Journal: Signal Transduction and Targeted Therapy

Article Title: PKC-eta promotes breast cancer metastasis by regulating the Hippo–YAP signaling pathway

doi: 10.1038/s41392-026-02572-0

Figure Lengend Snippet: PKCη promotes YAP phosphorylation at Ser128 and attenuates the interaction of YAP at Ser127 with the 14-3-3 proteins. a , b Coimmunoprecipitation assays using 4T1 and MDA-MB-231 cell lysates demonstrated the interaction between PKCη and YAP (a mouse PKCη antibody was used for YAP pulldown, and a rabbit YAP antibody was used for PKCη pulldown). c Surface representation of the YAP1-PKCη docked pose. A structural model depicting the docked pose of YAP1-PKCη reveals the potential binding probability between the catalytic domain of PKCη and the TEAD-binding domain of YAP, highlighting the Ser128 and Ser127 phosphorylation sites. d ELISA depicting the binding affinity of YAP for PKCη (the YAP concentration was set at 1 µg/ml, and the PKCη concentration was tested between 0.1 and 1 µg/ml). e ELISAs were performed to determine the binding affinities of PKCη with YAP-derived wild-type and mutated peptides (at S128) (peptide concentrations were maintained at 1 µg/mL in the presence of varying PKCη levels (0.1 to 1 µg/mL)). f Immunoblotting depicting YAP phosphorylation at Ser109, Ser127, Ser128, Ser397 and TAZ Ser89 in 4T1 and MDA-MB-231 control cells and in cells devoid of PKCη. g Immunohistochemical (IHC) analysis of phosphorylated YAP (Ser127) in primary tumor tissues from xenografts of 4T1 and MDA-MB-231 cells (control and PKCη KO ). Quantification revealed that pYAP (Ser127) expression was increased in PKCη KO tumors compared with control tumors. The scale bar indicates 50 μm. h Kinase activity assay of PKCη phosphorylation on recombinant YAP. Phosphorylation is represented in relative fluorescence units (RFUs). i Kinase activity assay of PKCη phosphorylation of YAP-derived peptides: wild-type and Ser128-mutated peptides. j HEK293FT cells were transfected with YAP alone (control), YAP + wild-type PKCη (active), or YAP + kinase-dead PKCη (PKCη KD ). Western blot analysis revealed that YAP S128 phosphorylation occurred only in cells overexpressing YAP together with active PKCη but not with PKCη KD , demonstrating that PKCη kinase activity is essential for this phosphorylation event. k Coimmunoprecipitation assays using 4T1 cell lysates demonstrated an interaction between YAP and 14-3-3 (a mouse YAP antibody was used for 14-3-3 pull-down). l Schematic representation of YAP activation by PKCη. We propose that PKCη expression leads to YAP activation (pYAP Ser128) and nuclear retention, disrupting the phosphorylation-dependent interaction between YAP (pYAP Ser127) and 14-3-3. Statistical significance was determined via two-way ANOVA, where * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001

Article Snippet: The antibodies used in this study were against YAP (CST, #14074), MST1 (CST, #3682), pMST1 (CST, #3681), LATS1 (CST, #3477), 14-3-3 ζ/δ (CST, #7413), pLATS1 (CST, #8654), pYAP (Ser127) (CST, #13008), pYAP (Ser109) (CST, #53749), pYAP (Ser397) (CST, #13619), pTAZ (Ser89) (CST, #75275), TAZ (CST, #83669), PTEN (CST, #9559) Lamin B1 (CST, #13435), AKT (pan) (CST, #4691), AXL (CST, #8661), Pan-TEAD (CST, #13295), CYR61 (CST, 14479), IGFBP3 (CST, #25864), HA-Tag (CST, #3724), FLAG (CST, #14793), pAKT (Ser473) (CST, #4060), and Anti-rabbit IgG (CST, # 7074).

