Janet M. Lee

A three-dimensional microenvironment alters protein expression and chemosensitivity of epithelial ovarian cancer cells in vitro.

For many cancers, there is a real need for more effective therapies. Although many drugs show promising results in vitro, most fail to translate into an in vivo model system, and only ∼5% show anti-tumor activity in clinical trials. It remains a significant challenge to accurately replicate in vitro the complex in vivo microenvironment in which cancers thrive, but this will be key to increasing the success of translating novel therapies into clinical practice. Three-dimensional (3D) cell culture models may better mimic primary tumors in vivo than traditional two-dimensional (2D) cultures. Therefore, we established and characterized 3D in vitro models of 31 epithelial ovarian cancer (EOC) cell lines, compared their biological and molecular features with 2D cultures and primary tumors, and tested their efficacy as models for evaluating chemoresponse. When cultured in 3D using polyhydroxoethylamethacrylate-coated plastics, EOC lines formed multicellular aggregates that could be classified as ‘large dense’, ‘large loose’, and ‘small’, based on size, light permeability, and proportion of cells incorporated into the complex structures. Features of histological differentiation characteristic of primary tumors that were not present in 2D cultures were restored in 3D. For many cell lines, the transition from a 2D to 3D microenvironment induced changes in the expression of several biomarkers relevant to disease. Generally, EOC cell lines proliferated more slowly and were more chemoresistant in 3D compared with 2D culture. In summary, 3D models of EOCs better reflect the histological, biological, and molecular features of primary tumors than the same cells cultured using traditional 2D techniques; 3D in vitro models also exhibit different sensitivities to chemotherapeutic agents compared with 2D models, which may have a significant impact on the success of drug testing pipelines for EOC. These findings could also impact in vitro modeling approaches and drug development strategies for other solid tumor types.

Molecular Analysis of Mixed Endometrioid and Serous Adenocarcinoma of the Endometrium

Background The molecular biology and cellular origins of mixed type endometrial carcinomas (MT-ECs) are poorly understood, and a Type II component of 10 percent or less may confer poorer prognoses. Methodology/Principal Findings We studied 10 cases of MT-EC (containing endometrioid and serous differentiation), 5 pure low-grade endometrioid adenocarcinoma (EAC) and 5 pure uterine serous carcinoma (USC). Endometrioid and serous components of the MT-ECs were macrodissected and the expression of 60 candidate genes compared between MT-EC, pure USC and pure EAC. We found that four genes were differentially expressed when MT-ECs were compared to pure low-grade EAC: CDKN2A (P = 0.006), H19 (P = 0.010), HOMER2 (P = 0.009) and TNNT1 (P = 0.006). Also while we found that even though MT-ECs closely resembled the molecular profiles of pure USCs, they also exhibit lower expression of PAX8 compared to all pure cases combined (P = 0.035). Conclusion Our data suggest that MT-EC exhibits the closest molecular and epidemiological similarities to pure USC and supports clinical observations that suggest patients with MT-EC should receive the same treatment as patients with pure serous carcinoma. Novel specific markers of MT-EC could be of diagnostic utility and could represent novel therapeutic targets in the future.

Identification of novel candidate biomarkers of epithelial ovarian cancer by profiling the secretomes of three‐dimensional genetic models of ovarian carcinogenesis

Epithelial ovarian cancer (EOC) is still considered the most lethal gynecological malignancy and improved early detection of ovarian cancer is crucial to improving patient prognoses. To address this need, we tested whether candidate EOC biomarkers can be identified using three‐dimensional (3D) in vitro models. We quantified changes in the abundance of secreted proteins in a 3D genetic model of early‐stage EOC, generated by expressing CMYC and KRASG12V in TERT‐immortalized normal ovarian epithelial cells. Cellular proteins were labeled in live cells using stable isotopic amino acid analogues, and secreted proteins identified and quantified using liquid chromatography‐tandem mass spectrometry. Thirty‐seven and 55 proteins were differentially expressed by CMYC and CMYC+KRASG12V expressing cells respectively (p < 0.05; >2‐fold). We evaluated expression of the top candidate biomarkers in ∼210 primary EOCs: CHI3L1 and FKBP4 are both expressed by >96% of primary EOCs, and FASN and API5 are expressed by 86 and 75% of cases. High expression of CHI3L1 and FKBP4 was associated with worse patient survival (p = 0.042 and p = 0.002, respectively). Expression of LGALS3BP was positively associated with recurrence (p = 0.0001) and suboptimal debulking (p = 0.018) suggesting that these proteins may be novel prognostic biomarkers. Furthermore, within early stage tumours (I/II), high expression of API5, CHI3L1 and FASN was associated with high tumour grade (p = 3 × 10−4, p = 0.016, p = 0.010, respectively). We show in vitro cell biology models of early‐stage cancer development can be used to identify novel candidate biomarkers for disease, and report the identification of proteins that represent novel potential candidate diagnostic and prognostic biomarkers for this highly lethal disease.

