TY - JOUR
AU - Levanon,, Keren
AB - Abstract The fallopian tube secretory epithelial cells (FTSECs) are the cell-of-origin of most high-grade serous ovarian carcinomas (HGSOC). FTSECs are repeatedly exposed to inflammation induced by follicular fluid (FF) that is released with every ovulation cycle throughout a woman’s reproductive years. Uninterrupted ovulation cycles are an established risk factor for HGSOC. Stimuli present in the FF induce an inflammatory environment which may cause DNA damage eventually leading to serous tumorigenesis. With the aim of elucidating possible mechanistic pathways, we established an ‘ex vivo persistent ovulation model’ mimicking the repeated exposure of human benign fallopian tube epithelium (FTE) to FF. We performed mass spectrometry analysis of the secretome of the ex vivo cultures as well as confirmatory targeted expressional and functional analyses. We demonstrated activation of the NF-κB pathway and upregulation of miR-155 following short-term exposure of FTE to human FF. Increased expression of miR-155 was also detected in primary HGSOC tumors compared with benign primary human FTE and corresponded with changes in the expression of miR-155 target genes. The phenotype of miR-155 overexpression in FTSEC cell line is of increased migratory and altered adhesion capacities. Overall, activation of the NF-κB-miR-155 axis in FTE may represent a possible link between ovulation-induced inflammation, DNA damage, and transcriptional changes that may eventually lead to serious carcinogenesis. Introduction Although inflammation is a normal and essential part of the host defense mechanisms, as a chronic state, the inflammatory process can promote tumor initiation and progression (1). This results from induction of a tumor-supporting microenvironment and, possibly, from release of nitrogen and reactive oxygen species which cause DNA damage (2). The fallopian tube epithelium (FTE), and more specifically the fallopian tube secretory epithelial cell (FTSEC), is the cell-of-origin of most high-grade serous ovarian carcinomas (HGSOCs) (3,4). These cells are repeatedly exposed to systemic hormonal fluctuations with every ovulation cycle throughout a woman’s reproductive years and are subject to local stimuli inflicted by exposure to follicular fluid (FF). Proteomic analysis of FF reveals over 700 different, mostly unexplored proteins (5). Among the proteins known to play a role in HGSOC development are luteinizing hormone, follicle-stimulating hormone (6), as well as IGF binding proteins, metalloproteinases and Serpins, transforming growth factor beta family members and various growth factors and inflammatory proteins [such as interleukin (IL)-1α and tumor necrosis factor alpha (TNFα)] (5,7). Non-protein factors such as estradiol, luekotrienes and radical oxygen species have also been described (5–7). The inflammatory insult induced by the physiological process of ovulation is suspected to be a main contributor to the neoplastic transformation in this tissue (8). We previously reported that a single short-term exposure of FTE to FF results in profound DNA damage and altered transcriptomic profile (9). Similar findings have been shown in similar models (10–12). Moreover, most epidemiological studies indicate that risk factors for HGSOC are associated with uninterrupted ovulation, including nulliparity, early menarche, late menopause and lack of use of oral contraceptives (13). Although the original ‘incessant ovulation theory’ was published almost half a century ago (14), the molecular events leading to the initiation and progression of the malignancy in the FTE of the fimbria still remain elusive after all these years. One of the best studied chronic inflammatory master regulators linked to cancer initiation and progression is the nuclear factor-κB (NF-κB) transcription factor (15). NF-κB activation (by translocation of the active subunits from the cytoplasm to the nucleus) induces pro-proliferative and anti-apoptotic genes and affects many other signaling pathways, including those involving STAT3, AP1, interferon regulatory factors, NRF2, Notch, WNT-β-catenin and p53. Its inhibition in HGSOC results in reduced cell proliferation, anoikis resistance and induced apoptotic cell death, as well as synergistically increases chemotherapy efficacy (16). MicroRNAs (miRs) are well established as gene regulators involved in various cancer-associated biological processes, including cell cycle, inflammation and regulation of tumor suppressor genes (17,18). MiR-155 is a key regulator of the immune system, and its expression has been associated with pathologic chronic inflammation (19) as well as with various types of cancer, including breast, colon and endometrioid ovarian cancer (20,21). In myeloid cells, inflammatory stimuli lead to the activation of NF-κB, which transiently activates miR-155 transcription (20). The rapidly and highly transcribed miR-155 amplifies NF-κB activation in a positive feed forward loop which is subsequently negated by other miRNAs (22). Several studies implicate miR-155 in genomic instability, including downregulation of the core mismatch repair proteins MLH1, MSH2 and MSH6 (23) and decreased homologous recombination efficiency by downregulation of RAD51 (24). HGSOC share several unique molecular and pathological features: almost 100% of tumors harbor a TP53 mutation (25), ~50% exhibit defective homologous recombination DNA repair due to genetic and epigenetic alterations (26) and the tendency of malignant cells to exfoliate from their basement membrane at an early stage, escape anoikis and adhere to the peritoneal