Relaxin‐2 Inhibits Cervical Cancer Growth by Downregulating Kruppel‐Like Factor 5 (KLF5) Through the Inhibition of Hippo‐YAP and JAK2/STAT3 Signaling Pathways

Cervical cancer continues to pose significant health problems globally, often fueled by the persistent activity of KLF5, a prototypical oncogenic transcription factor responsible for enhanced cell proliferation, survival, and extensive tumor growth. Relaxin-2 (RLN2), which is part of the relaxin peptide hormone family, is known to exert a wide range of biological effects such as remodeling tissues and having anti-fibrotic effects. There is growing concern that RLN2 has anti-cancer activity; nevertheless, its function as well as the molecular mechanisms involved in cervical cancer is largely unknown. The goal of this research was to analyze the anti-tumor effect of RLN2 on cervical cancer and to determine its molecular mechanism, with particular emphasis on KLF5 and the cross-talks modulated by Hippo-YAP and JAK2/STAT3 signaling pathways. SiHa and C33A human cervical cancer cell lines were treated with recombinant RLN2 at different concentrations and times. The expression level of KLF5 along with the expression of constituent components of the cell cycle (Cyclin D1, PCNA, CDK4), apoptotic indicators (Bax, Bcl-2, cleaved caspase 3, PARP), and proteins of Hippo-YAP (YAP, pYAP, TEAD1/2) and JAK2/STAT3 (phospho-JAK2, phospho-STAT3) were evaluated by Western blotting. To validate KLF5's mediating role, analysis of proliferation and apoptosis-associated proteins was done after siRNA-induced silencing of KLF5. In addition, cell proliferation was further confirmed using the Sulforhodamine B (SRB) assay. Likewise, the wound healing assay was performed to evaluate the migratory potential in RLN2-treated SiHa cells compared to controls. The anti-tumor effect of RLN2 was assessed in vivo using a SiHa xenograft model in nude mice. The mice received intraperitoneal injections of RLN2 (20 μg/kg/day) for 21 days. Throughout the duration, tumor growth parameters were monitored, and upon excision, the tumors were subjected to immunohistochemical staining for Ki-67 (a proliferation marker). KLF5 protein expression was markedly reduced in both the cervical cancer cell lines following RLN2 treatment. This was coupled with a reduction of cell growth markers Cyclin D1, PCNA, and CDK4 which suggested G1 phase cell cycle arrest. There was a pronounced induction of apoptosis as indicated by increased Bax/Bcl-2 ratios along with greater amounts of cleaved caspase-3 and cleaved PARP 3. Consistent with these findings, the SRB assay confirmed a robust inhibition of cell proliferation, while the wound healing assay supported the strong anti-migratory potential of RLN2. In terms of mechanism, RLN2 blocked the Hippo-YAP pathway by promoting phosphorylation and sequestration of YAP into the cytoplasm, and downregulation of TEAD1/2 transcription factors. STAT3 and JAK2/2 signaling pathways were simultaneously inhibited as evidenced by decreased phosphorylation of JAK2 and STAT3, key oncogenic transcription drivers. KLF5 knockdown by siRNA showed effect opposite to RLN2 treatment which suggests KLF5 is indeed acting a mediator of these anti-tumor effects. In vivo, the addition of RLN2 resulted in substantial suppression of tumor volume and weight while causing no body weight changes. Examination of tumor tissue showed decreased Ki67 staining. This study makes it very clear that RLN2 has strong anti-cancer properties in cervical cancer by increasing the expression of the KLF5. The anti-proliferative and pro-apoptotic activities are achieved by the simultaneous inhibition of the Hippo-YAP and JAK2/STAT3 signaling pathways. These results underline the potential of RLN2 as a therapeutic target for treatment of advanced cervical cancer and necessitate further applied research to investigate its clinical use.