Techniques: Phospho-proteomics, Binding Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay, Derivative Assay, Western Blot, Control, Immunohistochemical staining, Expressing, Kinase Assay, Recombinant, Fluorescence, Transfection, Activity Assay, Activation Assay

Journal: Signal Transduction and Targeted Therapy

Article Title: PKC-eta promotes breast cancer metastasis by regulating the Hippo–YAP signaling pathway

doi: 10.1038/s41392-026-02572-0

Figure Lengend Snippet: The uORF2-encoded peptide (uPEP2) upstream of PKCη degrades PKCη and downregulates the YAP-Hippo pathway both in vitro and in vivo. a Compared with control cells, 4T1 and MDA-MB-231 cells treated with uPEP2 presented decreased migration and invasion capabilities. Migration was evaluated via a Boyden chamber assay. Invasion was assessed using Matrigel-coated Boyden chamber chambers. The scale bar indicates 50 μm. b uPEP2 inhibits sphere cell invasion through Matrigel. The area of the invaded zone was measured after 24 h, and the results are shown ( n = 5). The scale bar indicates 20 μm. c Effects of uPEP2 treatment on EMT markers in 4T1 and MDA-MB-231 cells. d uPEP2 treatment of 4T1 and MDA-MB-231 cells downregulated PKCη and YAP expression and affected members of the Hippo pathway and their phosphorylation. e Immunohistochemical analysis of PKCη, YAP, and pYAP (S127) expression in uPEP2-treated MDA-MB-231 xenograft primary tumors. Representative images and quantitative analyses are presented. Scale bar, 100 μm. f Schematic of the effect of uPEP2 on PKCη and its downstream functions. uORF2-encoded uPEP2 (blue) inhibits the catalytic activity of PKCη (yellow) and causes PKCη degradation, leading to reduced cell proliferation, EMT, stemness, migration, invasion, and metastasis. The data are expressed as the means ± SEMs ( n = 3). Statistical significance was determined via two-way ANOVA, where * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001

Article Snippet: The antibodies used in this study were against YAP (CST, #14074), MST1 (CST, #3682), pMST1 (CST, #3681), LATS1 (CST, #3477), 14-3-3 ζ/δ (CST, #7413), pLATS1 (CST, #8654), pYAP (Ser127) (CST, #13008), pYAP (Ser109) (CST, #53749), pYAP (Ser397) (CST, #13619), pTAZ (Ser89) (CST, #75275), TAZ (CST, #83669), PTEN (CST, #9559) Lamin B1 (CST, #13435), AKT (pan) (CST, #4691), AXL (CST, #8661), Pan-TEAD (CST, #13295), CYR61 (CST, 14479), IGFBP3 (CST, #25864), HA-Tag (CST, #3724), FLAG (CST, #14793), pAKT (Ser473) (CST, #4060), and Anti-rabbit IgG (CST, # 7074).

Techniques: In Vitro, In Vivo, Control, Migration, Boyden Chamber Assay, Expressing, Phospho-proteomics, Immunohistochemical staining, Activity Assay

Journal: Advanced Science

Article Title: EM2, a Natural Product MST1/2 Kinase Activator, Suppresses Non‐Small Cell Lung Cancer via Hippo Pathway Activation