mesothelium (27). Shedding of malignant cells occurs already at the intraepithelial carcinoma (STIC) stage (28). Full understanding of molecular mechanisms, specifically those related to ovulation, that inflict these biological aberrations in FTSECs is not yet available. Here we present the most in-depth investigation of the effect of FF on human FTSECs using ex vivo models of single and persistent ovulation and propose a molecular mechanism linking ovulation to inflammation and serous carcinogenesis through activation of the NF-κB-miR155 axis. Materials and methods Primary tissues processing Fresh benign FT fimbriae were allocated from the Chaim Sheba Medical Center institutional tissue bank with appropriate ethical approvals. The participating patients underwent salpingectomy due to gynecological conditions not affecting the FTE, including early-stage cervical or endometrial cancer, uterine leiomyoma, risk reduction and uterine prolapse. The range of tissue donors’ age was 30–83. The clinical data of the tissue donors are presented in Supplementary Table S1 (available at Carcinogenesis online). FTE was harvested and cultured on Transwell inserts, as described before (9,29). FF experiments FF samples were obtained from women undergoing oocyte retrieval for in vitro fertilization treatments due to various conditions, in accordance to ethical committee approvals. The age range of FF donors was 27–45. The indications for oocyte aspiration included male infertility, pre-gestational genetic diagnosis and mechanical infertility. Women with hormonal abnormalities were excluded. In each experiment, we used a pool of 10 FF samples mixed in even proportions. The FF was centrifuged to remove blood cells and frozen in aliquots. Before use the FF was heat-inactivated at 56°C for 30 min. Ultroser G serum substitute (PALL Life Sciences, Cergy Saint Christophe, France) was added at a final concentration of 1% to both FF and culture medium, which was used as control in FF experiments (9). The ‘persistent ex vivo ovulation model’ was developed to recapitulate the cyclic nature of ovulation with repeated short-term exposures to FF followed by recovery phases. Overall, FTEs from 10 healthy donors were cultured until confluence on the upper compartment of 0.4 µm pore polyester membrane Transwell inserts (Corning, Corning, NY), coated with human collagen IV (Sigma–Aldrich, St. Louis, MO), forming 10 independent experiments. Feeding DMEM/F12 medium (Biological Industries, Beit Ha’Emek, Israel) was supplied to the bottom compartment, mimicking the blood supply to the basal aspect of the FTE monolayer in vivo. As previously reported (29), FF or culture medium (as control) was applied to the upper compartment representing the apical/luminal aspect of the FTE monolayer for 4 h, then washed thoroughly with phosphate-buffered saline. One hundred microliter fresh phosphate-buffered saline was then applied to the upper compartment for 18–24 h to allow accumulation of secreted proteins. The secretome-containing phosphate-buffered saline was collected before repeating the experiment daily for up to 5 weeks. At the end of the experiment, cells were harvested and RNA was extracted. The percentage of secretory cells in a FTE ex vivo culture system was previously reported (29) to be in the range of 50%–70%, with minor changes during up to 15 days in culture, well after confluence has been reached (at day 6 approximately). In this research, ex vivo cultures were confluent before initiation of the experiment, and the proportion of FTSECs remained stable. Proteomic analysis The secreted protein fractions were lysed in 8 M urea in 100 mM Tris–HCl (pH 8.5), followed by overnight in-solution trypsin digestion. Resulting peptides were purified on C18StageTips (3M Empore™, St. Paul, MN). Peptides were analyzed by liquid-chromatography using the EASY-nLC1000 HPLC coupled with high-resolution mass spectrometric analysis on the Q-Exactive Plus mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). Peptides were separated on 50 cm EASY-spray columns (Thermo Fisher Scientific) with a single run of 240 min. Mass spectrometry (MS) acquisition was performed in a data-dependent mode with selection of the top 10 peptides from each MS spectrum for fragmentation and MS/MS analysis. Raw MS files were analyzed in the MaxQuant software and the Andromeda search engine (30). A database search was performed using the Uniprot database and included carbamidomethyl-cysteine as a fixed modification, and N-terminal acetylation and methionine oxidation as variable modifications. A reverse decoy database was used to determine false discovery rate (FDR) of 1% at the peptide and protein levels. The label-free algorithm in MaxQuant was used to retrieve the quantitative information. All the statistical analyses were performed on the Perseus software (31). The data were filtered to include proteins with valid values in at least 70% of the samples. Missing values were then imputed to represent low abundance proteins by replacing them with random, low-intensity values that form a normal distribution. Student’s t-test was performed (FDR 0.05) to distinguish the significantly regulated proteins between the control and FF-treated samples. Paired t-test was performed in MATLAB with FDR 0.05. Pathway enrichment analysis was performed using Fisher’s exact test (FDR 0.02), and protein–protein interaction networks were visualized in STRING database. MiRNA microarray All experiments were performed using Affymetrix miR arrays (Affymetrix, Santa Clara, CA), according to the manufacturer’s instructions. Total RNA from each sample was