may contribute to the poorer survival in Blacks. The objective of this investigation was to determine whether gene expression among endometrial cancers is different between Blacks and Whites. METHODS: Fresh frozen tumors from 25 Black patients were matched by stage, grade, and histology to endometrial cancer specimens from 25 White patients. Each case was macrodissected to produce specimens possessing a minimum of 75% cancer cellularity. A subset of 10 matched pairs was also prepared using laser microdissection (LMD) to produce specimens possessing a minimum of 95% cancer cells. Total RNA isolated from each sample was analyzed using the Affymetrix Human Genome U133 Plus 2.0 arrays. Data were analyzed using principal component analysis and binary class comparison analyses. RESULTS: Unsupervised analysis of the 50 endometrial cancers failed to identify global gene expression profiles unique to Black or White patients. In a subset analysis of 10 matched pairs from Blacks and Whites prepared using LMD and macrodissection, unsupervised analysis did not reveal a unique gene expression profile associated with race in either set, but associations were identified that relate to sample preparation technique, histology and stage. CONCLUSIONS: Our microarray data revealed no global gene expression differences and identified few individual gene differences between endometrial cancers from Blacks and Whites. More comprehensive methods of transcriptome analysis could uncover RNAs that may underpin the disparity of outcome or prevalence of endometrial cancers in Blacks and Whites.

PPP2R1A mutations are common in the serous type of endometrial cancer.

Recently unbiased sequencing efforts identified PPP2R1A mutations in clear cell ovarian cancers (OCC). Similar mutations were also noted with high frequency in uterine serous carcinoma. Because the endometrium develops from the same developmental precursors we further examined the hypothesis that PPP2R1A mutations might also occur in diverse histologic subtypes of uterine cancer. We sequenced the PPP2R1A in 22 cell line models of uterine cancer and 10 primary cancers. We found no mutations in the cell lines originally derived from endometrioid (n = 13), undifferentiated (n = 3), clear cell (n = 1), and carcinosarcoma (n = 3) cancers. However, we found a CCC (Pro) to CGC (Arg) codon 179 mutation in the ACI-158 serous carcinoma cell line, a CCC (Pro) to CTC (Leu) in a primary serous carcinoma as well as a CGC (Arg) to CAC (His) codon 258 mutation in a poorly differentiated endometrioid cancer. We sequenced a large panel of endometrial malignancies (n = 181) and found 12 mutants. Importantly, we confirmed a high frequency of mutation in 8 of 25 (32%) serous carcinomas a subtype with well-recognized poor prognosis. Mutations were infrequent in endometrioid cancer and absent in clear cell and carcinosarcoma subtypes. The PPP2R1A mutation regions are conserved among species and known to interact with the regulatory subunits of the PP2A enzyme. PPP2R1A mutant endometrial cancers may represent good candidates for personalized drug therapies particularly for women with the lethal serous histologic variant of uterine cancer.

Evaluation of a Patient With Non-Myoinvasive Uterine Serous Carcinoma Confined to a Polyp and Positive Peritoneal Washings With Somatic ARHGAP35 and KRAS Mutations.

Currently, the application of peritoneal washings as a diagnostic tool for endometrial cancer staging is not well defined. The case described aims to highlight the current ambiguity surrounding the use of peritoneal washings in clinical practice.  A 69-year-old G3P3003 presented to her gynecologist with complaints of new-onset heavy vaginal bleeding. The patient sought an endometrial biopsy, which suggested serous endometrial intraepithelial carcinoma (EIC) focally suspicious for invasive carcinoma, with the involvement of polyps. Based on these results, a robotic-assisted total laparoscopic hysterectomy, bilateral salpingo-oophorectomy, bilateral sentinel lymph node dissection, and omentectomy were performed. Results from her final pathology exhibited a stage IA uterine serous carcinoma (USC) involving a polyp (4.2 cm in greatest dimension) with no myometrial or lymphovascular invasion, but washings were positive for adenocarcinoma. Based on her family history of malignancy, the patient underwent germline panel testing. The patient's somatic tumor testing demonstrated proficient DNA mismatch repair status, microsatellite stability, low tumor mutational burden (4 mut/Mb), low loss of heterozygosity (9%), amplification of the ERBB2 (HER2/neu) gene by both immunohistochemistry (3+, 20% positive) and fluorescence in-situ hybridization. Her tumor also had weakly positive estrogen receptor expression (1+, 10% positive); furthermore, some pathogenic variants in KRAS (c.37G>T), PIK3CA (c.263G>A), and TP53 (c.743G>A) were identified. Given the incongruent findings found with the positive peritoneal washing and negative lymph node involvement in addition to molecular testing, management for this patient was unclear. Ultimately, this case highlights a number of advances within the field of gynecological oncology but also emphasizes the persistent ambiguity and incongruency in the management of patients with early-stage high-risk histologies. Moving forward it will become increasingly important to be able to develop a more standardized process to assess how these diagnostic tools should inform prognosis and treatment plans.