Src as a novel therapeutic target for endometriosis.

BACKGROUND Endometriosis is a common condition that is associated with an increased risk of developing ovarian carcinoma. Improved in vitro models of this disease are needed to better understand how endometriosis, a benign disease, can undergo neoplastic transformation, and for the development of novel treatment strategies to prevent this progression. METHODS We describe the generation and in vitro characterization of novel TERT immortalized ovarian endometriosis epithelial cell lines (EEC16-TERT). RESULTS Expression of TERT alone was sufficient to immortalize endometriosis epithelial cells. TERT immortalization induces an epithelial-to-mesenchymal transition and perturbation in the expression of genes involved in the development of ovarian cancer. EEC16-TERT was non-tumorigenic when xenografted into immunocompromised mice but grew in anchorage-independent growth assays in an epidermal growth factor and hydrocortisone dependent manner. Colony formation in agar was abolished by inhibition of Src, and the Src pathway was found to be activated in human endometriosis lesions. CONCLUSIONS This new in vitro model system mimics endometriosis and the early stages of neoplastic transformation in the development of endometriosis associated ovarian cancer. We demonstrate the potential clinical relevance of this model by identifying Src activation as a novel pathway in endometriosis that could be targeted therapeutically, perhaps as a novel strategy to manage endometriosis clinically, or to prevent the development of endometriosis-associated ovarian cancer.

The PAX8 cistrome in epithelial ovarian cancer.

PAX8 is a lineage-restricted transcription factor that is expressed in epithelial ovarian cancer (EOC) precursor tissues, and in the major EOC histotypes. Frequent overexpression of PAX8 in primary EOCs suggests this factor functions as an oncogene during tumorigenesis, however, the biological role of PAX8 in EOC development is poorly understood. We found that stable knockdown of PAX8 in EOC models significantly reduced cell proliferation and anchorage dependent growth in vitro, and attenuated tumorigenicity in vivo. Chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) and transcriptional profiling were used to create genome-wide maps of PAX8 binding and putative target genes. PAX8 binding sites were significantly enriched in promoter regions (p < 0.05) and superenhancers (p < 0.05). MEME-ChIP analysis revealed that PAX8 binding sites overlapping superenhancers or enhancers, but not promoters, were enriched for JUND/B and ARNT/AHR motifs. Integrating PAX8 ChIP-seq and gene expression data identified PAX8 target genes through their associations within shared topological association domains. Across two EOC models we identified 62 direct regulatory targets based on PAX8 binding in promoters and 1,330 putative enhancer regulatory targets. SEPW1, which is involved in oxidation-reduction, was identified as a PAX8 target gene in both cell line models. While the PAX8 cistrome exhibits a high degree of cell-type specificity, analyses of PAX8 target genes and putative cofactors identified common molecular targets and partners as candidate therapeutic targets for EOC.

