Journal: Cell Communication and Signaling : CCS

Article Title: THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours

doi: 10.1186/s12964-022-00928-x

Figure Lengend Snippet: High CDK7 protein expression is associated with a poor prognosis of GIST. A Based on data from the GSE136755 dataset, the mRNA levels of CDK4, CDK7 and CDK9 were relatively higher than those of other CDKs for CDK1-10. The mRNA level was calculated via GEO2R. B CDK7 expression was significantly elevated in high-risk GISTs based on the data from GSE136755. C Representative scanned images of GIST samples with low or high CDK7 protein expression, as determined by IHC. D Kaplan–Meier survival curves with a risk table showing that high CDK7 protein expression was significantly positively related to poor recurrence-free survival in GIST patients ( P = 0.044)

Article Snippet: IHC staining was performed with anti-CDK7 antibody (#2916, CST).

Techniques: Expressing

Journal: Cell Communication and Signaling : CCS

Article Title: THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours

doi: 10.1186/s12964-022-00928-x

Figure Lengend Snippet: Knockdown of CDK7 decreases cell viability and proliferation and induces cell cycle arrest. A Immunoblotting analysis of CDK7 expression after targeting siRNA-mediated CDK7 knockdown in GIST-T1 and GIST-882 cells. A nontargeting siRNA and two independent siRNAs (siRNA1 and siRNA2) are represented by siNC, siCDK7-1, and siCDK7-2. B CCK-8 cell viability assay after CDK7 knockdown in GIST-T1 and GIST-882 cells. C Colony formation assays of GIST-T1 and GIST-882 cells after CDK7 knockdown. D Flow cytometry analysis was used to detect and analyse the cell cycle distribution after CDK7 knockdown. E Immunoblotting analysis of cyclin-D1, CDK4 and γH2AX expression after CDK7 knockdown

Article Snippet: IHC staining was performed with anti-CDK7 antibody (#2916, CST).

Techniques: Knockdown, Western Blot, Expressing, CCK-8 Assay, Viability Assay, Flow Cytometry

Journal: Cell Communication and Signaling : CCS

Article Title: THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours

doi: 10.1186/s12964-022-00928-x

Figure Lengend Snippet: CDK7 inhibition leads to the suppression of RNA transcription in GIST cells. A Heatmap showing the change in global active transcripts in GIST-T1 cells following treatment with 50 nmol THZ1 and DMSO for 6 h. B Heatmap showing the transcriptional changes in cell cycle-related genes from the gene set (GOBP_CELL_CYCLE.v7.5.1) in GIST-T1 cells after THZ1 treatment. C The enriched GO functional terms of the transcripts that were reduced over twofold in GIST-T1 cells following treatment with 50 nmol/L THZ1 for 6 h. D Immunoblot analyses of RNAPII, RNAPII CTD phosphorylation (S2, S5, and S7), and CDK7 in GIST-T1 and GIST-882 cells treated either with THZ1 or DMSO at the indicated concentrations for 24 h

Article Snippet: IHC staining was performed with anti-CDK7 antibody (#2916, CST).

Techniques: Inhibition, Functional Assay, Western Blot, Phospho-proteomics

Journal: Cell Communication and Signaling : CCS

Article Title: THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours

doi: 10.1186/s12964-022-00928-x

Figure Lengend Snippet: CDK7 inhibition leads to the suppression of c-KIT transcriptional activity and protein expression in GIST cells. A, B qRT–PCR and immunoblotting analysis of c-KIT expression after siRNA-mediated CDK7 knockdown in GIST-T1 and GIST-882 cells. C , D qRT–PCR and immunofluorescence assays showed that c-KIT expression changed after THZ1 treatment in GIST-T1 and GIST-882 cells. E Immunoblotting analysis showed that the expression of c-KIT and downstream ERK and AKT signalling pathway components was inhibited following THZ1 treatment at the indicated concentration in GIST-T1 and GIST-882 cells for 24 h. F Immunoblotting analysis indicated that combination treatment with imatinib and THZ1 enhanced c-KIT expression inhibition in GIST-T1 and GIST-882 cells for 24 h

Article Snippet: IHC staining was performed with anti-CDK7 antibody (#2916, CST).

