Anti-Tumoral Effect of Chemerin on Ovarian Cancer Cell Lines Mediated by Activation of Interferon Alpha Response

Abstract

The pleiotropic adipokine chemerin affects tumor growth primarily as anti-tumoral chemoattractant inducing immunocyte recruitment. However, little is known about its effect on ovarian adenocarcinoma. In this study, we examined chemerin actions on ovarian cancer cell lines in vitro and intended to elucidate involved cell signaling mechanisms. Employing three ovarian cancer cell lines, we observed differentially pronounced effects of this adipokine. Treatment with chemerin (huChem-157) significantly reduced OVCAR-3 cell numbers (by 40.8% on day 6) and decreased the colony and spheroid growth of these cells by half. The spheroid size of SK-OV-3 ovarian cancer cells was also significantly reduced upon treatment. Transcriptome analyses of chemerin-treated cells revealed the most notably induced genes to be interferon alpha (IFNα)-response genes like IFI27, OAS1 and IFIT1 and their upstream regulator IRF9 in all cell lines tested. Finally, we found this adipokine to elevate IFNα levels about fourfold in culture medium of the employed cell lines. In conclusion, our data for the first time demonstrate IFNα as a mediator of chemerin action in vitro. The observed anti-tumoral effect of chemerin on ovarian cancer cells in vitro was mediated by the notable activation of IFNα response genes, resulting from the chemerin-triggered increase of secreted levels of this cytokine.

Keywords: adipokine; cell line; chemerin; interferon alpha; ovarian cancer.

Figure 1

Western blot analysis of the chemerin receptors CMKLR1 and GPR1 in ovarian cancer cell lines employed in this study. As reference, β-actin levels were detected. For preparation of cell lysates, cells were lysed in RIPA buffer and aliquots containing 10 µg protein were resolved by SDS–PAGE, followed by electrotransfer to a PVDF membrane. Antibodies used were anti-CMKLR1 (Abcam, ab64881, 1:500), anti-GPR1 (antibodies-online, ABIN516152, 1:200), and anti-ACTB (Abcam, ab8226, 1:500) and a horseradish peroxidase conjugated secondary antibody (1:20,000) which was detected using ECL system as described in the methods section. Western blot results from three independent experiments were densitometrically analyzed (ImageJ software, NIH). Shown are representative Western blots (left) and diagrams of mean band density (n = 3). * p < 0.001 vs. OVCAR-3. Full pictures of the Western blots are presented in File S1.

Figure 2

Effects of chemerin (recombinant huChem-157) (0, 100 and 400 ng/mL) on relative cell numbers of the indicated ovarian cancer cell lines as assessed by means of the fluorometric Cell Titer Blue assay (Promega). Cells were treated on day 0 and cultured for 6 days in medium supplemented with 10% FBS (n = 4). * p < 0.05; ** p < 0.001; *** p < 0.0001 vs. vehicle control.

Figure 3

(a) Effects of chemerin (huChem-157) (0 and 400 ng/mL) on the colony sizes of OVCAR-3 cells grown for three weeks in soft agar. Colony numbers were not significantly affected. Cells were treated with chemerin once a week. (n = 3). * p < 0.01 vs. control. (b) Effects of chemerin (huChem-157) (0 and 400 ng/mL) on growth of spheroids of the indicated cell lines, which displayed a specific phenotype and density, generated using “5D Sphericalplates” (tebu-bio, Kugelmeiers, Erlenbach, Switzerland). Upper panel: Photomicrographs of the spheroids or clusters grown in these plates generated by means of bright field microscopy. Bottom panel: For determination of the mean size of the spheroids or clusters, 50 per cell line were measured using ImageJ software (NIH). ** Spheroid generation of this cell line resulted in loose, irregular formed cell clusters.