doi: 10.1002/advs.202510508

Figure Lengend Snippet: The role of YAP activity in EM2 anti‐NSCLC. A,B) After EM2 treatment with H520 and A549, representative images A) and quantification B) of YAP localization were detected by IF staining. C,D) Cytoplasmic and nuclear distributions of YAP in H520 and A549 cells treated with or without EM2 for 12 h. Histone 2B and GAPDH were used as endogenous references for nuclear and cytosolic fractions C). The indicated protein expression levels were quantified D). E) Transcriptional activity of YAP/TEAD complex in H520 and A549 cells. Cells were transfected with the reporter plasmids and treated with EM2 at the indicated concentrations for 24 h. F) mRNA levels of C CN1 and CCN2 in H520 cells treated with EM2. RT‐qPCR data were normalized to GAPDH levels and presented as fold‐change compared with control cells. G) Western blotting was used to detect the expressions of YAP and p‐YAP in YAP WT and YAP S127A H520 cells. H) Cell viability of A549 and H520 cells transfected with YAP WT and YAP S127A were treated with 0, 2.5, 5, 10, and 20 µM EM2 for 24 h, and the cell viability was detected by MTT. I,J) Representative images and quantification of EM2 on the migration I) and invasion J) in H520 cells transfected with YAP WT and YAP S127A. Following treatment with indicated concentrations of EM2, the cells were subjected to Transwell migration and invasion assays. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Antibodies against MST1 (#3682), LATS1 (#3477), YAP (#12395), YAP/TAZ (#8418), phospho‐MST1 (Thr183)/MST2 (Thr180) (#49332), phospho‐LATS1 (Ser909) (#9157), phospho‐YAP (Ser127) (#13008), phospho‐TAZ (Ser89) (#59971), phospho‐Mob1 (Thr35) (#8699), CTGF (#86641), CYR61 (#14479) Phospho‐Rb(Ser780) (#2808), CDK4 (#12790), CDK6 (#3136), and Cyclin D1 (#55506) were purchased from Cell Signaling Technology.

Techniques: Activity Assay, Staining, Expressing, Transfection, Quantitative RT-PCR, Control, Western Blot, Migration

Journal: Advanced Science

Article Title: EM2, a Natural Product MST1/2 Kinase Activator, Suppresses Non‐Small Cell Lung Cancer via Hippo Pathway Activation

doi: 10.1002/advs.202510508

Figure Lengend Snippet: The anti‐tumor activity of EM2 was counteracted by MST1/2 inhibitor in vitro. A) Cell viability of H520 and A549 cells treated by EM2 (8 µM) with or without XMU‐MP‐1 (3 µM). B,C) Representative images B) and quantification C) of colony formation assay of H520 and A549 cells treated by EM2 (2 µM) with or without XMU‐MP‐1 (0.5 µM). D) EdU assay of H520 and A549 cells treated by EM2 (2 µM) with or without XMU‐MP‐1 (0.5 µM). E) Quantification of Edu positive in H520 and A549 cells. F) Immunoblot analysis of p‐MST, p‐LATS1/2, p‐YAP, and p‐TAZ in H520 and A549 cells treated by EM2 (8 µM) with or without XMU‐MP‐1 (3 µM). β‐tubulin served as the loading control. G) Quantification of the protein levels indicated in H520 and A549 cells. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Antibodies against MST1 (#3682), LATS1 (#3477), YAP (#12395), YAP/TAZ (#8418), phospho‐MST1 (Thr183)/MST2 (Thr180) (#49332), phospho‐LATS1 (Ser909) (#9157), phospho‐YAP (Ser127) (#13008), phospho‐TAZ (Ser89) (#59971), phospho‐Mob1 (Thr35) (#8699), CTGF (#86641), CYR61 (#14479) Phospho‐Rb(Ser780) (#2808), CDK4 (#12790), CDK6 (#3136), and Cyclin D1 (#55506) were purchased from Cell Signaling Technology.

Techniques: Activity Assay, In Vitro, Colony Assay, EdU Assay, Western Blot, Control

Journal: Advanced Science

Article Title: EM2, a Natural Product MST1/2 Kinase Activator, Suppresses Non‐Small Cell Lung Cancer via Hippo Pathway Activation

