Staurosporine targets the Hippo pathway to inhibit cell growth

Staurosporine targets the Hippo pathway to inhibit cell growth Dear Editor, The Hippo signaling pathway was discovered to control organ size in Drosophila through regulating cell proliferation and apoptosis (Yu et al., 2015). Arising studies over the past two decades have defined the core components and regulation mechanisms of the pathway in both Drosophila and mammals. The core components and fundamental function of the Hippo pathway are highly conserved in mammals. Active Ste20-like kinases 1/2 (MST1/2) phosphorylates adaptor protein Sav family WW domain-containing protein 1 (SAV1) and MOB1A/B, and phosphorylated MOB1A/B could recruit large tumor suppressor 1/2 (LATS1/2) for phosphorylation by MST1/2 at the hydrophobic motif (HM), followed by LATS1/2 autophosphorylation at the activation loop (AL) and full activation (Meng et al., 2016). Activated LATS1/2 then phosphorylates Yes-associated protein (YAP)/PDZ-binding motif (TAZ) and sequesters them in the cytoplasm for further phosphorylation and degradation (Zhao et al., 2007, 2010). Unphosphorylated YAP/TAZ translocates into the nucleus and associates with the TEAD family transcription factors to activate target genes expression (Zhao et al., 2008). Numerous studies have shown that the hippo pathway plays an important role in tumorigenesis. Overexpression of Yap or deletion of core components of the pathway (Nf2, Mst1/2, Sav1, and Mob1a/b) in liver can cause liver overgrowth and liver cancer (Camargo et al., 2007; Zhou et al., 2009; Yu et al., 2015). Increased YAP expression and activity were found in squamous cell carcinoma and other tumors (Muramatsu et al., 2011; Yu et al., 2014). Considering the important roles of the Hippo pathway, manipulation of Hippo pathway activity might be potential therapeutic means for diseases related with Hippo pathway dysfunction. Compounds targeting the Hippo pathway directly or indirectly have gained much investigation. Verteporfin is the first compound found to inhibit YAP/TEAD interaction and liver overgrowth induced by Yap overexpression or Nf2 deletion (Liu-Chittenden et al., 2012). XMU-MP-1 was reported to inhibit Mst1/2 kinase activities to activate YAP and augment tissue repair and regeneration (Fan et al., 2016). Here we find that Staurosporine (STS) regulates the activity and function of YAP, an effector of the Hippo pathway. Mechanistically, STS induces phosphorylation of Hippo pathway kinases MST1/2 and LATS1/2, which in turn leads to YAP phosphorylation and cytoplasmic localization, thus decreases the expression of YAP target genes. MST1/2 double knockout (dKO) partially inhibits whereas LATS1/2 dKO largely blocks the effect of STS on YAP phosphorylation and target gene expression. In addition, LATS1/2 dKO partially rescues the inhibitory effect of STS on cell growth and colony formation. Together, our results indicate that STS is a negative regulator of YAP, and its anti-tumor property is at least in part mediated by the Hippo signaling pathway. STS, isolated from Streptomyces staurosporeus, is a broad-spectrum kinase inhibitor. STS in high concentrations (100 nM–100 μM) was frequently used to induce apoptosis in multiple cell lines (Deshmukh and Johnson, 2000). We first tested the effect of gradient concentrations of STS on Hippo pathway activity in Hela cells. Hela cells were treated with increasing concentrations of STS for 4 h and subjected to western blot and real-time PCR, respectively. Western blot showed that 2 nM or higher concentrations of STS promoted MST1/2 phosphorylation dose-dependently, and both 5 and 10 nM STS upregulated LATS1/2-HM phosphorylation (Figure 1A). Consistently, YAP phosphorylation was upregulated in phos-tag gels by low concentrations of STS (2–10 nM) (Figure 1A). We next checked