used to prepare biotinylated target RNA. The target RNA was processed using an Affymetrix GeneChip Instrument System. Robust Multi-chip Average (RMA) was used for primary analysis. Details of quality control measures can be found at http://www.ncbi.nlm.nih.gov/geo/. The probe sets contained in the Affymetrix miRarray oligonucleotide arrays were analyzed using RMA algorithm (PARTEK). Seventeen tumor samples (both frozen and FFPE solid tumors and HGSOC tumor cells from patients’ ascites) were compared with 8 normal FTE control samples. The comparison generated a list of ‘active miRs’ representing probe sets changed at least 2-fold. Hierarchical clustering was performed using Spotfire DecisionSite for Functional Genomics (Somerville, MA). Cell lines and plasmids iFTSECs (primary cells infected with hTERT and SV40 T-antigen-expressing retroviruses) designated FT190 and FT194 were kindly provided by the Drapkin lab, Dana-Farber Cancer Institute, Boston, MA (32). Pooled population of miR-155 overexpressing FTSECs (FT194-MG155) was generated by retroviral infection of iFTSECs with MG-155 construct, kindly provided by the lab of David Baltimore [Addgene plasmid # 26529, (33)], followed by selection based on GFP reporter gene expression. Control cells for miR-155 overexpression (FT194-MG) were produced by retroviral infection of iFTSECs with MG vector, also kindly provided by the lab of David Baltimore [Addgene plasmid # 26528, (33)]. Cell culture experiments BAY 11–7082 (Santa Cruz Biotechnology, Dallas, TX) at a final concentration of 100 µM was added to the cells 30 min prior to exposure to FF or TNFα (at a final concentration of 50 ng/ml, Sigma–Aldrich). Cell proliferation was assessed using CellTiter-Glo luminescent cell viability assay (Promega, Madison, WI) according to manufacturer’s instructions. For the migration assay, 105 FT194-MG155 and control cells were plated on the upper compartment of 8 µm pore diameter ThinCert cell culture inserts (Greiner-Bio-One, Kremsmünster, Austria). After 48 h cells were fixated with 4% paraformaldehyde and stained with 0.1% crystal violet for detection of migratory cells. Protein extraction and western blotting Subcellular fractionation protocol was used to extract cytoplasmic and nuclear proteins. Extracts were separated on sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes and reacted with the primary antibodies anti NF-κB RelA-p65 (sc-372, Santa Cruz Biotechnology), anti-β-tubulin (ab52901, Abcam, Burlingame, CA) and anti-Emerin (sc-25284, Santa Cruz Biotechnology) followed by a secondary antibody and chemiluminescent reaction. The signal intensity was quantified using ImageJ software tools. qRT- PCR Total RNA was extracted using QIAzol reagent (Qiagen, Valencia, CA) and RNeasy clean-up kit (Qiagen) according to manufacturer’s instructions. mRNA levels were assessed using FastStart Universal SYBR Green Master (ROX) (Roche Applied Science, Indianapolis, IN) with the following primers: RAD51: (FWD) 5′-CAACCCATTTCACGGTTAGAGC-3′ and (REV) 5′-GCTTTGGCTTCACTAATTCCCTT-3′, WEE1: (FWD) 5′-CACACGCCCAAGAGTTTGCT-3′ and (REV) 5′-ACACTGTCCTGAGGAATGAAGCA-3′, FOXO3a: (FWD) 5′-CCCAACCAGCTCCTTTAACA-3′ and (REV) 5′-GAGTCCGAAGTGAGCAGGTC-3′, AICDA: (FWD) 5′-GGGAACCCCAACCT CAGTCT-3′ and (REV) 5′- CCTTGCGGTCCTCACAGAAG-3′, IL-6: (FWD) 5′-GGTACATCCTCGACGGCATCT-3′ and (REV) 5′-GTGCCTCTTTGCTGCTTTCAC-3′, B2M: (FWD) 5′-TTCTGGCCTGGAGGCTATC-3′ and (REV) 5′-TCAGGAAATTTGACTTTCCATTC-3′ (Sigma–Aldrich). For miRNA evaluation, we extracted RNA as described above from either HGSOC patients’ ascites cell pellet, normal FTE or fresh frozen HGSOC tumors. HGSOC tumor cells were harvested from patients’ ascites as adopted from Barker et al. (34). Briefly, ascites that were drained for palliative causes from HGSOC patients were allocated by the institutional tissue bank, with the appropriate ethical approvals. Cells were collected using centrifugation at 1200 rpm for 10 min in 4°C, followed by brief resuspension in 0.2% NaCl solution for RBC lysis, and repeat centrifugation at 1200 rpm for 10 min in 4°C. This process was repeated until the pellet appeared RBC free. Normal FTE was harvested from fresh, grossly normal fallopian tube fimbriae as previously described (9). Ten nanogram total RNA was reverse transcribed using miRCURY LNATM Universal RT microRNA PCR Polyadenylation kit (Exiqon, Vedbaek, Denmark). MiRNA levels were assessed using miRCURY LNATM microRNA PCR ExiLENT SYBR Green master mix with primers for hsa-miR-155-5p and hsa-SNORD48, as control (Exiqon). Adhesion assay 105 FT194-MG155 and control cells were seeded on an extracellular matrix (ECM)-coated array plate containing seven different human ECM proteins (ECM Cell Adhesion Array Kit, colorimetric [cat# ECM540], Merck-Millipore, Burlington, MA). Adhesion capacity was assessed according to manufacturer’s instructions. Results The ex vivo persistent ovulation model To mimic the long-term cumulative effects of ovulation on FTE, we developed an ‘ex vivo persistent ovulation model’. In this model, benign primary human FTE from 10 different donors were cultured on Transwell inserts, incubated with pooled human FF samples daily for 4 h, washed and allowed to recover for 20 h daily for up to 4–5 weeks. The secretome of the FF-treated and control cells was recovered from the upper/apical side of the polarized FTE culture and was profiled using MS. Clinical features of the donors of human samples are presented in Supplementary Table S1 (available at Carcinogenesis online). We examined the differential expression of secreted proteins in response to recurrent FF exposure at three time points: early (days 1–4; n = 10), intermediate (days 10–16; n = 7) and late (days 25–30; n = 3). The samples were analyzed using label-free algorithms, which were normalized relative to the overall amount of protein in each sample (assuming equal amounts in all samples). A total of 4827 different proteins were identified across all samples, with an average of ~1700 proteins per sample. Several samples representing the late time point (and their matched counterpart samples) were excluded from the analyses due to very low protein count. Principal component analysis (PCA) showed clear separation between control and FF-treated samples (Figure 1A). Figure 1. Open in new tabDownload slide Proteomics of the ‘ex vivo persistent ovulation model’ secretome. Normal FTE was cultured in an air–liquid interface system and exposed to human FF repeatedly daily. The proteins secreted from the luminal aspect of the cultures were sampled at short-, intermediate- and long-term time points. (A) PCA and (B) heatmap representation of FTE ex vivo cultures repeatedly treated with FF compared with FTE treated with culture medium as control. String interaction plots of (C) over-represented and (D) under-represented proteins following repeated exposure to FF, compared with FTE treated with culture medium as control. Figure 1. Open in new tabDownload slide Proteomics of the ‘ex vivo persistent ovulation model’ secretome. Normal FTE was cultured in an air–liquid interface system and exposed to human FF repeatedly daily. The proteins secreted from the luminal aspect of the cultures were sampled at short-, intermediate- and long-term time points. (A) PCA and (B) heatmap representation of FTE ex vivo cultures repeatedly treated with FF compared with FTE treated with culture medium as control. String interaction plots of (C) over-represented and (D) under-represented proteins following repeated exposure to FF, compared with FTE treated with culture medium as control. The dataset was imputed and comparison between control and FF-treated FTE yielded 550 significantly different proteins (T-test, FDR = 0.05 and s0 = 0), of which 173 proteins were upregulated and 377 proteins were downregulated following FF treatment. The heatmap shows both separate clustering of the control versus the FF-treated FTE, and also two independent clusters within each treatment group (Figure 1B). The full data for individual proteins are available in Supplementary Table S2 (available at Carcinogenesis online). Fisher enrichment analyses show multiple functional networks that were over-represented upon repeated FF stimulation, especially cell adhesion pathways, inflammatory pathways, complement activation and lipid transport and metabolism, at all three time points. String interaction plot is presented in Figure 1C (see also Supplementary Table S3, available at Carcinogenesis online). A set of networks were under-expressed along the course of the experiment (Figure 1D and Supplementary Table S3, available at Carcinogenesis online): Ubiqitin-proteosomal proteins were under-represented at all three time points, as well as proteins of cell-cycle checkpoints and nucleotide metabolic pathways. Additionally, at the early time point, DNA unwinding and RNA stabilizing complexes were downregulated, possibly suggesting cell-cycle arrest and decreased transcription/translation. At the intermediate time point, protein translation and unfolded protein response networks were downregulated, whereas at the late time point, we encountered downregulation of DNA damage response pathways. Overall, this data reveals a multitude of signaling changes induced by repeated exposures to FF, resembling uninterrupted ovulation cycles, which may significantly perturb the FTE homeostasis and may amount to carcinogenic changes. FF induces NF-κB activation and miR-155 upregulation Next, we searched for a single, dominant mechanism that links inflammation and multiple aspects of the observed expressional changes. We previously reported strong upregulation of both IL-8 and AICDA in FTE following single short-term incubation with FF, both established products of NF-κB activation (9,35). Additionally, the above proteomic analyses highlighted additional individual proteins and networks that are putatively regulated by NF-κB, including complement factors (36), adhesion molecules such as fibronectin (37), apolipoproteins (38) and proteasomal proteins (39). Hence, we hypothesized that NF-κB activation is induced upon FF stimulation. To test this hypothesis, we compared the translocation of RelA (p65), one of NF-κB subunits, to the nucleus following incubation of FT194 immortalized FTSECs (iFTSECs) with FF or TNFα (an established activator of NF-κB pathway, as positive control) or culture medium (as negative control). The experiment was repeated with vs. without pre-incubation with BAY 11–7082, an inhibitor of IkBα phosphorylation, as control. Incubation of FTE with effective concentrations of BAY 11–7082 for a duration of 4 h resulted in >50% cell death, further highlighting the central role of the NF-κB pathway in this cell line, however limiting the duration of the experiments (data not shown). Exposure to FF and TNFα treatments resulted in increased translocation of RelA-p65 to the nucleus, as shown by western blot analysis of nuclear and cytoplasmic protein fractions (Figure 2A and B). Presumably, cell death, and the associated nuclear membrane disintegration, caused by BAY 11–7082 precluded isolation of pure cytoplasmic and nuclear fractions. This effect can already be seen following short incubation (30 min and 1 h, Supplementary Figure S2, available at