doi: 10.1002/advs.202510508

Figure Lengend Snippet: The role and mechanism of EM2 in inhibiting the tumor growth of NSCLC in vivo. Representative images of the subcutaneous tumor model in mice of control and EM2 (5 or 10 mg kg −1 ) group. B) Tumor volume in control and EM2 (5 or 10 mg kg −1 ) group. C) Quantification and analysis of the tumor weight in control and EM2 (5 or 10 mg kg −1 ) group. D) The expression levels of Ki67, YAP, CTGF, and CYR61 in tumors assayed by IHC in control and EM2 (5 or 10 mg kg −1 ) group. E) Expression levels of MST1, LATS1, and YAP and their phosphorylation forms along with downstream protein CTGF and CYR61 were examined by Western blotting in control and EM2 (5 or 10 mg kg −1 ) group. F) Representative images of tumors in subcutaneous mouse model, grouped by treatment with EM2 with or without XMU‐MP‐1. G) Tumor volume in each group of EM2 with or without XMU‐MP‐1. H) Quantification and analysis of the tumor weight in each group of EM2 with or without XMU‐MP‐1. I,J) Representative images I) and quantification J) of the expression levels of Ki67, YAP, CTGF, and CYR61 in tumors assayed by IHC in each group of EM2 with or without XMU‐MP‐1. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Antibodies against MST1 (#3682), LATS1 (#3477), YAP (#12395), YAP/TAZ (#8418), phospho‐MST1 (Thr183)/MST2 (Thr180) (#49332), phospho‐LATS1 (Ser909) (#9157), phospho‐YAP (Ser127) (#13008), phospho‐TAZ (Ser89) (#59971), phospho‐Mob1 (Thr35) (#8699), CTGF (#86641), CYR61 (#14479) Phospho‐Rb(Ser780) (#2808), CDK4 (#12790), CDK6 (#3136), and Cyclin D1 (#55506) were purchased from Cell Signaling Technology.

Techniques: In Vivo, Control, Expressing, Phospho-proteomics, Western Blot

Journal: Advanced Science

Article Title: EM2, a Natural Product MST1/2 Kinase Activator, Suppresses Non‐Small Cell Lung Cancer via Hippo Pathway Activation

doi: 10.1002/advs.202510508

Figure Lengend Snippet: Schematic illustration of EM2‐induced suppression of cancer through targeting MST1/2 and activation of the Hippo signaling pathway. EM2 directly targets MST1/2, enhancing its kinase activity to promote LATS and YAP phosphorylation. This cascade reduces YAP nuclear translocation and diminishes CTGF and CYR61 mRNA translation, thereby exerting a potent inhibitory effect on NSCLC progression.

Article Snippet: Antibodies against MST1 (#3682), LATS1 (#3477), YAP (#12395), YAP/TAZ (#8418), phospho‐MST1 (Thr183)/MST2 (Thr180) (#49332), phospho‐LATS1 (Ser909) (#9157), phospho‐YAP (Ser127) (#13008), phospho‐TAZ (Ser89) (#59971), phospho‐Mob1 (Thr35) (#8699), CTGF (#86641), CYR61 (#14479) Phospho‐Rb(Ser780) (#2808), CDK4 (#12790), CDK6 (#3136), and Cyclin D1 (#55506) were purchased from Cell Signaling Technology.

Techniques: Activation Assay, Activity Assay, Phospho-proteomics, Translocation Assay

Journal: Cell reports

Article Title: Mutant RIT1 cooperates with YAP to drive an EMT-like lung cancer state