Pharmacological targeting of polyamine and hypusine biosynthesis reduces tumour activity of endometrial cancer.

Endometrial cancer (EC) is a common and deadly cancer in women and novel therapeutic approaches are urgently needed. Polyamines (putrescine, spermidine, spermine) are critical for mammalian cell proliferation and MYC coordinately regulates polyamine metabolism through ornithine decarboxylase (ODC). ODC is a MYC target gene and rate-limiting enzyme of polyamine biosynthesis and the FDA-approved anti-protozoan drug α-difluoromethylornithine (DFMO) inhibits ODC activity and induces polyamine depletion that leads to tumour growth arrest. Spermidine is required for the hypusine-dependent activation of eukaryotic translation initiation factors 5A1 (eIF5A1) and 5A2 (eIF5A2) and connects the MYC/ODC-induced deregulation of spermidine to eIF5A1/2 protein translation, which is increased during cancer cell proliferation. We show that eIF5A1 is significantly upregulated in EC cells compared to control cells (p=.000038) and that combined pharmacological targeting of ODC and eIF5A hypusination with cytostatic drugs DFMO and N1-guanyl-1,7-diaminoheptane (GC7), respectively, reduces eIF5A1 activation and synergistically induces apoptosis in EC cells. In vivo, DFMO/GC7 suppressed xenografted EC tumour growth in mice more potently than each drug alone compared to control (p=.002) and decreased putrescine (p=.045) and spermidine levels in tumour tissues. Our data suggest DFMO and GC7 combination therapy may be useful in the treatment or prevention of EC.

Peptide ancestry informative markers in uterine neoplasms from women of European, African, and Asian ancestry.

Characterization of ancestry-linked peptide variants in disease-relevant patient tissues represents a foundational step to connect patient ancestry with disease pathogenesis. Nonsynonymous single-nucleotide polymorphisms encoding missense substitutions within tryptic peptides exhibiting high allele frequencies in European, African, and East Asian populations, termed peptide ancestry informative markers (pAIMs), were prioritized from 1000 genomes. In silico analysis identified that as few as 20 pAIMs can determine ancestry proportions similarly to >260K SNPs (R2 = 0.99). Multiplexed proteomic analysis of >100 human endometrial cancer cell lines and uterine leiomyoma tissues combined resulted in the quantitation of 62 pAIMs that correlate with patient race and genotype-confirmed ancestry. Candidates include a D451E substitution in GC vitamin D-binding protein previously associated with altered vitamin D levels in African and European populations. pAIMs will support generalized proteoancestry assessment as well as efforts investigating the impact of ancestry on the human proteome and how this relates to the pathogenesis of uterine neoplasms.

Improving Risk Assessment for Metastatic Disease in Endometrioid Endometrial Cancer Patients Using Molecular and Clinical Features: An NRG Oncology/Gynecologic Oncology Group Study.

Objectives: A risk assessment model for metastasis in endometrioid endometrial cancer (EEC) was developed using molecular and clinical features, and prognostic association was examined. Methods: Patients had stage I, IIIC, or IV EEC with tumor-derived RNA-sequencing or microarray-based data. Metastasis-associated transcripts and platform-centric diagnostic algorithms were selected and evaluated using regression modeling and receiver operating characteristic curves. Results: Seven metastasis-associated transcripts were selected from analysis in the training cohorts using 10-fold cross validation and incorporated into an MS7 classifier using platform-specific coefficients. The predictive accuracy of the MS7 classifier in Training-1 was superior to that of other clinical and molecular features, with an area under the curve (95% confidence interval) of 0.89 (0.80-0.98) for MS7 compared with 0.69 (0.59-0.80) and 0.71 (0.58-0.83) for the top evaluated clinical and molecular features, respectively. The performance of MS7 was independently validated in 245 patients using RNA sequencing and in 81 patients using microarray-based data. MS7 + MI (myometrial invasion) was preferrable to individual features and exhibited 100% sensitivity and negative predictive value. The MS7 classifier was associated with lower progression-free and overall survival (p ≤ 0.003). Conclusion: A risk assessment classifier for metastasis and prognosis in EEC patients with primary tumor derived MS7 + MI is available for further development and optimization as a companion clinical support tool.

Proteomic analysis of stage I endometrial cancer tissue: identification of proteins associated with oxidative processes and inflammation.