the effect of STS on YAP targets, as phosphorylated YAP should be sequestered in the cytoplasm and thus could not activate the expression of YAP target genes. As expected, real-time PCR results showed that the expression of YAP target genes CTGF and Cyr61 decreased in accordance with YAP phosphorylation levels upon STS treatment (Figure 1B). Western blot also showed that STS could downregulate CYR61 protein levels (Figure 1A). Meanwhile, immunostaining showed that 10 nM STS drove YAP cytosol localization at low cell density, consistent with YAP phosphorylation level (Figure 1C). Taken together, our results indicate that STS affects the activity of the Hippo pathway in a dose-dependent manner. Figure 1 View largeDownload slide STS inhibits cell growth in part mediated by the Hippo signaling pathway in Hela cells. (A) Hela cells were treated with increasing concentrations of STS for 4 h and subjected to western blot analysis with the indicated antibodies. The results showed that STS regulated MST1/2, LATS1/2, and YAP phosphorylation and CYR61 expression dose-dependently. (B) Real-time PCR results showed that STS regulated CTGF and Cyr61 expression dose-dependently. (C) Hela cells were cultured at low or high density and treated with the indicated concentrations of STS for 4 h before subjected to immunostaining for YAP and DAPI. Results showed that 10 nM STS drove YAP into cytosol at low density. (D) WT, Mst1/2 dKO, or Lats1/2 dKO Hela cells were treated with the indicated concentrations of STS. Western blot results showed that Mst1/2 dKO partially inhibited the upregulation of LATS and YAP phosphorylation induced by STS, while Lats1/2 dKO completely abolished YAP phosphorylation with or without STS treatment. (E) WT, Mst1/2 dKO, or Lats1/2 dKO Hela cells were treated with DMSO or 10 nM STS. Real-time PCR result showed that Lats1/2 dKO but not Mst1/2 dKO completely blocked the downregulation of Hippo target genes induced by 10 nM STS. (F) Mst1/2 dKO partially rescued YAP cytosol localization induced by 10 nM STS at low density, while Lats1/2 dKO completely blocked YAP cytosol localization. (G) Quantification of the percentage of cells with differential YAP localization shown in F. More than three fields were randomly chosen for each group. (H) Hela cells were treated with increasing concentrations of STS. MTT assay showed that STS inhibited Hela cell proliferation dose-dependently with an IC50 of ~10 nM. (I) WT, Mst1/2 dKO, or Lats1/2 dKO Hela cells were treated with DMSO or 10 nM STS. MTT assay showed that Lats1/2 dKO partially rescued the inhibitory effect of STS. (J) Soft agar assay with WT, Mst1/2 dKO, or Lats1/2 dKO Hela cells treated with DMSO or 10 nM STS showed that STS treatment decreased the clone number dramatically and Lats1/2 dKO not only increased the clone number but also blocked the effect of STS obviously. (K) Quantification of the clone number in J with triplicates for each group. All data are shown as mean ± SD. Statistical significance was determined using Student’s t-test. ***P < 0.001, **P < 0.01, *P < 0.05. Figure 1 View largeDownload slide STS inhibits cell growth in part mediated by the Hippo signaling pathway in Hela cells. (A) Hela cells were treated with increasing concentrations of STS for 4 h and subjected to western blot analysis with the indicated antibodies. The results showed that STS regulated MST1/2, LATS1/2, and YAP phosphorylation and CYR61 expression dose-dependently. (B) Real-time PCR results showed that STS regulated CTGF and Cyr61 expression dose-dependently. (C) Hela cells were cultured at low or high density and treated with the indicated concentrations of STS for 4 h before subjected to immunostaining for YAP and DAPI. Results showed that 10 nM STS drove YAP into cytosol at low density. (D) WT, Mst1/2 dKO, or Lats1/2 dKO Hela