Hippo pathway inhibition increases YAP activity and augments liver regeneration in murine partial hepatectomy

Abstract

Presenter: Ryan Watkins MD | Mayo Clinic, Rochester Background: Impaired liver regeneration following hepatectomy is associated with poor outcomes. Augmenting the Hippo pathway effector, Yes-associated protein (YAP), has been associated with improved liver regeneration. We show that YAP transcriptional co-activity is increased by inhibiting MST1/2 with a novel inhibitor mCLC846 (mCLC), leading to improved liver regeneration in murine partial hepatectomy. Methods: Normal human cholangiocyte (NHC) cells were treated with mCLC. Cell viability was evaluated via an ATP based assay and YAP transcriptional co-activity was assessed by quantitative RT-PCR. 12-15-week-old male C57BL/6J mice underwent standard 2/3 partial hepatectomy (PH). Mice were treated with mCLC (5, 15, 50, and 100mg/kg) or vehicle 8-12 hours pre-hepatectomy and then twice daily until euthanasia. Liver regeneration was evaluated by liver to body weight ratio at 40 and 72 hours following PH, proliferation was characterized by immunoblot for proliferating cellular nuclear antigen (PCNA) and quantified by densitometry, and YAP target gene expression by quantitative RT-PCR in whole liver lysates. Additional mice were aged to 34 weeks and then underwent the same experiments as the 12-15 week old mice. Results: NHC cell culture treated with mCLC showed statistically increased cell viability following 72 hours of treatment. YAP target genes Ctgf, Cyr61, and Nuak2 significantly increased following 24 hours of treatment as compared to vehicle treated cells. Mice undergoing PH treated with mCLC showed statistically significant increases in liver to body weight ratio at both 40 and 72 hours following PH. Liver to body weight ratio at 72 hours increased as mCLC doses increased from 5 to 100 mg/kg. YAP target genes Ctgf, Cyr61 and Ankrd1 in liver lysates 40 hours post hepatectomy were significantly compared to controls. PCNA levels 40 hours following PH were 5 fold higher when compared to vehicle treated mice. Aged mice have impaired regeneration as indicated by the liver to body weight ratio at 72 hours. Aged mice treated with mCLC resulted in significant increases in liver to body weight ratio when compared to both vehicle treated mice and the vehicle treated standard cohort. Conclusion: Our data suggests that inhibiting MST1/2 with mCLC leads to cholangiocyte proliferation and increased YAP co-transcriptional activity. Additionally, MST1/2 inhibition causes an increase in whole liver YAP activity, increased cellular proliferation, and accelerated liver regeneration in murine partial hepatectomy. Aged mice have impaired regenerative capacity following hepatectomy however, mCLC remains efficacious in promoting liver regeneration.