OBJECTIVE: The present study aimed to identify differentially expressed proteins employing a high resolution mass spectrometry (MS)-based proteomic analysis of endometrial cancer cells harvested using laser microdissection. METHODS: A differential MS-based proteomic analysis was conducted from discrete epithelial cell populations gathered by laser microdissection from 91 pathologically reviewed stage I endometrial cancer tissue samples (79 endometrioid and 12 serous) and 10 samples of normal endometrium from postmenopausal women. Hierarchical cluster analysis of protein abundance levels derived from a spectral count analysis revealed a number of proteins whose expression levels were common as well as unique to both histologic types. An independent set of endometrial cancer specimens from 394 patients were used to externally validate the differential expression of select proteins. RESULTS: 209 differentially expressed proteins were identified in a comparison of stage I endometrial cancers and normal post-menopausal endometrium controls (Q<0.005). A number of differentially abundant proteins in stage I endometrial cancer were identified and independently validated by western blot and tissue microarray analyses. Multiple proteins identified with elevated abundance in stage I endometrial cancer are functionally associated with inflammation (annexins) and oxidative processes (peroxiredoxins). PRDX1 and ANXA2 were both confirmed as being overexpressed in stage I cancer compared to normal endometrium by independent TMA (Q=0.008 and Q=0.00002 respectively). CONCLUSIONS: These data provide the basis for further investigation of previously unrecognized novel pathways involved in early stage endometrial carcinogenesis and provide possible targets for prevention strategies that are inclusive of both endometrioid and serous histologic subtypes.

An immune competent orthotopic model of endometrial cancer with metastasis.

Endometrial cancer is the most common gynecologic malignancy in the U.S. with metastatic disease remaining the major cause of patient death. Therapeutic strategies have remained essentially unchanged for decades. A significant barrier to progression in treatment modalities stems from a lack of clinically applicable in vivo models to accurately mimic endometrial cancer; specifically, ones that form distant metastases and maintain an intact immune system. To address this problem, we have established the first immune competent murine orthotopic tumor model for metastatic endometrial cancer by creating a green fluorescent protein labeled cell line from an endometrial cancer that developed in a Pgr cre/+ Pten f/f Kras G12D genetically engineered mouse. These cancer cells were grafted into the abraded uterine lumen of ovariectomized recipient mice treated with estrogen and subsequently developed local and metastatic endometrial tumors. We noted primary tumor formation in 59% mixed background and 86% of C57BL/6 animals at 4 weeks and distant lung metastases in 78% of mice after 2 months. This immunocompetent orthotopic tumor model closely resembles some human metastatic endometrial cancer, modeling both local metastasis and hematogenous spread to lung and has significant potential to advance the study of endometrial cancer and its metastasis.

Ubiquitin Carboxyl-Terminal Hydrolase L1 (UCHL1) Promotes Uterine Serous Cancer Cell Proliferation and Cell Cycle Progression.

Uterine serous carcinoma (USC) is the most aggressive form of endometrial cancer, with poor survival rates and high recurrence risk. Therefore, the purpose of this study was to identify therapeutic targets that could aid in the management of USC. By analyzing endometrial cancer samples from The Cancer Genome Atlas (TCGA), we found Ubiquitin Carboxyl-Terminal Hydrolase L1 (UCHL1) to be highly expressed in USC and to correlate with poorer overall survival. UCHL1 silencing reduced cell proliferation in vitro and in vivo, cyclin B1 protein levels and cell cycle progression. Further studies showed that UCHL1 interacts with cyclin B1 and increases cyclin B1 protein stability by deubiquitination. Treatment of USC-bearing mice with the UCHL1-specific inhibitor reduced tumor growth and improved overall survival. Our findings suggest that cyclin B1 is a novel target of UCHL1 and targeting UCHL1 is a potential therapeutic strategy for USC.

Human Ovarian Cancer Tumor Formation in Severe Combined Immunodeficient (SCID) Pigs.

Ovarian cancer (OvCa) is the most lethal gynecologic malignancy, with two-thirds of patients having late-stage disease (II-IV) at diagnosis. Improved diagnosis and therapies are needed, yet preclinical animal models for ovarian cancer research have primarily been restricted to rodents, for data on which can fail to translate to the clinic. Thus, there is currently a need for a large animal OvCa model. Therefore, we sought to determine if pigs, being more similar to humans in terms of anatomy and physiology, would be a viable preclinical animal model for OvCa. We injected human OSPC-ARK1 cells, a chemotherapy-resistant primary ovarian serous papillary carcinoma cell line, into the neck muscle and ear tissue of four severe combined immune deficient (SCID) and two non-SCID pigs housed in novel biocontainment facilities to study the ability of human OvCa cells to form tumors in a xenotransplantation model. Tumors developed in ear tissue of three SCID pigs, while two SCID pigs developed tumors in neck tissue; no tumors were detected in non-SCID control pigs. All tumor masses were confirmed microscopically as ovarian carcinomas. The carcinomas in SCID pigs were morphologically similar to the original ovarian carcinoma and had the same immunohistochemical phenotype based on expression of Claudin 3, Claudin 4, Cytokeratin 7, p16, and EMA. Confirmation that OSPC-ARK1 cells form carcinomas in SCID pigs substantiates further development of orthotopic models of OvCa in pigs.