Figure 4

Effects of chemerin (huChem-157) (0 and 400 ng/mL) treatment for 48 h on cellular apoptosis. (a) Western blot analysis of PARP-1 cleavage, cleaved caspase 3 and caspase 8. Cell lysate from U937 cells treated with TNF/CHX (cycloheximide) (100 ng/mL; 0.5 µg/mL) was loaded as positive control. (b) FACS analysis by means of (Guava^® Muse^® Cell Analyzer, Luminex, Austin, TX, USA), using the Muse^® Annexin V & Dead Cell Kit. The indicated cell lines were treated with chemerin (400 ng/mL) for 48 h or as a positive control with TNF/BV6 (100 ng/mL; 2.5 µM). EA = early apoptosis, LA = late apoptosis.

Figure 5

Effects of chemerin (huChem-157) (400 ng/mL) on the transcriptome of the indicated ovarian cancer cell lines as assessed by means of Affymetrix Clariom S human arrays. Cells were treated with chemerin for 48 h, and isolated RNA was processed and hybridized as described in the methods section. Venn diagrams show the number of genes with at least 2.5-fold change in mRNA expression.

Figure 6

Verification of the Affymetrix DNA microarray data. Expression of the indicated genes induced upon chemerin (huChem-157) (400 ng/mL) treatment was analyzed by Western blot or RT-qPCR. Shown is their expression in the indicated ovarian cancer cell lines in (a) representative Western blots or (b) in fold-change of mRNA levels normalized to β-actin (ACTB) expression compared to untreated cells (RT-qPCR). For comparison, the -fold change values after chemerin treatment assessed by microarray analyses are indicated. * p < 0.01 (n = 3). Full pictures of the Western blots are presented in File S1.

Figure 7

Pathway analysis of the genes being up-regulated in all chemerin (huChem-157)-treated ovarian cancer cell lines after 48 h as assessed with Affymetrix Clariom S human microarrays. All genes with elevated transcript levels (red) are type I interferon response genes, mostly induced by IFNα-triggered assembly of upstream regulator and transcription factor complex ISGF3, consisting of IRF9 and STAT1/2 (ISGF3 effects are not shown here). Also indicated is the cellular localization of the gene products. Broken arrows indicate transcriptional activation, other arrows protein interaction. The intensity of red color represents the measured grade of transcript up-regulation after chemerin treatment (Ingenuity pathway analysis (IPA) software, Qiagen).

Figure 8

Pathway analysis of key genes being up- or down-regulated on the transcript level upon 48 h of chemerin (huChem-157) treatment in OVCAR-3 cells as assessed by Affymetrix Clariom S human microarrays. Most of the up-regulated genes (red) are IFNα-response genes, with IRF9 as the key mediator of IFNα-induced gene regulation (as part of the ISGF3 complex, not shown). The observed regulation pattern is predicted to lead to growth inhibition of tumor cells (Ingenuity pathway analysis (IPA) software, Qiagen) (see discussion section). Broken arrows indicate transcriptional activation, other arrows protein interaction. The color intensity represents the measured grade of mRNA up- (red) or down-regulation (green).

Figure 9

Network of genes regulated after 48 h of chemerin (huChem-157)-treatment in at least two ovarian cancer cell lines, and connection to tumor growth inhibition. Activation of the network of interferon response genes is mediated by IFNα-induced formation of the upstream regulator and transcription factor ISGF3 (ISGF3 target genes are indicated by an asterisk). Broken arrows indicate transcriptional activation, other arrows protein interaction. Connection of genes to growth inhibition is indicated using solid arrows. The color intensity represents the measured grade of mRNA up- (red) or –down-regulation (green). * Type I interferon response genes (Ingenuity pathway analysis (IPA) software, Qiagen).

Figure 10

Interferon alpha (IFNα) concentration (ng/mL) in cell culture supernatants of the indicated cell lines after treatment with huChem-157 (400 ng/mL) for 24 or 48 h as assessed via ELISA. * p < 0.05; ** p < 0.01.

Figure 11

Associations of expression of the indicated interferon response genes markedly up-regulated upon huChem-157 treatment in all ovarian cancer cell lines with overall survival (OS) of 347 ovarian cancer patients. Open-source RNA-seq data in combination with patients´ survival data were provided and examined on the KMplotter website and its online tools at https://kmplot.com/analysis/ (accessed on 20 January 2022) [36].