doi: 10.1016/j.celrep.2025.116185

Figure Lengend Snippet: (A) Growth in low attachment assay of isogenic SALE cells expressing RIT1 M90I and YAP1 8SA alone or in combination analyzed by CellTiterGlo. Data shown are the mean ± SD of 14–16 technical replicates. (B) Heatmap of gene expression data derived from bulk RNA-seq of isogenic SALE cells. Each row is a differentially expressed gene, and each column is a replicate of the cell line variant indicated. The top 100 up- and downregulated genes distinguishing combined RIT1 M90I /YAP1 8SA (RY) cells from YAP1 8SA cells were determined by marker selection based on the mean difference of RY replicates compared to YAP1 8SA replicates and then genes and samples were clustered using one minus Pearson correlation. (C) Volcano plot of differentially expressed genes from RNA-seq in SALE-RY cells versus parental SALE cells. p values shown were calculated by t tests of three biological replicates per cell line. Multiple hypothesis testing was performed using the false discovery rate (FDR) method. Red points: log 2 fold change (log 2 FC) > 1.5, false discovery rate (FDR) < 0.1; blue points: log 2 FC < −1.5, FDR < 0.1. (D) MSigDB overlap analysis of up- and downregulated genes from RNA-seq in RY cells compared to parental. The FDR is the false discovery rate analog of the hypergeometic p value after correction for multiple hypothesis testing. (E) Motif analysis of up- and downregulated genes from (C) using the MSigDB Transcription Factor Target (TFT) gene set. AP-1 and ZEB1 (AREB6) motifs are shown in bold. FDR was calculated as in (D). (F) Western blot of isogenic SALE cells using antibodies against RIT1 and AP-1 transcription factors cJUN and FOSL1/FRA1. Vinculin is used as a loading control. (G) Quantification of biological replicates ( n = 3) of cJUN abundance determined by western blot, normalized to loading control and parental cJUN abundance. Data shown are mean ± SEM. P, parental; R, RIT1 M90I ; Y, YAP1 8SA ; RY, RIT1 M90I/ YAP1 8SA . (H) Quantification of biological replicates ( n = 3) of FRA1 abundance determined by western blot, normalized to loading control and parental FRA1 abundance. Data shown are mean ± SEM. Labeling as in (G). See also and . ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 by unpaired two-tailed t test.

Article Snippet: YAP1 (D8H1X) (WB) , Cell Signaling Technology , Cat #14710; RRID: AB_2798483.

Techniques: Expressing, Gene Expression, Derivative Assay, RNA Sequencing, Variant Assay, Marker, Selection, Western Blot, Control, Labeling, Two Tailed Test

Journal: Cell reports

Article Title: Mutant RIT1 cooperates with YAP to drive an EMT-like lung cancer state

doi: 10.1016/j.celrep.2025.116185

Figure Lengend Snippet: (A) Immunofluorescence and confocal images of SALE cells fixed and stained with anti-YAP and anti-cJUN antibodies and counterstained with DAPI. Scale bar, 100 μm. Inset, 50 μm. (B) Quantification of nuclear YAP fluorescence intensity determined by confocal microscopy. Data shown are mean ± SEM of four biological replicates. P, parental; R, RIT1 M90I ; Y, YAP1 8SA ; RY, RIT1 M90I /YAP1 8SA (C) Quantification of nuclear cJUN fluorescence intensity determined by confocal microscopy. Data shown are mean ± SEM of four biological replicates. Labeling as in (B). (D) Dual p-AP1 luciferase reporter assay in HEK293T cells transiently transfected with control vectors (V), RIT1 M90I (R), or YAP1 WT and YAP1 8SA in the presence or absence of RIT1 M90I . p-RL renilla luciferase was co-transfected and used for normalization. PMA was used as a positive control. Data shown are mean ± SEM of 3 technical replicates. Data are representative of at least three independent experiments. (E) Western blot of SALE-RY cells showing doxycycline-regulated induction of cJUN or a dominant-negative cJUN (TAM67). (F) In vivo xenograft tumor growth on doxycycline of the cells shown in (E). Data shown are mean ± SEM of 8–10 tumors per group. * P < 0.05 by one-way ANOVA. (G) Tumor weights of tumors shown in (F). Data shown are mean ± SEM. The cJUN wild-type group was not analyzed due to early euthanasia for tumor ulceration. ns, not significant; * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 by unpaired two-tailed t test unless otherwise specified. See also .

Article Snippet: YAP1 (D8H1X) (WB) , Cell Signaling Technology , Cat #14710; RRID: AB_2798483.

Techniques: Immunofluorescence, Staining, Fluorescence, Confocal Microscopy, Labeling, Luciferase, Reporter Assay, Transfection, Control, Positive Control, Western Blot, Dominant Negative Mutation, In Vivo, Two Tailed Test