Journal: Advanced Science

Article Title: Excessive MYC Orchestrates Macrophages induced Chromatin Remodeling to Sustain Micropapillary‐Patterned Malignancy in Lung Adenocarcinoma

doi: 10.1002/advs.202403851

Figure Lengend Snippet: MYC in collaboration with M2‐like macrophages gains unique binding sites that exhibit alternative transcriptional regulatory activity. A) The DNase I hypersensitivity site identified via DNase I treated‐PCR in promoters of MP‐pattern genes in E‐V cells, M‐V cells, E‐M2 cells and M‐M2 cells (A549 cell based). B) HOMER de novo motif analysis on altered H3K27ac occupied sites in AC‐subtype tissue versus MP‐subtype tissue, and M‐V cells versus M‐M2 cells. C) Western Blots of FOSL2 and JUN in cells with different treatment. Three biological replicates were performed for each cell. D) ChIP‐PCR of IgG, MYC and FOSL2 for binding promoters of MP‐pattern genes in A549 cells with different treatment. E) The DNase I hypersensitivity site identified via DNase I treated‐PCR in promoters of MP‐pattern genes in A549 cells with different treatment. The dependence experiment revealed that the high DNase I sensitivity of MP‐pattern gene promoters induced by M2‐like macrophages depends on TGFβ‐FOSL2 axis. F) ChIP‐PCR of IgG, MYC and FOSL2 for binding promoters of MP‐pattern genes in A549 cells with different treatment. The dependence experiment revealed that MYC binding MP‐pattern gene promoters in M‐M2 A549 cells depends on TGFβ‐FOSL2 axis. G) The DNase I hypersensitivity site identified via DNase I treated‐PCR in promoters of MP‐pattern genes in A549 cells with different treatment. The rescue experiment revealed that the high DNase I sensitivity of MP‐pattern gene promoters in solely MYC‐overexpression A549 cells were rescued by FOSL2 overexpression or exogenous addition of TGFβ. H) ChIP‐PCR of IgG, MYC and FOSL2 for binding promoters of MP‐pattern genes in A549 cells with different treatment. The rescue experiment revealed that MYC binding MP‐pattern gene promoters in M‐V A549 cells were rescued by FOSL2 overexpression or exogenous addition of TGFβ. I) Western Blots of FOSL2 in cancer cells with different time‐point of coculture with M2‐like macrophages. Three biological replicates were performed for each cell. J) Western Blots of FOSL2 in cancer cells coculture with M2‐like macrophages and several antibodies were used to neutralize cytokines. Three biological replicates were performed for each cell. K,L) Western Blots of FOSL2 in cancer cells exogenous addition of TGFβ with different concentration (K) and treatment time (L). M) Representative images of immunohistochemistry of FOSL2 in tissues micro‐array from MAPes cohort. Right: quantitative statistics. Scale bars: 100 µm. N) Relative expression of FOSL2 in SCISSOR+ cells (MP‐subtype proneness, mean expression: 0.4277) and SCISSOR‐ cells (AC‐subtype proneness, mean expression: 0.3369) from single‐cell RNA‐seq data in Figure . O) The correlation analysis in expression of MYC and expression of FOSL2 from TCGA‐LUAD mRNA dataset. P) Representative images of immunofluorescence of FOSL2 (green), MYC (red) and DAPI (blue) in M‐M2 cells, Scale bars: 1µm (Upper). The detected fluorescence intensity at the white line (Bottom). Q) Virtual docking was performed based on the protein structure of MYC and the tertiary structure of FOSL2 protein predicted by Alphafold2. R) Co‐immunoprecipitation of c‐Myc antibody and IgG antibody indicated that MYC binding FOSL2 after redundant expression when cancer cells were co‐cultured with M2‐like macrophages. For each group, n = 5. The p values were determined by Student's t ‐test (unpaired two‐tailed), n.s., not significant; * p < 0.05, ** p < 0.01, *** p < 0.001. Data are represented as mean ± SEM.

Article Snippet: For certain experiments, cells were transfected with a full‐length c‐Myc expression vector (pcDNA3‐cmyc, Addgene, #16011, RR_ID: Addgene_16011). pcDNA3 empty vector (Corues Biotechnology Co.), targeting FOSL2 short hairpin RNA and control scribble (Sangon Biotech), FOSL2‐expression vector (Addgene, #187907, RR_ID: Addgene_187907), pcDNA3.1 empty vector (Corues Biotechnology Co.), FOSL2 tet‐off system vector (pLV3rsv‐hPGK‐FOSL2‐Tetoff‐IRES‐Puro, Corues Biotechnology Co.), dual‐luciferase reporter system plasmid (Corues Biotechnology Co.), CASP3‐promoter‐dsRed tracing system plasmid (Corues Biotechnology Co.) and MYC‐truncated plasmid (Corues Biotechnology Co.).