Techniques: Inhibition, Activity Assay, Expressing, Quantitative RT-PCR, Western Blot, Knockdown, Immunofluorescence, Concentration Assay

Journal: Cell Communication and Signaling : CCS

Article Title: THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours

doi: 10.1186/s12964-022-00928-x

Figure Lengend Snippet: CDK7 inhibits c-KIT expression through OSR1 in GIST. A Volcano plot of RNA-seq data displaying the distribution of differential gene expression between THZ1 treatment and DMSO treatment. The upregulated and downregulated genes are highlighted in red and blue, respectively. The results illustrated that OSR1 was the top downregulated gene with the lowest P value. B , C qRT–PCR and immunoblotting analyses showed that OSR1 expression was inhibited by CDK7 knockdown or THZ1 treatment in a dose-dependent manner. OSR1 expression was inhibited by CDK7 knockdown in GIST-T1 and GIST-882 cells. D Gene expression profile in the Oncomine database. The results showed that OSR1 was significantly upregulated in GIST compared with normal gastric tissue. E Gene expression profile in the MediSapiens IST Online transcriptome database. The results showed that OSR1 was uniquely overexpressed in GIST and prostate cancer compared with other cancer types. F Immunoblot analyses of OSR1 expression in clinical sample tissues of GIST, gastrointestinal leiomyoma and schwannoma. The expression of OSR1 was significantly higher in GIST than in leiomyoma and schwannoma. G Data from the MediSapiens database showed that c-KIT and OSR1 expression levels were positively correlated (n = 77, r = 0.407, P < 0.001). H , I qRT–PCR and immunoblotting analyses showed that OSR1 knockdown significantly inhibited c-KIT mRNA and protein expression in GIST-T1 and GIST-882 cells. J Immunoblot analyses showed that OSR1 overexpression promoted c-KIT expression in GIST-T1 and GIST-882 cells. K The inhibitory effect of c-KIT expression was reversed when CDK7 siRNA and OSR1 plasmid were cotransfected into GIST-T1 and GIST-882 cells

Article Snippet: IHC staining was performed with anti-CDK7 antibody (#2916, CST).

Techniques: Expressing, RNA Sequencing, Gene Expression, Quantitative RT-PCR, Western Blot, Knockdown, Over Expression, Plasmid Preparation

Journal: Cell Communication and Signaling : CCS

Article Title: THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours

doi: 10.1186/s12964-022-00928-x

Figure Lengend Snippet: Relationship between CDK7 expression and clinicopathological characteristics of GIST patients

Article Snippet: IHC staining was performed with anti-CDK7 antibody (#2916, CST).

Techniques: Expressing, Modification

Journal: Cancer research

Article Title: Pharmacological targeting of TFIIH suppresses KRAS mutant pancreatic ductal adenocarcinoma and synergizes with TRAIL

doi: 10.1158/0008-5472.CAN-21-4222

Figure Lengend Snippet: A. Immunoblots of dose dependent TFIIIH inhibition on the expression of RPB1, CFLAR, and IAPs: MCL1, and XIAP in PDCLs: PancVH1, TKCC18-LO (Stable), TKCC16-LO (Scattered), TKCC27-LO (Unstable); drug concentrations (μM); 24hr; GAPDH loading control. B. Immunoblots of dose dependent TFIIIH inhibition on the expression of RPB1, FOSL1, and STAT3 in PDCLs; drug concentrations (μM); 24hr; GAPDH loading control. C. Proposed model of TFIIH inhibition on RNA polymerase II transcription. TFIIH complex: GTF2H1 (p62), GTF2H2 (p44), GTF2H3 (p34), GTF2H4 (p52), GTF2H5 (p8), ATP-dependent DNA helicase subunits XPD (ERCC2), and XPB (ERCC3), and the CAK that includes CDK7, Cyclin H, and MAT1, and inhibition via the covalent CDK7/12/13 inhibitor THZ1, the dual kinase CDK7/CDK9 inhibitor SNS-032, and XPB (subunit of TFIIH) inhibitor Triptolide. In this model, TFIIH inhibition results in the disruption of the spatiotemporal phosphorylation of the RNA polymerase II Subunit B1 carboxy terminal domain (CTD) heptapeptide repeat as well as the stability of the RPB1 protein. RNA polymerase II transcription initiation is therefore arrested leading to the downregulation of CFLAR, IAPs and transcriptional effectors of the KRAS mutant signaling cascade; RPB1 also acts as a substrate for transcriptional kinases CDK9/CyclinT (P-TEFb), essential to RNA polymerase II transcription elongation, and CDK13/CyclinK, involved in the coordination of RNA splicing.

