Inhibition of relaxin autocrine signaling confers therapeutic vulnerability in ovarian cancer
Helen E. Burston, … , Anne-Marie Mes-Masson, Robert Rottapel
Helen E. Burston, … , Anne-Marie Mes-Masson, Robert Rottapel
Published February 9, 2021
Citation Information: J Clin Invest. 2021;131(7):e142677. https://doi.org/10.1172/JCI142677.
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Research Article Oncology

Inhibition of relaxin autocrine signaling confers therapeutic vulnerability in ovarian cancer

Abstract

Ovarian cancer (OC) is the most deadly gynecological malignancy, with unmet clinical need for new therapeutic approaches. The relaxin peptide is a pleiotropic hormone with reproductive functions in the ovary. Relaxin induces cell growth in several types of cancer, but the role of relaxin in OC is poorly understood. Here, using cell lines and xenograft models, we demonstrate that relaxin and its associated GPCR RXFP1 form an autocrine signaling loop essential for OC in vivo tumorigenesis, cell proliferation, and viability. We determined that relaxin signaling activates expression of prooncogenic pathways, including RHO, MAPK, Wnt, and Notch. We found that relaxin is detectable in patient-derived OC tumors, ascites, and serum. Further, inflammatory cytokines IL-6 and TNF-α activated transcription of relaxin via recruitment of STAT3 and NF-κB to the proximal promoter, initiating an autocrine feedback loop that potentiated expression. Inhibition of RXFP1 or relaxin increased cisplatin sensitivity of OC cell lines and abrogated in vivo tumor formation. Finally, we demonstrate that a relaxin-neutralizing antibody reduced OC cell viability and sensitized cells to cisplatin. Collectively, these data identify the relaxin/RXFP1 autocrine loop as a therapeutic vulnerability in OC.

Authors

Helen E. Burston, Oliver A. Kent, Laudine Communal, Molly L. Udaskin, Ren X. Sun, Kevin R. Brown, Euihye Jung, Kyle E. Francis, Jose La Rose, Joshua Lowitz, Ronny Drapkin, Anne-Marie Mes-Masson, Robert Rottapel

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Figure 5

The relaxin promoter is activated by STAT3 and NF-κB.

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The relaxin promoter is activated by STAT3 and NF-κB. (A) Schematic of t...
(A) Schematic of the genomic region proximal to the RLN2 transcriptional start site (UCSC genome browser-human GRCh37/hg19). Species conservation is indicated. Boundaries of 3 relaxin promoter (RP) constructs, RP-1, RP-2, and RP-3, are mapped. Predicted binding sites for STAT3, NF-κB, and SOX9 are indicated. (B) Luciferase activity of the indicated RP constructs compared with empty vector control (EV) in OVCAR8 and SKOV3. Luciferase activity is normalized to Renila activity. For this and subsequent experiments, error bars indicate mean ± SEM. n = 3. (C) Genomic region of the RLN2 promoter (RP-3) compared with the RLN1 promoter. Peaks indicate species conservation. Red bars in the RP-3 sequence indicate single nucleotide differences in RLN1 compared with RLN2, and the open box indicates a small sequence not present in RLN1. Predicted binding sites for STAT3, NF-κB, and SOX9 are indicated. (D) RP-3 luciferase activity in cells transfected with control siRNA (siCON) or siRNA targeting STAT3 or SOX9. n = 3. (E) RP-3 luciferase activity in cells expressing shGFP or hairpins targeting NFκB1 or NFκB2 subunits (sh-NFκB1 and sh-NFκB2). n = 3. (F) Relaxin expression and STAT3 phosphorylation (pY705) in OVCAR8 treated for 48 hours with small molecule inhibitors of STAT3 (STATTIC, 1 μM) or NF-κB (QNZ, 5 nM) compared with mock-treated (–) cells. (G) RP3-luciferase activity in OVCAR8 and SKOV3 treated with 1%FBS, IL-6 (50 ng/mL), or TNF-α (50 ng/mL) for 24 hours compared with untreated cells. n = 3. (H) Relaxin levels and STAT3 phosphorylation (pY705) in OVCAR8 treated with IL-6 (50 ng/mL) or control (–) 24 hours after treatment with the JAK1/2 inhibitor Ruxolitinib (+Rux) compared with DMSO. (I and J) ChIP analysis of TF occupancy at the RLN1 promoter (I) and RLN2 promoter (J). ChIP signals are shown as fold enrichment over IgG. n = 3.