Carcinogenesis online). Short-term BAY 11–7082 treatment reduced the translocation to the nucleus by more than 45% and 60%, respectively (n = 3, Wilcoxon test P = 0.063, Figure 2A). Figure 2. Open in new tabDownload slide FF induces the NF-κB-miR-155 axis. FT194 cell line was pre-treated with BAY 11–7082 (IkBα phosphorylation inhibitor) followed by treatment with either FF, TNFα or no treatment. Cytoplasmic and nuclear protein fractions were separately used for sodium dodecyl sulfate–polyacrylamide gel electrophoresis separation and western blot analysis using anti-p65 antibody. Anti-tubulin (cytoplasmic marker) and anti-Emerin (nuclear marker) antibodies were used as controls of fractionation. (A) Nuclear fraction. NF-κB translocates to the nucleus upon FF and TNFα treatments. The effect is blocked by BAY 11–7082. (B). Cytoplasmic fraction, no significant change in p65 levels. (C) MiR-155 induction in primary FTE following short- or long-term exposure to FF. (D) MiR-155 induction in immortalized FTSECs following incubation with FF. Bars represent mean ± SEM. Figure 2. Open in new tabDownload slide FF induces the NF-κB-miR-155 axis. FT194 cell line was pre-treated with BAY 11–7082 (IkBα phosphorylation inhibitor) followed by treatment with either FF, TNFα or no treatment. Cytoplasmic and nuclear protein fractions were separately used for sodium dodecyl sulfate–polyacrylamide gel electrophoresis separation and western blot analysis using anti-p65 antibody. Anti-tubulin (cytoplasmic marker) and anti-Emerin (nuclear marker) antibodies were used as controls of fractionation. (A) Nuclear fraction. NF-κB translocates to the nucleus upon FF and TNFα treatments. The effect is blocked by BAY 11–7082. (B). Cytoplasmic fraction, no significant change in p65 levels. (C) MiR-155 induction in primary FTE following short- or long-term exposure to FF. (D) MiR-155 induction in immortalized FTSECs following incubation with FF. Bars represent mean ± SEM. Next, we searched for the signaling pathway linking between FF effects and NF-κB activation. A possible candidate is miR-155 (20,40), which is transcriptionally regulated by NF-κB and at the same time serves as an amplifier of the NF-κB pathway by downregulating inhibitory genes (22,41). MiR-155 was upregulated in primary benign FTE incubated with FF for 4 or 24 h with an increase of 2.02-fold (n = 4, Wilcoxon test P < 0.05) and 1.69-fold (n = 4, Wilcoxon test P < 0.05), respectively (Figure 2C). Recurrent exposure to FF for 6 days in the ‘ex vivo persistent ovulation model’ revealed a 46% increase in miR-155 expression (n = 3) and a 67% increase after a period of ~30 days (n = 2; both not statistically significant) (Figure 2C). Similarly, we treated FT190 and FT194 iFTSECs with either FF or culture medium (as control) for 4, 24 or 48 h. Elevated miR-155 expression levels were observed at all three time points with 2.16-, 3.21- and 2.13-fold increase, respectively (n = 4, Wilcoxon test P < 0.05, Figure 2D). Interestingly, short-term incubation with BAY 11–7082 before the incubation with FF resulted in a 33% decrease in miR-155 expression (n = 4, Wilcoxon test P < 0.05, data not shown). This indicates that NF-κB regulates miR-155 expression in FTSEC, consistent with previous data from innate immune cells (22). Applying TargetScanHuman 7.2 miRNA target gene prediction tool (42) on the secretome profiles of the ‘ex vivo persistent ovulation models’, we identified 17 known miR-155 target proteins among the 377 downregulated secreted proteins (SEPT11, BCL10, CARHSP1, CD47, CDC37, CKAP5, EEF2, G3BP2, GATAD2B, HOOK1, MYLK, PICALM, SMS, STRN3, TIAL1, USP14, WNK1, all with P < 0.005). This finding provides further evidence that miR-155 is functional in FTE. MiR-155 is upregulated in HGSOC compared with normal FTE MiR-155 has been previously implicated in multiple types of cancer, but its role to HGSOC is not clear. To further test the hypothesis that miR-155 is indeed involved in serous carcinogenesis, we profiled the expression of miRNAs in fresh frozen benign primary FTE from 8 healthy donors and 17 advanced–stage solid HGSOC tumors [fresh frozen (n = 4) and FFPE (n = 13)] using Affymetrix miRNA expression microarrays. The clinical characteristics of the samples are shown in Supplementary Table S1 (available at Carcinogenesis online). The miRNA expression levels are shown in Supplementary Table S4 (available at Carcinogenesis online), and the CEL files are found in GEO repository (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE131790). PCA and hierarchical clustering indicate that the normal FTE samples differ significantly from tumor samples (Figure 3A and B). However, FFPE samples clustered separately from fresh frozen tumors (Supplementary Figure S1A, available at Carcinogenesis online); hence, differential expression analyses were performed both for the entire cohort and separately for the fresh frozen samples (Supplementary Figure S1B and Table S4, available at Carcinogenesis online). Figure 3. Open in new tabDownload slide MiR-155 is overexpressed in HGSOC compared with FTE. MiRNA profiling was done using Affymetrix microarrays on 17 HGSOC tumor samples and 8 normal FTE samples. (A) PCA and (B) heatmap representation of the miRNA landscape. (C) MiR-155 expression levels in HGSOC solid tumors and tumor cells from patients’ ascites compared with normal FTE from benign fimbria. Extracted RNA was quantified using RT–PCR. Bars represent mean + SEM. Figure 3. Open in new tabDownload slide MiR-155 is overexpressed in HGSOC compared with FTE. MiRNA profiling was done using Affymetrix microarrays on 17 HGSOC tumor samples and 8 