Figure 7.

(A) Summary of cell lines diagram. Expression of the Hippo kinases was lost at the protein level in 0% (LATS1) to 58% (MST1) of the sarcoma cell lines, indicated by (+). Accumulation of the Hippo kinases with treatment with MG132, indicated by (+), was noted only for MST2, indicating that proteosomal degradation is an important mechanism by which MST2 expression is lost. Loss of expression at the RNA level was identified for MST1 (42%) and MST2 (25%) of sarcoma cell lines. Loss of expression at the RNA level for LATS1 and LATS2 was negligible. Deletions of the Hippo kinases were essentially absent from the sarcoma cell lines with the exception of LATS2, where 1 of the 12 sarcoma cell lines (8%) demonstrated a deletion. Treatment with 10 μM 5-azacytidine resulted in a modest increase in expression in 8–17% of the sarcoma cell lines. Treatment with 0.5 μM TSA resulted in a reversal of expression in a higher percentage of cell lines, predominantly with MST1 and MST2. Treatment with TSA and 5-azacytidine showed an additive effect with regards to re-expressing the Hippo kinases in some cell lines. (B) Expression of the Hippo kinases is regulated by at least three different mechanisms shown in this model, potentially targetable by different therapeutic interventions. Promoter (CpG island) hypermethylation is one mechanism that appears to modestly regulate the expression of predominantly MST1 and MST2. Histone deacetylation can also promote silencing the expression of the Hippo kinases, again particularly MST1 and MST2, and to a lesser degree LATS2. Proteosomal degradation plays a role in in regulating the expression of MST2 and could be targeted as well.