Article Snippet: Biochemical purification of the CAK complex in PancVH1 and HPNE was performed via immunoprecipitation (IP) and immunoblotting (IB) with antibodies to CDK7 (2916), Cyclin H (2927), Mat1 (sc-135981), and Protein A/G PLUS-Agarose (sc-2003 Santa Cruz) via standard protocol.

Techniques: Western Blot, Inhibition, Expressing, Control, Disruption, Phospho-proteomics, Mutagenesis

Journal: Cancer Research

Article Title: Pharmacologic Targeting of TFIIH Suppresses KRAS-Mutant Pancreatic Ductal Adenocarcinoma and Synergizes with TRAIL

doi: 10.1158/0008-5472.can-21-4222

Figure Lengend Snippet: Figure 4. TFIIH inhibition disrupts the protein stability of RPB1, resulting in the downregulation of IAPs and transcrip- tional effectors of oncogenic signaling in KRAS TP53 mutant PDCLs. A, Immunoblots of dose dependent TFIIIH inhibition on the expression of RPB1, CFLAR, and IAPs: MCL1, and XIAP in PDCLs: PancVH1, TKCC18-LO (Stable), TKCC16-LO (Scattered), TKCC27-LO (Unstable); drug concen- trations (mmol/L); 24 hour assay; GAPDH, loading control. B, Immuno- blots of dose dependent TFIIIH inhi- bition on the expression of RPB1, FOSL1, and STAT3 in PDCLs; drug concentrations (mmol/L); 24 hour assay; GAPDH, loading control. C, Proposed model of TFIIH inhibition on RNA polymerase II transcription. TFIIH complex: GTF2H1 (p62), GTF2H2 (p44), GTF2H3 (p34), GTF2H4 (p52), GTF2H5 (p8), ATP-dependent DNA helicase subunits XPD (ERCC2), and XPB (ERCC3), and the CAK that includes CDK7, cyclin H, and MAT1, and inhibition via the covalent CDK7/ 12/13 inhibitor THZ1, the dual kinase CDK7/CDK9 inhibitor SNS-032, and XPB (subunit of TFIIH) inhibitor trip- tolide. In this model, TFIIH inhibition results in the disruption of the spa- tiotemporal phosphorylation of the RNA polymerase II subunit B1 car- boxy terminal domain (CTD) hepta- peptide repeat as well as the stability of the RPB1 protein. RNA polymerase II transcription initiation is therefore arrested, leading to the downregula- tion of CFLAR, IAPs, and transcriptional effectors of the KRAS mutant signaling cascade; RPB1 also acts as a substrate for transcriptional kinases CDK9/Cyclin T (P-TEFb), essential to RNA polymer- ase II transcription elongation, and CDK13/Cyclin K, involved in the coor- dination of RNA splicing.

Article Snippet: For immunoprecipitation and immunoblotting, CDK7 (2916), cyclin H (2927), Mat1 (sc-135981), BID (2002), and Protein A/G PLUSAgarose (sc-2003 Santa Cruz).

Techniques: Inhibition, Mutagenesis, Western Blot, Expressing, Control, Disruption, Phospho-proteomics, Polymer