Techniques: Binding Assay, Activity Assay, Western Blot, Over Expression, Concentration Assay, Immunohistochemistry, Microarray, Expressing, RNA Sequencing, Immunofluorescence, Fluorescence, Immunoprecipitation, Cell Culture, Two Tailed Test

Journal: Advanced Science

Article Title: Excessive MYC Orchestrates Macrophages induced Chromatin Remodeling to Sustain Micropapillary‐Patterned Malignancy in Lung Adenocarcinoma

doi: 10.1002/advs.202403851

Figure Lengend Snippet: Inhibition of the TGFβ‐FOSL2 axis effectively diminishes the malignancy associated with the MP‐pattern. A–D) The dependence experiment revealed that detachment‐induced cell death resistance (A), non‐anchored clonality ability (B), anti‐shearing force (C) and MP‐pattern genes expression in protein level (D) induced by M2‐like macrophages depends on TGFβ‐FOSL2 axis in cancer cell lines in A549 cells. For detachment‐induced cell death assay, Lung cancer cell lines were cultured attached or detached on TC‐treated plates or covalently bound hydrogel layer‐treaded plates. For non‐anchored clonality ability, indicated cells were allowed to grow in soft agar for 2 weeks and colonies were counted. E) An experimental illustration showing the rescue experiment in BALB/c Nude athymic mice with A549 cells xenograft, which transfected with FOSL2 tet‐off system. F,G) Tumor images (F) and growth kinetics (G) of subcutaneously implanted tumors in 2‐week‐after‐implanted FOSL2 tet‐off system transfected A549 cells and treated/not treated with doxycycline. n = 6. Scale bars: 1 cm. H) CTCs detected from venous blood in BALB/c Nude athymic mice 2‐week‐after‐implanted FOSL2 tet‐off system transfected A549 cells, which treated/not treated with doxycycline. Left: Representative images, right: quantitative statistics. n = 6. I) Dissemination index detected from venous blood in BALB/c Nude athymic mice bearing 2‐week‐after‐implanted FOSL2 tet‐off system transfected A549 cells, which treated/not treated with doxycycline. n = 6. J) An experimental illustration showing efficacy of Galunisertib against the malignancy of MP‐pattern with redundant expression of MYC via orthotopic lung injection. n = 5. K) Lung tumor growth was detected using in vivo imaging. Left: Representative images, right: quantitative statistics. n = 5. L,M) CTCs (L) and Dissemination index (M) detected from venous blood in BALB/c Nude athymic mice bearing MYC basal/redundant expression tumor, treated or not treated with Galunisertib. n = 5. N) Representative images of immunofluorescence staining of CTOS derived from indicated tumor tissue. Red, villin; blue, DAPI. Scale bars: 20 µm. O) Working model for MP/AC‐pattern malignancy orchestration. The p values were determined by Student's t ‐test (unpaired two‐tailed), n.s., not significant; * p < 0.05, ** p < 0.01, *** p < 0.001. Data are represented as mean ± SEM.

Article Snippet: For certain experiments, cells were transfected with a full‐length c‐Myc expression vector (pcDNA3‐cmyc, Addgene, #16011, RR_ID: Addgene_16011). pcDNA3 empty vector (Corues Biotechnology Co.), targeting FOSL2 short hairpin RNA and control scribble (Sangon Biotech), FOSL2‐expression vector (Addgene, #187907, RR_ID: Addgene_187907), pcDNA3.1 empty vector (Corues Biotechnology Co.), FOSL2 tet‐off system vector (pLV3rsv‐hPGK‐FOSL2‐Tetoff‐IRES‐Puro, Corues Biotechnology Co.), dual‐luciferase reporter system plasmid (Corues Biotechnology Co.), CASP3‐promoter‐dsRed tracing system plasmid (Corues Biotechnology Co.) and MYC‐truncated plasmid (Corues Biotechnology Co.).

Techniques: Inhibition, Expressing, Cell Culture, Transfection, Injection, In Vivo Imaging, Immunofluorescence, Staining, Derivative Assay, Two Tailed Test

Journal: Molecular cancer therapeutics

Article Title: Structure-Based Screen Identification of a Mammalian Ste20-like Kinase 4 (MST4) Inhibitor with Therapeutic Potential for Pituitary Tumors

doi: 10.1158/1535-7163.MCT-15-0703

Figure Lengend Snippet: Predicted MST4 inhibitors as determined by computational modelling

Article Snippet: LANCE (Perkin Elmer) Europium TR-FRET kinase binding assays were performed in white 384-well plates (Perkin Elmer, OptiPlate #6007299) using recombinant MST4 kinase (Carna Biosciences #07-119), ULight PKC substrate (Perkin Elmer #TRF0108), ATP (Sigma Aldrich # {"type":"entrez-protein","attrs":{"text":"A26209","term_id":"93012","term_text":"pir||A26209"}} A26209 ) and LANCE Eu-anti-PKC (Ala25Ser) antibody (Perkin Elmer #TRF0207).