cells were treated with the indicated concentrations of STS. Western blot results showed that Mst1/2 dKO partially inhibited the upregulation of LATS and YAP phosphorylation induced by STS, while Lats1/2 dKO completely abolished YAP phosphorylation with or without STS treatment. (E) WT, Mst1/2 dKO, or Lats1/2 dKO Hela cells were treated with DMSO or 10 nM STS. Real-time PCR result showed that Lats1/2 dKO but not Mst1/2 dKO completely blocked the downregulation of Hippo target genes induced by 10 nM STS. (F) Mst1/2 dKO partially rescued YAP cytosol localization induced by 10 nM STS at low density, while Lats1/2 dKO completely blocked YAP cytosol localization. (G) Quantification of the percentage of cells with differential YAP localization shown in F. More than three fields were randomly chosen for each group. (H) Hela cells were treated with increasing concentrations of STS. MTT assay showed that STS inhibited Hela cell proliferation dose-dependently with an IC50 of ~10 nM. (I) WT, Mst1/2 dKO, or Lats1/2 dKO Hela cells were treated with DMSO or 10 nM STS. MTT assay showed that Lats1/2 dKO partially rescued the inhibitory effect of STS. (J) Soft agar assay with WT, Mst1/2 dKO, or Lats1/2 dKO Hela cells treated with DMSO or 10 nM STS showed that STS treatment decreased the clone number dramatically and Lats1/2 dKO not only increased the clone number but also blocked the effect of STS obviously. (K) Quantification of the clone number in J with triplicates for each group. All data are shown as mean ± SD. Statistical significance was determined using Student’s t-test. ***P < 0.001, **P < 0.01, *P < 0.05. To test whether the effect of STS on the Hippo pathway is dependent on MST1/2 or LATS1/2, we generated Mst1/2 dKO and Lats1/2 dKO Hela cells using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology and checked the effect of STS on these cell lines. Western blot showed that Mst1/2 dKO could partially inhibit the upregulation of LATS and YAP phosphorylation induced by STS (Figure 1D). Lats1/2 dKO not only abolished the basal level of YAP phosphorylation but also completely blocked the effect of STS on YAP phosphorylation (Figure 1D). Real-time PCR results also showed that Mst1/2 dKO only partially blocked, while Lats1/2 dKO completely rescued the downregulation of CTGF and Cyr61 expression by 10 nM STS (Figure 1E). Consistently, immunostaining showed that 10 nM STS induced YAP cytosol localization, which was completely blocked in Lats1/2 dKO cells, but only partially blocked in Mst1/2 dKO cells (Figure 1F and G). Collectively, these results suggest that MST1/2 are involved in STS-induced YAP phosphorylation and LATS1/2 are required for the effect of STS on the Hippo pathway. STS was reported to induce apoptosis and inhibit cell proliferation (Deshmukh and Johnson, 2000). We tested the effect of STS on Hela cell proliferation with MTT assay and found that STS inhibited cell proliferation dose-dependently with an IC50 of ~10 nM (Figure 1H). We next found that Lats1/2 dKO partially rescued the inhibitory effect of 10 nM STS on cell proliferation, while Mst1/2 dKO did not show any obvious effect (Figure 1I). Moreover, STS dramatically inhibited the colony formation ability of WT Hela cells. Lats1/2 dKO not only obviously enhanced the colony formation ability of Hela cells but also strikingly blocked the effect of STS, while Mst1/2 dKO did not exhibit any obvious effect (Figure 1J and K). Overall, our results suggest that STS acts through LATS1/2 but not MST1/2 to inhibit cell proliferation. The Hippo pathway plays critical roles in tumorigenesis, including promoting cancer cell proliferation and invasion and inhibiting apoptosis. Especially high Yap expression and activity were detected in some tumors, making YAP an attractive target for anticancer drug development. Here, we