normal FTE samples. (A) PCA and (B) heatmap representation of the miRNA landscape. (C) MiR-155 expression levels in HGSOC solid tumors and tumor cells from patients’ ascites compared with normal FTE from benign fimbria. Extracted RNA was quantified using RT–PCR. Bars represent mean + SEM. In this small cohort of fresh samples, miR-155 was increased 9.8-fold (P = 0.006) and was among the top most significantly upregulated miRNAs in HGSOC tumors. This finding was validated by RT-PCR on an independent set of fresh frozen primary solid HGSOC tumors (n = 6) and primary tumor cells harvested from fresh patient’s ascites of HGSOC patients (n = 6) compared with normal FTE (n = 11), with a significant 4.3-fold and 7-fold increase, respectively (Wilcoxon test P < 0.005 for both; Figure 3C). MiR-155 induces downregulation of DNA repair and cell-cycle proteins in FF-treated FT epithelial cells Next, we examined the effects of miR-155 overexpression in FTSECs on putative target genes, with particular focus on genes involved in DNA damage repair, cell cycle and adhesion (all hallmarks of HGSOC). We stably overexpressed miR-155 in FT194 cells (FT194-MG155), achieving a 164-fold increase in miR-155 expression (n = 5, Wilcoxon test P < 0.005). RAD51, a key factor in homologous recombination pathway, was mildly reduced in FT194-MG155 cells at the mRNA and at the protein levels compared with control (15% and 20%, respectively, n = 4, Wilcoxon test P = 0.28); Figure 4A, protein data not shown). RAD51 foci formation is reduced in FT194-MG155 cells, as seen by immunofluorescent staining (Supplementary Figure S3, available at Carcinogenesis online). MiR-155 also regulates WEE1, a kinase which inhibits G2/M cell-cycle progression, and FOXO3a, a tumor suppressive transcription factor which is lost in early stages of HGSOC development. Both genes were downregulated following miR-155 overexpression by 40% (n = 4, Wilcoxon test P < 0.05) and 32% (n = 4, Wilcoxon test P < 0.05, Figure 4A), respectively. FT194-MG155 had 2.64-fold increase in AICDA mRNA levels (n = 4, Wilcoxon test P < 0.05, Figure 4B), in accordance with our previous report describing in vitro effects of FF (35). IL-6, a pro-inflammatory cytokine and activator of the STAT3 pathway in HGSOC (43,44), was increased 1.69-fold in FT194-MG155 compared with control cells (n = 4, Figure 4B). Of note, ATM/ATR are DNA damage response enzymes known to be altered a small fraction of HGSOC cases (25). However, our experimental system does not identify transcriptional alterations in these two genes, nor are they predicted to be targets of miR-155. Nonetheless, the expression of mismatch repair genes MSH2 and MSH6 in miR-155 overexpressing FT194 compared with control FT194 cell decreased by 21% (n = 4, Wilcoxon test P < 0.05) and 27% (n = 4, Wilcoxon test P < 0.05), respectively. A decrease in the mRNA levels of MLH1 was not significant. Figure 4. Open in new tabDownload slide MiR-155 regulatory effects on putative target genes in FTE. MiR-155 was overexpressed (OE) in FTSEC line (FT194-MG155). Downregulated (A) and upregulated (B) miR-155 target genes in miR-155 over-expressing cells compared with control FTSEC. Downregulated (C) and upregulated (D) miR-155 target genes in primary FTE treated with FF for either 4 or 24 h versus controls. Extracted RNA was quantified using RT-PCR. Bars represent mean + SEM. Figure 4. Open in new tabDownload slide MiR-155 regulatory effects on putative target genes in FTE. MiR-155 was overexpressed (OE) in FTSEC line (FT194-MG155). Downregulated (A) and upregulated (B) miR-155 target genes in miR-155 over-expressing cells compared with control FTSEC. Downregulated (C) and upregulated (D) miR-155 target genes in primary FTE treated with FF for either 4 or 24 h versus controls. Extracted RNA was quantified using RT-PCR. Bars represent mean + SEM. Overall, these results support the activity of miR-155 in DNA damage, deregulation of cell cycle and pro-neoplastic inflammatory networks in FTE. Next, we wanted to test whether these target genes are also differentially expressed in primary FTE following exposure to FF, in our short-term ovulation model. After a single 4 h incubation with FF, we observed a 43% decrease in mRNA levels of RAD51, whereas WEE1 and FOXO3a were downregulated by 25% and 27%, respectively (n = 4, Wilcoxon test P < 0.05, Figure 4C). AICDA and IL-6 mRNA levels were increased by 25-fold and 8.7-fold, respectively (n = 4, Wilcoxon test P < 0.05, Figure 4D). Although less profound, a 24 h incubation with FF resulted in a 2-fold and 8.25-fold increase of AICDA and IL-6 mRNA levels in FTE, respectively (n = 4, Wilcoxon test P < 0.05, Figure 4D), while RAD51 and WEE1 mRNA levels decreased by 32% and 19%, respectively (n = 4, Wilcoxon test P < 0.05, Figure 4C). In addition, in primary FTE that were exposed to single 4 h incubation with FF, we observed a decrease in MLH1 and MSH2 by 36% and 40%, respectively, though not statistically significant (n = 4, Wilcoxon test P = 0.28). Overall, these results indicate that miR-155 regulates an extensive transcriptional network in FTSECs with implications to serous carcinogenesis and that short-term exposure to FF inflicts similar changes to those induced by experimental overexpression of miR-155. MiR-155 induces migration and adhesion of benign FTSECs Next, we searched for additional tumorigenic phenotypic changes in FTSECs that over-express miR-155. Cell proliferation was non-significantly, yet persistently, increased in FT194-MG155 compared with control cells (16% increase, n = 3) at both 24 and 48 h. In vitro migration capacity, examined using