Journal: Molecular Medicine Reports

Article Title: Synergistic antitumor effects of CDK inhibitor SNS-032 and an oncolytic adenovirus co-expressing TRAIL and Smac in pancreatic cancer

doi: 10.3892/mmr.2017.6472

Figure Lengend Snippet: Characterization of oncolytic adenovirus ZD55-TIS and structure of SNS-032. (A) Schematic diagram of the oncolytic adenovirus ZD55-TIS. (B) Identification of recombinant adenovirus by western blot analysis. ZD55-TIS (8 MOI), SNS-032 (160 ng/ml), or ZD55-TIS plus SNS-032 was used to treat PANC-1 cells for 48 h. Cell proteins were collected to determine the expression of E1A, Smac and TRAIL. Untreated cells were used as a control group. Differences in protein expression level is semi-quantitatively determined by densitometry and expressed as a value relative to untreated cells. GAPDH was used as a protein loading control. (C) Schematic diagram of SNS-032 (C 17 H 24 N 4 O 2 S 2 ). ZD55-TIS, ZD55-TRAIL-IETD-Smac; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; IETD, isoleucine-aspartate-threonine-glutamate; Smac, second mitochondria-derived activator of caspase; MW, molecular weight.

Article Snippet: Antibodies targeting CDK-2 (cat. no. 2546S), CDK-7 (cat. no. 2916), and CDK-9 (cat. no. 2316S) were from Cell Signaling Technology, Inc., and antibodies targeting E1A (cat. no. sc-374663), TRAIL (cat. no. sc-4547), and Smac (cat. no. sc-136302) were from Santa Cruz Biotechnology, Inc.

Techniques: Recombinant, Western Blot, Expressing, Control, Derivative Assay, Molecular Weight

Journal: Molecular Medicine Reports

Article Title: Synergistic antitumor effects of CDK inhibitor SNS-032 and an oncolytic adenovirus co-expressing TRAIL and Smac in pancreatic cancer

doi: 10.3892/mmr.2017.6472

Figure Lengend Snippet: Combination of ZD55-TIS and SNS-032 enhances suppression of tumor cell proliferation. Pancreatic cancer cells (A) PANC-1 and (B) BxPC-3 were treated with ZD55-TIS (1, 2, 4, 8 and 16 MOI), SNS-032 (5, 10, 20, 40 and 80 ng/ml), or ZD55-TIS plus SNS-032 for 48 h. The image represents three independent experiments. Cell viability was evaluated by MTT assay and the synergistic effect of ZD55-TIS combined with SNS-032 on PANC-1 and BxPC-3 was quantified by CI analysis and expressed as log (CI) vs. fractional effect. Where calculable, 95% confidence intervals are shown. **P<0.05. ZD55-TIS, ZD55-TRAIL-IETD-Smac; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; IETD, isoleucine-aspartate-threonine-glutamate; Smac, second mitochondria-derived activator of caspase; CI, combination index.

Article Snippet: Antibodies targeting CDK-2 (cat. no. 2546S), CDK-7 (cat. no. 2916), and CDK-9 (cat. no. 2316S) were from Cell Signaling Technology, Inc., and antibodies targeting E1A (cat. no. sc-374663), TRAIL (cat. no. sc-4547), and Smac (cat. no. sc-136302) were from Santa Cruz Biotechnology, Inc.

Techniques: MTT Assay, Derivative Assay

Journal: Molecular Medicine Reports

Article Title: Synergistic antitumor effects of CDK inhibitor SNS-032 and an oncolytic adenovirus co-expressing TRAIL and Smac in pancreatic cancer

doi: 10.3892/mmr.2017.6472

Figure Lengend Snippet: SNS-032 enhances ZD55-TIS-induced apoptosis and cell cycle arrest in pancreatic cancer cells. PANC-1 were infected with ZD55-TIS (8 MOI), treated with SNS-032 (160 ng/ml) or ZD55-TIS (8 MOI) plus SNS-032 (160 ng/ml). (A) After 72 h, PANC-1 cells were stained with Hoechst 33342 and observed under an inverted fluorescence microscope. White arrows indicate positive apoptotic cells. Original magnification, ×200. (B) Western blotting was performed to detect cleaved caspase-9 and −3, and PARP. GAPDH was used as the loading control and the level in the untreated group was set as 1. At 48 h after treatment, (C) apoptosis and (D) the cell cycle were analyzed by flow cytometry and the % of cells in the G2 phase was calculated. Uninfected cells served as control. ZD55-TIS, ZD55-TRAIL-IETD-Smac; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; IETD, isoleucine-aspartate-threonine-glutamate; Smac, second mitochondria-derived activator of caspase; Comb, combination. PI, propidium iodide; FITC, fluorescein isothiocyanate; PARP, poly(ADP-ribose) polymerase.

