Mammalian Mst1 and Mst2 kinases play essential roles in organ size control and tumor suppression

Abstract

Control of organ size by cell proliferation and survival is a fundamental developmental process, and its deregulation leads to cancer. However, the molecular mechanism underlying organ size control remains elusive in vertebrates. In Drosophila, the Hippo (Hpo) signaling pathway controls organ size by both restricting cell growth and proliferation and promoting cell death. Here we investigated whether mammals also require the Hpo pathway to control organ size and adult tissue homeostasis. We found that Mst1 and Mst2, the two mouse homologs of the Drosophila Hpo, control the sizes of some, but not all organs, in mice, and Mst1 and Mst2 act as tumor suppressors by restricting cell proliferation and survival. We show that Mst1 and Mst2 play redundant roles, and removal of both resulted in early lethality in mouse embryos. Importantly, tumors developed in the liver with a substantial increase of the stem/progenitor cells by 6 months after removing Mst1 and Mst2 postnatally. We show that Mst1 and Mst2 were required in vivo to control Yap phosphorylation and activity. Interestingly, apoptosis induced by TNFalpha was blocked in the Mst1 and Mst2 double-mutant cells both in vivo and in vitro. As TNFalpha is a pleiotropic inflammatory cytokine affecting most organs by regulating cell proliferation and cell death, resistance to TNFalpha-induced cell death may also contribute significantly to tumor formation in the absence of Mst1 and Mst2.

Fig. 1.

 Removal of Mst1 and Mst2 in the liver. (A) Livers from 1-month-old mice. (B) H&E staining of liver sections. The boxed region is shown in higher magnification in the lower panel. The cells in the Mst1^Δ/Δ;Mst2^c/Δ;Albumin-Cre liver were smaller and the density was higher. (C) Cell proliferation shown by Ki67 (green, arrow) and BrdU staining (brown, Lower). More Ki67- or BrdU-positive cells were found in the Mst1^Δ/Δ;Mst2^c/Δ;Albumin-Cre liver. Autofluorescence blood cells were indicated by arrowhead. (Right) Statistical analysis. (D) TUNEL assay of liver sections. A slight increase in cell death (arrow) was detected in the mutant liver as quantified in the right panel.

Fig. 2.

 Liver tumors form in the absence of Mst1 and Mst2. (A) Livers from 6-month-old TM-injected wild-type and mutant mice. Two of the mutant livers with tumor nodules are shown. (B) H&E staining of liver sections from the liver samples in A. Boxed regions are shown in the lower panel in higher magnification. Both hepatocyte carcinoma (HCC) and chonlangiocyte carcinoma (CC) were observed. Multilayered epithelial cells are indicated by arrows.

Fig. 3.

 Increased cell proliferation and reduced Yap phosphorylation in the liver tumor. (A) Ki67 (green) and BrdU staining (brown) of liver sections from 6-month-old TM-injected wild-type and Mst1^Δ/Δ;Mst2^c/Δ;CAGGCre-ER mice. In the HCC regions, both Ki67- and BrdU-positive cell numbers were increased as shown by quantification. DAPI (blue) stains the nucleus. Autofluorescence blood cells were indicated by arrowhead. (B) TUNEL assay of liver sections as in A. Cell death (green) was increased in the HCC region as shown by quantification. (C) Western blot of samples from normal (wild-type) and tumor (Mst1^Δ/Δ;Mst2^c/Δ;CAGGCre-ER)-bearing livers. GAPDH was control for total protein, and Yap phosphorylation levels were normalized by the total Yap protein. The phosphorylated YAP to total YAP protein ratio was significantly reduced in the mutant as quantified by densitometry. (D and E) Immunohistochemistry of Yap (green) on the liver sections. Yap protein levels and nuclear localization (arrow) were increased in the mutants. (Lower) Nucleus stained red by propidium iodide (PPI, red).

Fig. 4.

 TNFα-induced liver toxicity was blocked by loss of Mst1 and Mst2. (A) Survival of Cre-adenovirus-injected wild-type mice (n = 10), Mst1^Δ/Δ;Mst2^c/+ mice (n = 10), and Mst1^Δ/Δ;Mst2^c/Δ mice (n = 10) after treatment with GalN and TNFα. (B) TUNEL analysis of liver tissue isolated 6 h after administration of GalN and TNFα. Results represent the staining of four mice from each group. (C) Serum concentration of liver enzymes in nontreated mice or at 6 h and 24 h after treatment with GalN and TNFα (n = 5 mice per group). ALT, alanine aminotransferase; AST, aspartate aminotransferase.