Boyden chamber assay, was increased 1.76-fold after 48 h (n = 3, Wilcoxon test P = 0.0636; Figure 5A). Figure 5. Open in new tabDownload slide Overexpression of miR-155 effects migration and adhesion. (A) Migration of FT194-MG155 versus control FTSECs was evaluated using Boyden chamber assays followed by crystal violet stain. Representative image after 48 h shows increased migration in miR-155 overexpressing cells. (B) FT194-MG155 versus control cells were plated on seven various ECM protein-coated wells. Quantification was performed using the ECM cell adhesion array kit. (C) Differential expression of various integrin’s subunits in FTSEC-overexpressing miR-155 compared with control FTSEC. (D) Differential expression of the integrin’s subunits in primary FTE repeatedly exposed to FF for 6 days versus control. Extracted RNA was quantified using RT-PCR. Bars represent mean + SEM. Figure 5. Open in new tabDownload slide Overexpression of miR-155 effects migration and adhesion. (A) Migration of FT194-MG155 versus control FTSECs was evaluated using Boyden chamber assays followed by crystal violet stain. Representative image after 48 h shows increased migration in miR-155 overexpressing cells. (B) FT194-MG155 versus control cells were plated on seven various ECM protein-coated wells. Quantification was performed using the ECM cell adhesion array kit. (C) Differential expression of various integrin’s subunits in FTSEC-overexpressing miR-155 compared with control FTSEC. (D) Differential expression of the integrin’s subunits in primary FTE repeatedly exposed to FF for 6 days versus control. Extracted RNA was quantified using RT-PCR. Bars represent mean + SEM. Next, consistent with the unique predilection of FTE to epithelial cell shedding and re-implantation, we evaluated the effect of miR-155 on the ability of FTSECs to adhere to seven abundant ECM components using a commercial assay. FT194-MG155 cells displayed increased binding properties to most ECM components compared with control FTSECs, with 2-fold increase in binding capacity to vitronectin and 1.4-fold increase in binding to collagen I (n = 3, t-test P < 0.05, Figure 5B). Both vitronectin and collagen I were implicated in HGSOC cell adhesion to the peritoneum and metastasis (45–48). We then compared the expression of specific integrin molecules in FT194-MG155 compared with control cells. As shown in Figure 5C, mRNA expression of ITGA4 and ITGAV subunits were significantly upregulated with a 6.8- and a 1.44-fold increase, respectively (n = 4, Wilcoxon test, P < 0.05). Similarly, mRNA from ‘ex vivo persistent ovulation model’ disclosed induction of integrin molecules’ expression with a 3.9-, 1.3- and 1.68-fold increase in ITGA4, ITGB1 and ITGB3 subunits, respectively (n = 4, Wilcoxon test P < 0.05, Figure 4D). Proteomic data from the ‘ex vivo persistent ovulation model’ showed significant downregulation of ITGA6 at the short- and intermediate-term time points (−Log Student’s t-test P-value = 3.396). This data illuminate a new possible mechanism through which ovulation-associated inflammatory and pro-tumorigenic signals may interact with adhesion molecules signaling. Discussion Ample data have been gathered in the past decade about the origin of HGSOC in the FTE and about genomic and transcriptional changes occurring in advanced stages of the disease (25). However, the molecular events leading to the initiation and progression of this cancer are not yet clear. The main challenge to defining these events remains developing genuine in vivo and in vitro models for transformation of the FTE. In this study, we describe an ’ex vivo persistent ovulation model’, designed to mimic repeated cyclic exposure of human FTE to FF. Due to technical limits, the process had to be ‘fast-forwarded’, with a daily short exposure of the cells to FF, followed by a recovery phase. This model does not recapitulate the cyclic systemic hormonal fluctuations, which also possess significant impact on FTE homeostasis (49). The secretome profiling approach, which has already been used in short-term FTE ex vivo cultures (29), provides a unique insight into the dynamic transcriptional changes using normal human primary FTE cells. Importantly, the possibility of contamination by medium or FF proteins, as well as transcriptional changes of prolonged ex vivo cells culture, are experimental biases that require implementation of bioinformatics solutions. Nonetheless, we discovered perturbations in diverse cancer-associated networks, including several that are regulated by the NF-κB canonical pathway. We also show upregulation of miR-155 in this model, as well as in HGSOC tumors compared with normal FTE. The feedback loop connecting NF-κB and miR-155 has been described in immune cells (22). Using an NF-κB inhibitor (BAY 11–7082) we now show direct regulation of miR-155 expression in FTSEC. This has been suggested for BRCA1-deficient FTE before (50), however we show a more generalized phenomenon. Our next aim was to tie the ovulation-associated activation of the NF-κB-miR-155 axis to the two most distinctive features of HGSOC: genomic instability and mode of metastasis through shedding. HGSOC is characterized by genomic instability due to a homologous recombination defect in over 50% of the cases, while germline BRCA1/2 mutations account for the minority of cases (51). Enhanced DNA damage following incubation of FTE with human FF and defective DNA repair kinetics in these FTSECs have been previously reported (9,29). Our current data suggest that miR-155-induced downregulation of RAD51 may contribute to this phenomenon. Shedding of (pre-)malignant