JUN-Mediated downregulation of EGFR signaling is associated with resistance to gefitinib in EGFR-mutant NSCLC cell lines

Mutations or deletions in exons 18–21 in the EGFR) are present in approximately 15% of tumors in patients with non–small cell lung cancer (NSCLC). They lead to activation of the EGFR kinase domain and sensitivity to molecularly targeted therapeutics aimed at this domain (gefitinib or erlotinib). These drugs have demonstrated objective clinical response in many of these patients; however, invariably, all patients acquire resistance. To examine the molecular origins of resistance, we derived a set of gefitinib-resistant cells by exposing lung adenocarcinoma cell line, HCC827, with an activating mutation in the EGFR tyrosine kinase domain, to increasing gefitinib concentrations. Gefitinib-resistant cells acquired an increased expression and activation of JUN, a known oncogene involved in cancer progression. Ectopic overexpression of JUN in HCC827 cells increased gefitinib IC50 from 49 nmol/L to 8 μmol/L (P < 0.001). Downregulation of JUN expression through shRNA resensitized HCC827 cells to gefitinib (IC50 from 49 nmol/L to 2 nmol/L; P < 0.01). Inhibitors targeting JUN were 3-fold more effective in the gefitinib-resistant cells than in the parental cell line (P < 0.01). Analysis of gene expression in patient tumors with EGFR-activating mutations and poor response to erlotinib revealed a similar pattern as the top 260 differentially expressed genes in the gefitinib-resistant cells (Spearman correlation coefficient of 0.78, P < 0.01). These findings suggest that increased JUN expression and activity may contribute to gefitinib resistance in NSCLC and that JUN pathway therapeutics merit investigation as an alternate treatment strategy. Mol Cancer Ther; 16(8); 1645–57. ©2017 AACR.

Integrated Molecular Profiling Studies to Characterize the Cellular Origins of High-Grade Serous Ovarian Cancer

Historically, high-grade serous ovarian cancers (HGSOCs) were thought to arise from ovarian surface epithelial cells (OSECs) but recent data implicate fallopian tube secretory epithelial cells (FTSECs) as the major precursor. We performed transcriptomic and epigenomic profiling to characterize molecular similarities between OSECs, FTSECs and HGSOCs. Transcriptomic signatures of FTSECs were preserved in most HGSOCs reinforcing FTSECs as the predominant cell-of-origin; though an OSEC-like signature was associated with increased chemosensitivity (Padj = 0.03) and was enriched in proliferative-type tumors, suggesting a dualistic model for HGSOC origins. More super-enhancers (SEs) were shared between FTSECs and HGSOCs than between OSECS and HGSOCs (P < 2.2 × 10−16). SOX18, ELF3 and EHF transcription factors (TFs) coincided with HGSOC SEs and represent putative novel drivers of tumor development. Our integrative analyses support a predominantly fallopian origin for HGSOCs and indicate tumorigenesis may be driven by different TFs according to cell-of-origin.

Multi-Tissue Transcriptome-Wide Association Studies Identify 21 Novel Candidate Susceptibility Genes for High Grade Serous Epithelial Ovarian Cancer

Genome-wide association studies (GWASs) have identified about 30 different susceptibility loci associated with high grade serous ovarian cancer (HGSOC) risk. We sought to identify potential susceptibility genes by integrating the risk variants at these regions with genetic variants impacting gene expression and splicing of nearby genes. We compiled gene expression and genotyping data from 2,169 samples for 6 different HGSOC-relevant tissue types. We integrated these data with GWAS data from 13,037 HGSOC cases and 40,941 controls, and performed a transcriptome-wide association study (TWAS) across >70,000 significantly heritable gene/exon features. We identified 24 transcriptome-wide significant associations for 14 unique genes, plus 90 significant exon-level associations in 20 unique genes. We implicated multiple novel genes at risk loci, e.g. LRRC46 at 19q21.32 (TWAS P=1×10−9) and a PRC1 splicing event (TWAS P=9×10−8) which was splice-variant specific and exhibited no eQTL signal. Functional analyses in HGSOC cell lines found evidence of essentiality for GOSR2, INTS1, KANSL1 and PRC1; with the latter gene showing levels of essentiality comparable to that of MYC. Overall, gene expression and splicing events explained 41% of SNP-heritability for HGSOC (s.e. 11%, P=2.5×10−4), implicated at least one target gene for 6/13 distinct genome-wide significant regions and revealed 2 known and 26 novel candidate susceptibility genes for HGSOC. STATEMENT OF SIGNIFICANCE For many ovarian cancer risk regions, the target genes regulated by germline genetic variants are unknown. Using expression data from >2,100 individuals, this study identified novel associations of genes and splicing variants with ovarian cancer risk; with transcriptional variation now explaining over one-third of the SNP-heritability for this disease.