found that STS affected YAP localization dramatically and speculated that STS might modulate Hippo pathway activity, which was validated by the assays in Hela cells. Increasing low concentrations of STS promoted MST1/2 and LATS1/2 phosphorylation, which in turn regulated YAP phosphorylation and target gene expression. Results in Mst1/2 or Lats1/2 dKO Hela cells demonstrated that the effect of STS on the Hippo pathway required LATS1/2. Moreover, we found that Lats1/2 dKO could partially rescue the inhibitory effect of STS on cell proliferation. Taken together, our findings support the notion that STS acts through Hippo signaling to inhibit cell growth and provide a convenient way to modulate Hippo pathway activity. [This work was supported by the ‘Strategic Priority Research Program’ of the Chinese Academy of Sciences (XDB19000000), the National Key Research and Development Program of China (2017YFA0103601), the National Natural Science Foundation of China (31530043 and 31625017), the ‘Cross and Cooperation in Science and Technology Innovation Team’ Project of the Chinese Academy of Sciences, and the CAS/SAFEA International Partnership Program for Creative Research Teams.] References Camargo, F.D., Gokhale, S., Johnnidis, J.B., et al.  . ( 2007). YAP1 increases organ size and expands undifferentiated progenitor cells. Curr. Biol.  17, 2054– 2060. Google Scholar CrossRef Search ADS PubMed  Deshmukh, M., and Johnson, E.M., Jr. ( 2000). Staurosporine-induced neuronal death: multiple mechanisms and methodological implications. Cell Death Differ.  7, 250– 261. Google Scholar CrossRef Search ADS PubMed  Fan, F., He, Z., Kong, L.L., et al.  . ( 2016). Pharmacological targeting of kinases MST1 and MST2 augments tissue repair and regeneration. Sci. Transl. Med.  8, 352ra108. Google Scholar CrossRef Search ADS PubMed  Liu-Chittenden, Y., Huang, B., Shim, J.S., et al.  . ( 2012). Genetic and pharmacological disruption of the TEAD-YAP complex suppresses the oncogenic activity of YAP. Genes Dev.  26, 1300– 1305. Google Scholar CrossRef Search ADS PubMed  Meng, Z., Moroishi, T., and Guan, K.L. ( 2016). Mechanisms of Hippo pathway regulation. Genes Dev.  30, 1– 17. Google Scholar CrossRef Search ADS PubMed  Muramatsu, T., Imoto, I., Matsui, T., et al.  . ( 2011). YAP is a candidate oncogene for esophageal squamous cell carcinoma. Carcinogenesis  32, 389– 398. Google Scholar CrossRef Search ADS PubMed  Yu, F.X., Luo, J., Mo, J.S., et al.  . ( 2014). Mutant Gq/11 promote uveal melanoma tumorigenesis by activating YAP. Cancer Cell  25, 822– 830. Google Scholar CrossRef Search ADS PubMed  Yu, F.X., Zhao, B., and Guan, K.L. ( 2015). Hippo pathway in organ size control, tissue homeostasis, and cancer. Cell  163, 811– 828. Google Scholar CrossRef Search ADS PubMed  Zhao, B., Li, L., Tumaneng, K., et al.  . ( 2010). A coordinated phosphorylation by Lats and CK1 regulates YAP stability through SCF(β-TRCP). Genes Dev.  24, 72– 85. Google Scholar CrossRef Search ADS PubMed  Zhao, B., Wei, X., Li, W., et al.  . ( 2007). Inactivation of YAP oncoprotein by the Hippo pathway is involved in cell contact inhibition and tissue growth control. Genes Dev.  21, 2747– 2761. Google Scholar CrossRef Search ADS PubMed  Zhao, B., Ye, X., Yu, J., et al.  . ( 2008). TEAD mediates YAP-dependent gene induction and growth control. Genes Dev.  22, 1962– 1971. Google Scholar CrossRef Search ADS PubMed  Zhou, D., Conrad, C., Xia, F., et al.  . ( 2009). Mst1 and Mst2 maintain hepatocyte quiescence and suppress hepatocellular carcinoma development through inactivation of the Yap1 oncogene. Cancer Cell  16, 425– 438. Google Scholar CrossRef Search ADS PubMed  © The Author(s) (2018). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Molecular Cell Biology Oxford University Press

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Abstract