Techniques:

Journal: Molecular cancer therapeutics

Article Title: Structure-Based Screen Identification of a Mammalian Ste20-like Kinase 4 (MST4) Inhibitor with Therapeutic Potential for Pituitary Tumors

doi: 10.1158/1535-7163.MCT-15-0703

Figure Lengend Snippet: Hypoxia model confirms the effects of selected inhibitor candidates on cell survival under severe hypoxia. A, immunoblot analysis of MST4 protein levels in normal pituitary and pituitary adenomas [gonadotrope, prolactin (PRL), ACTH, and growth hormone]. GAPDH was used as a loading control. B, immunoblot analysis shows overexpression of MST4 in pcDNA3-MST4–stable transfectants. C, percentage of nonviable cells in vector control and MST4 cells in the presence of the mammalian target of rapamycin (mTOR) inhibitor PKI-587 (0, 0.1, 1, 10, 100, and 1,000 nmol/L). D, percentage of nonviable cells in vector control and MST4 cells in the presence of the Polo-like kinase 1 (PLK-1) inhibitor volasertib (0, 0.1, 1, 10, 100, and 1,000 nmol/L). E, percentage of nonviable cells in control and MST4 transfectants in the presence of hesperadin (0, 0.1, 1, 10, 100, and 1000 nmol/L). *, P < 0.01; #, P < 0.0001, MST4 transfectants compared with pcDNA3 vector control cells.

Article Snippet: LANCE (Perkin Elmer) Europium TR-FRET kinase binding assays were performed in white 384-well plates (Perkin Elmer, OptiPlate #6007299) using recombinant MST4 kinase (Carna Biosciences #07-119), ULight PKC substrate (Perkin Elmer #TRF0108), ATP (Sigma Aldrich # {"type":"entrez-protein","attrs":{"text":"A26209","term_id":"93012","term_text":"pir||A26209"}} A26209 ) and LANCE Eu-anti-PKC (Ala25Ser) antibody (Perkin Elmer #TRF0207).

Techniques: Western Blot, Over Expression, Plasmid Preparation

Journal: Molecular cancer therapeutics

Article Title: Structure-Based Screen Identification of a Mammalian Ste20-like Kinase 4 (MST4) Inhibitor with Therapeutic Potential for Pituitary Tumors

doi: 10.1158/1535-7163.MCT-15-0703

Figure Lengend Snippet: Hesperadin is identified as a potent inhibitor of MST4. A, the chemical structure of hesperadin. B, small molecule docking depicting the predicted interaction of hesperadin (cyan) with the ATP-binding domain of MST4. C, in vitro TR-FRET recombinant kinase assay demonstrates direct inhibition of the MST4 kinase by hesperadin at low nanomolar concentrations. PKI-587 and volasertib were also screened in the TR-FRET recombinant kinase assay and show no direct inhibition of MST4.

Article Snippet: LANCE (Perkin Elmer) Europium TR-FRET kinase binding assays were performed in white 384-well plates (Perkin Elmer, OptiPlate #6007299) using recombinant MST4 kinase (Carna Biosciences #07-119), ULight PKC substrate (Perkin Elmer #TRF0108), ATP (Sigma Aldrich # {"type":"entrez-protein","attrs":{"text":"A26209","term_id":"93012","term_text":"pir||A26209"}} A26209 ) and LANCE Eu-anti-PKC (Ala25Ser) antibody (Perkin Elmer #TRF0207).

Techniques: Binding Assay, In Vitro, Recombinant, Kinase Assay, Inhibition

Journal: Molecular cancer therapeutics

Article Title: Structure-Based Screen Identification of a Mammalian Ste20-like Kinase 4 (MST4) Inhibitor with Therapeutic Potential for Pituitary Tumors

doi: 10.1158/1535-7163.MCT-15-0703

Figure Lengend Snippet: Hesperadin blocks the effects of MST4 on cell survival under acute hypoxia (1% O2). A, representative immunocytochemistry of apoptotic cells as assessed by TUNEL in the presence or absence of hesperadin (40 nmol/L) under hypoxia for 17 hours. B, rates of apoptosis were expressed as a percentage of TUNEL-positive cells to total cells in the absence or presence of various concentrations of hesperadin (5, 10, 20, and 40 nmol/L). *, P = 0.01 (at 10 nmol/L); #, P = 0.04 (at 20 nmol/L); **, P < 0.001, MST4 transfectants compared with pcDNA3 control cells.