Article Snippet: Antibodies targeting CDK-2 (cat. no. 2546S), CDK-7 (cat. no. 2916), and CDK-9 (cat. no. 2316S) were from Cell Signaling Technology, Inc., and antibodies targeting E1A (cat. no. sc-374663), TRAIL (cat. no. sc-4547), and Smac (cat. no. sc-136302) were from Santa Cruz Biotechnology, Inc.

Techniques: Infection, Staining, Fluorescence, Microscopy, Western Blot, Control, Flow Cytometry, Derivative Assay

Journal: Molecular Medicine Reports

Article Title: Synergistic antitumor effects of CDK inhibitor SNS-032 and an oncolytic adenovirus co-expressing TRAIL and Smac in pancreatic cancer

doi: 10.3892/mmr.2017.6472

Figure Lengend Snippet: Effect of SNS-032 combined with ZD55-TIS on pro-apoptotic signaling elements in PANC-1 cells. PANC-1 cells were treated with ZD55-TIS (8 MOI) or/and SNS-032 (160 ng/ml). The combination of ZD55-TIS and SNS-032 downregulated the protein expression of CDK-2, −7, and −9, and XIAP, Mcl-1 and Bcl-2. ZD55-TIS, ZD55-TRAIL-IETD-Smac; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; IETD, isoleucine-aspartate-threonine-glutamate; Smac, second mitochondria-derived activator of caspase; CDK, cyclin-dependent kinase; Mcl-1, Bcl-2 family apoptosis regulator; XIAP, X-linked inhibitor of apoptosis; Bcl-2, Bcl-2 apoptosis regulator.

Article Snippet: Antibodies targeting CDK-2 (cat. no. 2546S), CDK-7 (cat. no. 2916), and CDK-9 (cat. no. 2316S) were from Cell Signaling Technology, Inc., and antibodies targeting E1A (cat. no. sc-374663), TRAIL (cat. no. sc-4547), and Smac (cat. no. sc-136302) were from Santa Cruz Biotechnology, Inc.

Techniques: Expressing, Derivative Assay

Journal: Molecular Medicine Reports

Article Title: Synergistic antitumor effects of CDK inhibitor SNS-032 and an oncolytic adenovirus co-expressing TRAIL and Smac in pancreatic cancer

doi: 10.3892/mmr.2017.6472

Figure Lengend Snippet: Synergistic effects of ZD55-TIS and SNS-032 in vivo . (A) Tumor volume was measured at different times after treatment. Data are presented as the mean ± standard deviation (n=7). **P<0.05, with comparisons indicated by lines. (B) The image presents inhibitory effect of each group on tumor growth at the last time point (day 60) when the mice were sacrificed. (C) Subcutaneous BxPC-3 tumors were collected 7 days after injection, and sections were analyzed by different methods. H&E staining showed that tumor tissues treated with the combination of ZD55-TIS and SNS-032 exhibited the strongest cell death (original magnification, ×400). Immunohistochemistry analysis demonstrated there was strong expression of TRAIL and Smac in xenografts treated with ZD55-TIS and the combined therapy group. However, the combined therapy group had a more obvious downregulation of CDK-2, −7 and −9 expression compared with SNS-032 alone. TUNEL assay revealed enhanced cell apoptosis in the group treated with ZD55TIS plus SNS-032. ZD55-TIS, ZD55-TRAIL-IETD-Smac; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; IETD, isoleucine-aspartate-threonine-glutamate; Smac, second mitochondria-derived activator of caspase; H&E, hematoxylin and eosin; CDK, cyclin-dependent kinase; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.

Article Snippet: Antibodies targeting CDK-2 (cat. no. 2546S), CDK-7 (cat. no. 2916), and CDK-9 (cat. no. 2316S) were from Cell Signaling Technology, Inc., and antibodies targeting E1A (cat. no. sc-374663), TRAIL (cat. no. sc-4547), and Smac (cat. no. sc-136302) were from Santa Cruz Biotechnology, Inc.

Techniques: In Vivo, Standard Deviation, Injection, Staining, Immunohistochemistry, Expressing, TUNEL Assay, Derivative Assay