Dear Editor, The Hippo signaling pathway was discovered to control organ size in Drosophila through regulating cell proliferation and apoptosis (Yu et al., 2015). Arising studies over the past two decades have defined the core components and regulation mechanisms of the pathway in both Drosophila and mammals. The core components and fundamental function of the Hippo pathway are highly conserved in mammals. Active Ste20-like kinases 1/2 (MST1/2) phosphorylates adaptor protein Sav family WW domain-containing protein 1 (SAV1) and MOB1A/B, and phosphorylated MOB1A/B could recruit large tumor suppressor 1/2 (LATS1/2) for phosphorylation by MST1/2 at the hydrophobic motif (HM), followed by LATS1/2 autophosphorylation at the activation loop (AL) and full activation (Meng et al., 2016). Activated LATS1/2 then phosphorylates Yes-associated protein (YAP)/PDZ-binding motif (TAZ) and sequesters them in the cytoplasm for further phosphorylation and degradation (Zhao et al., 2007, 2010). Unphosphorylated YAP/TAZ translocates into the nucleus and associates with the TEAD family transcription factors to activate target genes expression (Zhao et al., 2008). Numerous studies have shown that the hippo pathway plays an important role in tumorigenesis. Overexpression of Yap or deletion of core components of the pathway (Nf2, Mst1/2, Sav1, and Mob1a/b) in liver can cause liver overgrowth and liver cancer (Camargo et al., 2007; Zhou et al., 2009; Yu et al., 2015). Increased YAP expression and activity were found in squamous cell carcinoma and other tumors (Muramatsu et al., 2011; Yu et al., 2014). Considering the important roles of the Hippo pathway, manipulation of Hippo pathway activity might be potential therapeutic means for diseases related with Hippo pathway dysfunction. Compounds targeting the Hippo pathway directly or indirectly have gained much investigation. Verteporfin is the first compound found to inhibit YAP/TEAD interaction and liver overgrowth induced by Yap overexpression or Nf2 deletion (Liu-Chittenden et al., 2012). XMU-MP-1 was reported to inhibit Mst1/2 kinase activities to activate YAP and augment tissue repair and regeneration (Fan et al., 2016). Here we find that Staurosporine (STS) regulates the activity and function of YAP, an effector of the Hippo pathway. Mechanistically, STS induces phosphorylation of Hippo pathway kinases MST1/2 and LATS1/2, which in turn leads to YAP phosphorylation and cytoplasmic localization, thus decreases the expression of YAP target genes. MST1/2 double knockout (dKO) partially inhibits whereas LATS1/2 dKO largely blocks the effect of STS on YAP phosphorylation and target gene expression. In addition, LATS1/2 dKO partially rescues the inhibitory effect of STS on cell growth and colony formation. Together, our results indicate that STS is a negative regulator of YAP, and its anti-tumor property is at least in part mediated by the Hippo signaling pathway. STS, isolated from Streptomyces staurosporeus, is a broad-spectrum kinase inhibitor. STS in high concentrations (100 nM–100 μM) was frequently used to induce apoptosis in multiple cell lines (Deshmukh and Johnson, 2000). We first tested the effect of gradient concentrations of STS on Hippo pathway activity in Hela cells. Hela cells were treated with increasing concentrations of STS for 4 h and subjected to western blot and real-time PCR, respectively. Western blot showed that 2 nM or higher concentrations of STS promoted MST1/2 phosphorylation dose-dependently, and both 5 and 10 nM STS upregulated LATS1/2-HM phosphorylation (Figure 1A). Consistently, YAP phosphorylation was upregulated in phos-tag gels by low concentrations of STS (2–10 nM) (Figure 1A). We next checked the effect of STS on YAP targets, as phosphorylated YAP should be sequestered in the cytoplasm and