Fig 2: Evaluation of proteosomal degradation of the Hippo kinases in vitroCell lines were treated with 10 µM MG132 for 12 hrs. A 1.5-fold or greater accumulation of protein was considered indicative of proteosomal degradation. (A) Western blot demonstrates accumulation of MST2, but not MST1, LATS1, or LATS2 with treatment with 10 µM MG132. This was validated quantitatively in 3 cell lines (SKLMS1, A204, and SJCRH30) in part (B). These three lines are indicated by an asterisk in part (A). Statistical significance determined by two-tailed t-test. * indicates p < 0.05; ** indicates p < 0.01; *** indicates p < 0.001.

Fig 3: Loss of expression of the Hippo kinases at the RNA level and reconstitution with 5-azacytidine in sarcoma cell lines evaluated by quantitative RT-PCRLoss of expression was defined as a two-fold or greater decrease of expression of the Hippo kinases (0.5 fold decrease in expression as compared to GCT). Increase in expression was defined as a two-fold or greater increase in expression after treatment with 10 µM 5-azacytidine. (A) MST1 expression is decreased at the RNA level in 5 of 12 sarcoma cell lines (42%). MST2 expression is decreased in 3 of 12 sarcoma cell lines (25%). LATS1 expression is decreased in 1 of 12 sarcoma cell lines (8%). No decrease in LATS2 expression is detected (0%). (B) TCGA data demonstrating that MST2 expression is inversely proportional to CpG island methylation (Spearman's correlation coefficient r = -0.4). (C) MST1 expression is increased after treatment with 5-azacytidine in 1 of 12 cell lines (8%). MST2 expression is increased after treatment in 2 of 12 cell lines (17%). Neither LATS1 nor LATS2 expression is increased in any (0%) of the cell lines after treatment with 5-azacytidine. Statistical analysis is shown for cell lines demonstrating a two-fold or greater decrease in expression. Statistical significance determined by two-tailed t-test. * indicates p < 0.05; ** indicates p < 0.01; *** indicates p < 0.001.

Fig 4: The Hippo kinases are rarely deleted in sarcomas(A) Evaluation of The Cancer Genome Atlas data set revealed less than 1% of sarcomas demonstrate deletions of MST2 and LATS2. (B–D) Fluorescence in situ hybridization (FISH) utilizing fluorescent labeled BAC probes hybridizing to metaphase chromosomes and interphase nuclei (inset). (B) FISH probes for MST1 (20q13) demonstrating two signals in metaphase chromosomes and two signals in the interphase nucleus in the A204 cell line, indicating no deletions of the MST1 gene region are present. (C) FISH probes for MST2 (8q22) demonstrating polyploidy, but no deletions of the MST2 gene region. (D) FISH probes for LATS1 (6q25) demonstrate polyploidy, but no deletions of the LATS1 gene region (E) FISH probes for LATS2 (13q12) demonstrate a normal disomic pattern in both the metaphase chromosomes and the interphase nucleus in the A204 cell line, indicating no deletions of the LATS2 gene region (F) FISH for LATS2 in the RD cell line, demonstrating disomic signal pattern in a polyploid cell. Although a deletion or loss of chromosome 13 is present, two copies of LATS2 are still present. (G) Table summarizing FISH results. No deletions were observed in MST1, MST2, or LATS1. A deletion was noted in RD, however the cell line is polyploid, and two copies of LATS2 were still present.

Fig 5: Expression of the Hippo kinases in clinical samplesImmunohistochemical evaluation of MST1, MST2, LATS1, and LATS2 in a sarcoma tissue microarray. (A) Myxofibrosarcoma demonstrating diffuse and strong expression and nuclear localization (activation) of TAZ, and a lack of expression of YAP. (B) Synovial sarcoma demonstrating a lack of expression of MST1, MST2, and LATS2 in a sarcoma with activated TAZ and YAP. LATS1 expression is maintained. (C) Table demonstrating range of Hippo kinase loss in YAP or TAZ activated sarcomas. Loss of expression ranged from 19% (LATS1) to 47% (MST1). (D) Table demonstrating the frequency of loss of at least one Hippo kinase as a function of sarcoma histological type in sarcomas demonstrating activated TAZ or YAP.