Article Snippet: LANCE (Perkin Elmer) Europium TR-FRET kinase binding assays were performed in white 384-well plates (Perkin Elmer, OptiPlate #6007299) using recombinant MST4 kinase (Carna Biosciences #07-119), ULight PKC substrate (Perkin Elmer #TRF0108), ATP (Sigma Aldrich # {"type":"entrez-protein","attrs":{"text":"A26209","term_id":"93012","term_text":"pir||A26209"}} A26209 ) and LANCE Eu-anti-PKC (Ala25Ser) antibody (Perkin Elmer #TRF0207).

Techniques: Immunocytochemistry, TUNEL Assay

Journal: Molecular cancer therapeutics

Article Title: Structure-Based Screen Identification of a Mammalian Ste20-like Kinase 4 (MST4) Inhibitor with Therapeutic Potential for Pituitary Tumors

doi: 10.1158/1535-7163.MCT-15-0703

Figure Lengend Snippet: Hesperadin blocks the effects of MST4 on proliferation and colony formation under chronic hypoxic stress (5% O2). A, representative immunocytochemistry of the rates of BrdUrd incorporation in pcDNA3 control and MST4 transfectants in the absence and presence of hesperadin (40 nmol/L) under chronic hypoxia (5% O2) for 7 days. B, hesperadin abolishes MST4 increased cell proliferation. In the absence or presence of various doses of hesperadin (0, 5, 10, 20, and 40 nmol/L), cell proliferation was measured by BrdUrd after exposure to chronic hypoxia (5% O2) for 7 days. C, photomicrograph of colony formation in vector and MST4 transfectants incubated with DMSO or hesperadin (20 nmol/L). D, hesperadin decreases the ability of MST4 to promote increased colony formation. Numbers of pcDNA3 and MST4 transfectant colonies were counted after exposure to chronic hypoxia (5%O2) for 7 days. *, P = 0.002; **, P < 0.001, MST4 transfectants compared with pcDNA3 vector cells; #, P < 0.01, MST4 transfectants with hesperadin treatment (20 nmol/L) compared with the cells treated with DMSO.

Article Snippet: LANCE (Perkin Elmer) Europium TR-FRET kinase binding assays were performed in white 384-well plates (Perkin Elmer, OptiPlate #6007299) using recombinant MST4 kinase (Carna Biosciences #07-119), ULight PKC substrate (Perkin Elmer #TRF0108), ATP (Sigma Aldrich # {"type":"entrez-protein","attrs":{"text":"A26209","term_id":"93012","term_text":"pir||A26209"}} A26209 ) and LANCE Eu-anti-PKC (Ala25Ser) antibody (Perkin Elmer #TRF0207).

Techniques: Immunocytochemistry, Plasmid Preparation, Incubation, Transfection

Journal: Molecular cancer therapeutics

Article Title: Structure-Based Screen Identification of a Mammalian Ste20-like Kinase 4 (MST4) Inhibitor with Therapeutic Potential for Pituitary Tumors

doi: 10.1158/1535-7163.MCT-15-0703

Figure Lengend Snippet: A, illustration of MST4 signaling pathways in response to hypoxic stress. B, hesperadin blocks MST4 downstream signaling effectors. Phosphorylation of AKT, p38 MAPK, and ERK were determined by immunoblot in the presence or absence of hesperadin (0, 20, and 40 nmol/L). C, MST4-induced HIF-1 activity is blocked by hesperadin. Control or MST4 cells were transfected with HRE-luciferase reporter constructs. After 24 hours of transfection, cells were subjected to normoxia or hypoxia (1% O2) for 17 hours. HRE-luciferase values were detected and normalized to Renilla control luciferase values. *, P < 0.001, MST4 transfectants compared with pcDNA3 vector cells; #, P = 0.002, MST4 transfectants with hesperadin treatment compared with DMSO-treated controls.

Article Snippet: LANCE (Perkin Elmer) Europium TR-FRET kinase binding assays were performed in white 384-well plates (Perkin Elmer, OptiPlate #6007299) using recombinant MST4 kinase (Carna Biosciences #07-119), ULight PKC substrate (Perkin Elmer #TRF0108), ATP (Sigma Aldrich # {"type":"entrez-protein","attrs":{"text":"A26209","term_id":"93012","term_text":"pir||A26209"}} A26209 ) and LANCE Eu-anti-PKC (Ala25Ser) antibody (Perkin Elmer #TRF0207).

Techniques: Western Blot, Activity Assay, Transfection, Luciferase, Construct, Plasmid Preparation