Antitumor T cells are subject to multiple mechanisms of negative regulation1–3. Recent findings that innate lymphoid cells (ILCs) regulate adaptive T cell responses4–6 led us to examine the regulatory potential of ILCs in the context of cancer. We identified a unique ILC population that inhibits tumor-infiltrating lymphocytes (TILs) from high-grade serous tumors, defined their suppressive capacity in vitro, and performed a comprehensive analysis of their phenotype. Notably, the presence of this CD56+CD3− population in TIL cultures was associated with reduced T cell numbers, and further functional studies demonstrated that this population suppressed TIL expansion and altered TIL cytokine production. Transcriptome analysis and phenotypic characterization determined that regulatory CD56+CD3− cells exhibit low cytotoxic activity, produce IL-22, and have an expression profile that overlaps with those of natural killer (NK) cells and other ILCs. NKp46 was highly expressed by these cells, and addition of anti-NKp46 antibodies to TIL cultures abrogated the ability of these regulatory ILCs to suppress T cell expansion. Notably, the presence of these regulatory ILCs in TIL cultures corresponded with a striking reduction in the time to disease recurrence. These studies demonstrate that a previously uncharacterized ILC population regulates the activity and expansion of tumor-associated T cells.

Liver kinase B1 (LKB1) is a tumor suppressor ubiquitously expressed serine/threonine protein kinase involved in energy metabolism and cellular polarity. In microarray experiments that compared normal tubal epithelium with high-grade serous carcinoma (HGSC), we observed a decrease in LKB1 mRNA expression in HGSC. In this study, we demonstrate that loss of cytoplasmic and nuclear LKB1 protein expression is frequently observed in tubal cancer precursor lesions as well as in both sporadic and hereditary HGSCs compared with other ovarian cancer histotypes. Bi-allelic genomic loss of LKB1 in HGSC did not account for the majority of cases with a decrease in protein expression. In vitro, shLKB1-fallopian tube epithelial (FTE) cells underwent premature cellular arrest and in ex vivo FTE culture, LKB1 loss and p53 mutant synergized to disrupt apical to basal polarity and decrease the number of ciliated cells. Overexpression of cyclin E1 allowed for bypass of LKB1-induced cellular arrest, and increased both proliferation and anchorage-independent growth of transformed FTE cells. These data suggest that LKB1 loss early in ovarian serous tumorigenesis has an integral role in tumor promotion by disrupting apical to basal polarity in the presence of mutated p53 in fallopian tube cells.

Background: CCAAT/enhancer binding protein delta (C/EBPδ,CEBPD), a gene part of the highly conserved basicleucine zipper (b-ZIP) domain of transcriptional factors, is downregulated in 65% of high grade serous carcinomas of the ovary (HGSC). Overexpression of C/EBPδ in different tumours, such as glioblastoma and breast cancer either promotes tumour progression or inhibits growth and has low expression in normal tissue until activated by cytotoxic stressors. Methods: Higher overall expression of C/EBPδ in the luteal phase of the menstrual cycle prompted us to investigate the role of C/EBPδ in carcinogenesis. In vitro experiments were conducted in fallopian tube cell samples and cancer cell lines to investigate the role of C/EBPδ in proliferation, migration, and the epithelial to mesenchymal transition. Findings: Expression of C/EBPδ induced premature cellular arrest and decreased soft agar colony formation. Loss of C/EBPδ in epithelial cancer cell lines did not have significant effects on proliferation, yet overexpression demonstrated downregulation of growth, similar to normal fallopian tube cells. C/EBPδ promoted a partial mesenchymal to epithelial (MET) phenotype by upregulating E-cadherin and downregulating Vimentin and N-cadherin in FTE cells and increased migratory activity, which suggests a regulatory role in the epithelial-mesenchymal plasticity of these cells. Interpretation: Our findings suggest that C/EBPδ regulates the phenotype of normal fallopian tube cells by acting on downstream regulatory factors that are implicated in the development of ovarian serous carcinogenesis.

Epithelial ovarian cancer represents a group of heterogeneous diseases with high grade serous cancer (HGSC) representing the most common histotype. Molecular profiles of precancerous lesions found in the fallopian tube have implicated this tissue as the presumptive site of origin of HGSC. Precancerous lesions are primarily found in the distal fallopian tube (fimbria), near the ovary relative to the proximal tissue (ampulla), nearer to the uterus. The proximity of the fimbria to the ovary and the link between ovulation, through follicular fluid release, and ovarian cancer risk led us to examine transcriptional responses of fallopian tube epithelia (FTE) at the different anatomical sites of the human fallopian tube. Gene expression profiles of matched FTE from the fimbria and from premenopausal women resulted in differentially expressed genes (DEGs): CYYR1, SALL1, FOXP2, TAAR1, AKR1C2/C3/C4, NMBR, ME1 and GSTA2. These genes are part of the antioxidant, stem and inflammation pathways. Comparisons between the luteal phase (post-ovulation) to the follicular phase (pre-ovulation) demonstrated greater differences in DEGs than a comparison between fimbria and fallopian tube anatomical differences alone. This data suggests that cyclical transcriptional changes experienced in pre-menopause are inherent physiological triggers that expose the FTE in the fimbria to cytotoxic stressors. These cyclical exposures induce transcriptional changes reflective of genotoxic and cytotoxic damage to the FTE in the fimbria which are closely related to transcriptional and genomic alterations observed in ovarian cancer.

Genetic and reproductive factors predicate epidemiological risk factors underlying epithelial ovarian cancer. The fallopian tube epithelia (FTE), the presumptive etiological site of high-grade serous ovarian cancer (HGSC), is a hormonal responsive tissue. Estrogen is known to promote cell proliferation and its metabolism produces reactive oxygen species that damage DNA and promote tumorigenesis. Estrogen receptor (ER) is rarely mutated, amplified or deleted in HGSC, yet only 10% of patients respond to anti-estrogen treatment, suggesting that intrinsic variables to the ER pathway contribute to this clinical outcome. TP53 mutations occur in almost 100% of HGSCs, indicating that mutated p53 supports a model as an early event in the pathogenesis of HGSC. We hypothesized that in the presence of dysfunctional p53, subsequent promiscuous binding of ER will yield aberrant signaling, ultimately significantly contributing to cellular transformation. Mutant p53 and ER co-localize in FTE cells, suggesting potential synergy. We established cell lines with p53 mutations and treated them with estradiol, an estrogen analog, to observe any changes in response. The genome binding sites of ER-regulated transcription factors were then identified and mapped by whole genome chromatin immunoprecipitation-deep sequencing (ChIP-Seq). The data generated will facilitate the development of gene signatures that will predict response to anti-estrogen therapy in serous ovarian cancer patients, and contribute to the discovery of biomarkers to more accurately identify patients who will benefit from hormonal therapies. Citation Format: Leah V. Dodds, Omar L. Nelson, Ramlogan Sowamber, Andres Rodrigues, Victoria de Castro, Wendell Henry, Guillermo Morales, Brian Slomovitz, Patricia Shaw, Sophia H. George. Estrogen receptor signaling in FTE of BRCA mutation carriers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1454. doi:10.1158/1538-7445.AM2017-1454

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<p>Microbial abundance by histotype</p>

<p>Additional statistical methods</p>