thus could not activate the expression of YAP target genes. As expected, real-time PCR results showed that the expression of YAP target genes CTGF and Cyr61 decreased in accordance with YAP phosphorylation levels upon STS treatment (Figure 1B). Western blot also showed that STS could downregulate CYR61 protein levels (Figure 1A). Meanwhile, immunostaining showed that 10 nM STS drove YAP cytosol localization at low cell density, consistent with YAP phosphorylation level (Figure 1C). Taken together, our results indicate that STS affects the activity of the Hippo pathway in a dose-dependent manner. Figure 1 View largeDownload slide STS inhibits cell growth in part mediated by the Hippo signaling pathway in Hela cells. (A) Hela cells were treated with increasing concentrations of STS for 4 h and subjected to western blot analysis with the indicated antibodies. The results showed that STS regulated MST1/2, LATS1/2, and YAP phosphorylation and CYR61 expression dose-dependently. (B) Real-time PCR results showed that STS regulated CTGF and Cyr61 expression dose-dependently. (C) Hela cells were cultured at low or high density and treated with the indicated concentrations of STS for 4 h before subjected to immunostaining for YAP and DAPI. Results showed that 10 nM STS drove YAP into cytosol at low density. (D) WT, Mst1/2 dKO, or Lats1/2 dKO Hela cells were treated with the indicated concentrations of STS. Western blot results showed that Mst1/2 dKO partially inhibited the upregulation of LATS and YAP phosphorylation induced by STS, while Lats1/2 dKO completely abolished YAP phosphorylation with or without STS treatment. (E) WT, Mst1/2 dKO, or Lats1/2 dKO Hela cells were treated with DMSO or 10 nM STS. Real-time PCR result showed that Lats1/2 dKO but not Mst1/2 dKO completely blocked the downregulation of Hippo target genes induced by 10 nM STS. (F) Mst1/2 dKO partially rescued YAP cytosol localization induced by 10 nM STS at low density, while Lats1/2 dKO completely blocked YAP cytosol localization. (G) Quantification of the percentage of cells with differential YAP localization shown in F. More than three fields were randomly chosen for each group. (H) Hela cells were treated with increasing concentrations of STS. MTT assay showed that STS inhibited Hela cell proliferation dose-dependently with an IC50 of ~10 nM. (I) WT, Mst1/2 dKO, or Lats1/2 dKO Hela cells were treated with DMSO or 10 nM STS. MTT assay showed that Lats1/2 dKO partially rescued the inhibitory effect of STS. (J) Soft agar assay with WT, Mst1/2 dKO, or Lats1/2 dKO Hela cells treated with DMSO or 10 nM STS showed that STS treatment decreased the clone number dramatically and Lats1/2 dKO not only increased the clone number but also blocked the effect of STS obviously. (K) Quantification of the clone number in J with triplicates for each group. All data are shown as mean ± SD. Statistical significance was determined using Student’s t-test. ***P < 0.001, **P < 0.01, *P < 0.05. Figure 1 View largeDownload slide STS inhibits cell growth in part mediated by the Hippo signaling pathway in Hela cells. (A) Hela cells were treated with increasing concentrations of STS for 4 h and subjected to western blot analysis with the indicated antibodies. The results showed that STS regulated MST1/2, LATS1/2, and YAP phosphorylation and CYR61 expression dose-dependently. (B) Real-time PCR results showed that STS regulated CTGF and Cyr61 expression dose-dependently. (C) Hela cells were cultured at low or high density and treated with the indicated concentrations of STS for 4 h before subjected to immunostaining for YAP and DAPI. Results showed that 10 nM STS drove YAP into cytosol at low density. (D) WT, Mst1/2 dKO, or Lats1/2 dKO Hela cells were treated with the indicated concentrations of STS. Western blot results showed that Mst1/2 