cells from the FTE and attachment on the mesothelium of the ovaries and peritoneum is mediated mainly by integrins which drive signaling pathways that modulate the metastatic sites and facilitate anchorage-independent survival of circulating tumor cells (52). Several integrins were reported to be overexpressed in HGSOC, including α5β1 (interacts with collagen, vitronectin and fibronectin), αvβ3 (interact with both vitronectin and fibronectin) and α4β1 (53–56). We show that miR-155 increases the migration capacity of benign FTSECs and affects their adhesion to specific ECM components, especially to vitronectin and collagen I. Vitronectin is an ECM glycoprotein associated with tumor invasion, metastasis and angiogenesis in different types of cancer (57,58). In ovarian cancer, it has been shown to promote adhesion of epithelial ovarian cancer cells to peritoneal mesothelium in vitro through uPAR receptor and αv integrin (46). We show that the latter is upregulated in miR-155 overexpressing FTSECs. Several publications suggest that even benign FTE may detach from the fimbria and re-integrated in the ovarian surface epithelium in the form of inclusion cysts (59). Our results indicate a role for miR-155 in aberrant expression of specific integrin subunits in FTSECs, though further research is needed to fully define the effect of this observation on signaling and anoikis escape in these cells. The carcinogenic process is a prolonged process during which the FTE is exposed to ovulation-associated inflammatory stimuli monthly for many years. Our study highlights the role of the NF-κB-miR-155 axis in linking ovulation, defective DNA repair, migration and altered adhesion capacity, which may cumulatively promote serous carcinogenesis. Abbreviations Abbreviations ECM extracellular matrix FF follicular fluid FTE fallopian tube epithelium FTSEC fallopian tube secretory epithelial cell HGSOCs high-grade serous ovarian carcinomas IL interleukin miRs microRNAs MS mass spectrometry NF-κB nuclear factor-κB PCA principal component analysis TNFα tumor necrosis factor alpha Supplementary material Supplementary data are available at Carcinogenesis online. Supplementary Figure S1. Principal component analysis of miRNA expression in HGSOC tumors and normal FTE. (A) PCA indicates separation of FFPE samples from fresh frozen samples. (B) PCA of fresh frozen samples highlights separation of tumor samples from unmatched normal FTE. Supplementary Figure S2. Early nuclear translocation of p65 in FTSEC in response to stimulation with TNFα or FF. FT194 cell line was treated with either FF, TNFα or no treatment for 30 minutes (A) or 1 hr (B). Cytoplasmic and nuclear protein fractions were separately used for SDS–PAGE separation and WB analysis using anti-p65 antibody. Supplementary Figure S3. MiR-155 overexpression results in reduced expression and activity of Rad51 protein. WB shows decrease in Rad51 level in FT194-MG155 cells vs. control (A). Representative images of immunofluorescent staining show decrease in Rad51 activity in FT194-MG155 cells vs. control demonstrated by decrease in foci formation (B). Supplementary Table S1. Clinical data of human samples. Clinical characteristics of HGSOC tumors and normal FT fimbriae. Supplementary Table S2. Label-free quantification (LFQ) intensities for all the identified proteins in the ex vivo persistent ovulation model secretome. The values are in log2 scale. The table lists the gene names followed by the LFQ intensities in different samples, UniProt IDs, protein and gene names. Table S3. Fisher enrichment of ex vivo persistent ovulation model proteomics. Pathway enrichment analysis of the ex vivo persistent ovulation model secretome data indicates networks and functions that are up- and down-regulated at the short (sheet #1), intermediate (sheet #2), and long term (sheet #3) exposure to human FF. Changes in expression in control cultures along time are controlled for. Table S4. HGSOC tumors vs. normal FTE miRNA microarray data. Normalized expression levels of all miRNAs in all HGSOC and normal samples are shown in sheet #1. Expressional fold-change, p-value and FDR, are shown in sheet #2. Similar analysis of fresh frozen samples only is shown in sheet #3. Funding This research was funded by the Israel Cancer Research Fund (ICRF)—Len and Susan Mark Initiative for Ovarian and Uterine/MMMT Cancers Grant; Israel Cancer Association Research Grants; Israel Science Foundation (ISF) (personal grant 1104/17); the Chaim Sheba Medical Center Talpiot Medical Leadership award. Acknowledgement We thank Dr Shira Galper, Institute of Oncology at Sheba Medical Center, for critical review of the manuscript. Conflict of Interest Statement: The authors declare no conflict of interests. References 1. Coussens , LM et al. ( 2002 ) Inflammation and cancer . Nature , 420 , 860 – 867 . Google Scholar Crossref Search ADS PubMed WorldCat 2. Kawanishi , S et al. ( 2017 ) Crosstalk between DNA damage and inflammation in the multiple steps of carcinogenesis . Int. J. Mol. Sci ., 18 , 1808 . Google Scholar Crossref Search ADS WorldCat 3. Levanon , K et al. ( 2008 ) New insights into the pathogenesis of serous ovarian cancer and its clinical impact . J. Clin. 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TI - NF-κB-miR-155 axis activation mediates ovulation-induced oncogenic effects in fallopian tube epithelium
JF - Carcinogenesis
DO - 10.1093/carcin/bgaa068
DA - 2020-12-31
UR - https://www.deepdyve.com/lp/oxford-university-press/nf-b-mir-155-axis-activation-mediates-ovulation-induced-oncogenic-CWCMd8ExIO
SP - 1703
EP - 1712
VL - 41
IS - 12
DP - DeepDyve
ER -