dKO partially inhibited the upregulation of LATS and YAP phosphorylation induced by STS, while Lats1/2 dKO completely abolished YAP phosphorylation with or without STS treatment. (E) WT, Mst1/2 dKO, or Lats1/2 dKO Hela cells were treated with DMSO or 10 nM STS. Real-time PCR result showed that Lats1/2 dKO but not Mst1/2 dKO completely blocked the downregulation of Hippo target genes induced by 10 nM STS. (F) Mst1/2 dKO partially rescued YAP cytosol localization induced by 10 nM STS at low density, while Lats1/2 dKO completely blocked YAP cytosol localization. (G) Quantification of the percentage of cells with differential YAP localization shown in F. More than three fields were randomly chosen for each group. (H) Hela cells were treated with increasing concentrations of STS. MTT assay showed that STS inhibited Hela cell proliferation dose-dependently with an IC50 of ~10 nM. (I) WT, Mst1/2 dKO, or Lats1/2 dKO Hela cells were treated with DMSO or 10 nM STS. MTT assay showed that Lats1/2 dKO partially rescued the inhibitory effect of STS. (J) Soft agar assay with WT, Mst1/2 dKO, or Lats1/2 dKO Hela cells treated with DMSO or 10 nM STS showed that STS treatment decreased the clone number dramatically and Lats1/2 dKO not only increased the clone number but also blocked the effect of STS obviously. (K) Quantification of the clone number in J with triplicates for each group. All data are shown as mean ± SD. Statistical significance was determined using Student’s t-test. ***P < 0.001, **P < 0.01, *P < 0.05. To test whether the effect of STS on the Hippo pathway is dependent on MST1/2 or LATS1/2, we generated Mst1/2 dKO and Lats1/2 dKO Hela cells using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology and checked the effect of STS on these cell lines. Western blot showed that Mst1/2 dKO could partially inhibit the upregulation of LATS and YAP phosphorylation induced by STS (Figure 1D). Lats1/2 dKO not only abolished the basal level of YAP phosphorylation but also completely blocked the effect of STS on YAP phosphorylation (Figure 1D). Real-time PCR results also showed that Mst1/2 dKO only partially blocked, while Lats1/2 dKO completely rescued the downregulation of CTGF and Cyr61 expression by 10 nM STS (Figure 1E). Consistently, immunostaining showed that 10 nM STS induced YAP cytosol localization, which was completely blocked in Lats1/2 dKO cells, but only partially blocked in Mst1/2 dKO cells (Figure 1F and G). Collectively, these results suggest that MST1/2 are involved in STS-induced YAP phosphorylation and LATS1/2 are required for the effect of STS on the Hippo pathway. STS was reported to induce apoptosis and inhibit cell proliferation (Deshmukh and Johnson, 2000). We tested the effect of STS on Hela cell proliferation with MTT assay and found that STS inhibited cell proliferation dose-dependently with an IC50 of ~10 nM (Figure 1H). We next found that Lats1/2 dKO partially rescued the inhibitory effect of 10 nM STS on cell proliferation, while Mst1/2 dKO did not show any obvious effect (Figure 1I). Moreover, STS dramatically inhibited the colony formation ability of WT Hela cells. Lats1/2 dKO not only obviously enhanced the colony formation ability of Hela cells but also strikingly blocked the effect of STS, while Mst1/2 dKO did not exhibit any obvious effect (Figure 1J and K). Overall, our results suggest that STS acts through LATS1/2 but not MST1/2 to inhibit cell proliferation. The Hippo pathway plays critical roles in tumorigenesis, including promoting cancer cell proliferation and invasion and inhibiting apoptosis. Especially high Yap expression and activity were detected in some tumors, making YAP an attractive target for anticancer drug development. Here, we found that STS affected YAP localization dramatically and speculated that STS might modulate Hippo pathway activity, which was validated by the assays in Hela cells. Increasing low concentrations of STS promoted MST1/2 and LATS1/2 phosphorylation, which in turn regulated YAP phosphorylation and target gene expression. Results in Mst1/2 or Lats1/2 dKO Hela cells demonstrated that the effect of STS on the Hippo pathway required LATS1/2. Moreover, we found that Lats1/2 dKO could partially rescue the inhibitory effect of STS on cell proliferation. Taken together, our findings support the notion that STS acts through Hippo signaling to inhibit cell growth and provide a convenient way to modulate Hippo pathway activity. [This work was supported by the ‘Strategic Priority Research Program’ of the Chinese Academy of Sciences (XDB19000000), the National Key Research and Development Program of China (2017YFA0103601), the National Natural Science Foundation of China (31530043 and 31625017), the ‘Cross and Cooperation in Science and Technology Innovation Team’ Project of the Chinese Academy of Sciences, and the CAS/SAFEA International Partnership Program for Creative Research Teams.] References Camargo, F.D., Gokhale, S., Johnnidis, J.B., et al.  . ( 2007). YAP1 increases organ size and expands undifferentiated progenitor cells. Curr. Biol.  17, 2054– 2060. Google Scholar CrossRef Search ADS PubMed  Deshmukh, M., and Johnson, E.M., Jr. ( 2000). Staurosporine-induced neuronal death: multiple mechanisms and methodological implications. Cell Death Differ.  7, 250– 261. Google Scholar CrossRef Search ADS PubMed  Fan, F., He, Z., Kong, L.L., et al.  . ( 2016). Pharmacological targeting of kinases MST1 and MST2 augments tissue repair and regeneration. Sci. Transl. Med.  8, 352ra108. Google Scholar CrossRef Search ADS PubMed  Liu-Chittenden, Y., Huang, B., Shim, J.S., et al.  . ( 2012). Genetic and pharmacological disruption of the TEAD-YAP complex suppresses the oncogenic activity of YAP. Genes Dev.  26, 1300– 1305. Google Scholar CrossRef Search ADS PubMed  Meng, Z., Moroishi, T., and Guan, K.L. ( 2016). Mechanisms of Hippo pathway regulation. Genes Dev.  30, 1– 17. Google Scholar CrossRef Search ADS PubMed  Muramatsu, T., Imoto, I., Matsui, T., et al.  . ( 2011). YAP is a candidate oncogene for esophageal squamous cell carcinoma. Carcinogenesis  32, 389– 398. Google Scholar CrossRef Search ADS PubMed  Yu, F.X., Luo, J., Mo, J.S., et al.  . ( 2014). Mutant Gq/11 promote uveal melanoma tumorigenesis by activating YAP. Cancer Cell  25, 822– 830. Google Scholar CrossRef Search ADS PubMed  Yu, F.X., Zhao, B., and Guan, K.L. ( 2015). Hippo pathway in organ size control, tissue homeostasis, and cancer. Cell  163, 811– 828. Google Scholar CrossRef Search ADS PubMed  Zhao, B., Li, L., Tumaneng, K., et al.  . ( 2010). A coordinated phosphorylation by Lats and CK1 regulates YAP stability through SCF(β-TRCP). Genes Dev.  24, 72– 85. Google Scholar CrossRef Search ADS PubMed  Zhao, B., Wei, X., Li, W., et al.  . ( 2007). Inactivation of YAP oncoprotein by the Hippo pathway is involved in cell contact inhibition and tissue growth control. Genes Dev.  21, 2747– 2761. Google Scholar CrossRef Search ADS PubMed  Zhao, B., Ye, X., Yu, J., et al.  . ( 2008). TEAD mediates YAP-dependent gene induction and growth control. Genes Dev.  22, 1962– 1971. Google Scholar CrossRef Search ADS PubMed  Zhou, D., Conrad, C., Xia, F., et al.  . ( 2009). Mst1 and Mst2 maintain hepatocyte quiescence and suppress hepatocellular carcinoma development through inactivation of the Yap1 oncogene. Cancer Cell  16, 425– 438. Google Scholar CrossRef Search ADS PubMed  © The Author(s) (2018). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

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Journal of Molecular Cell BiologyOxford University Press

Published: Mar 19, 2018

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