Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Histone deacetylase inhibitor enhances the efficacy of MEK inhibitor through NOXA-mediated MCL1 degradation in triple-negative and inflammatory breast cancer

doi: 10.1158/1078-0432.CCR-16-2622

Figure Lengend Snippet: Kaplan-Meier survival curves for overall survival (OS) and distant metastasis-free survival (DMFS) of breast cancer patients from the IBC World Consortium dataset, correlated to NOXA and MCL1 tumor mRNA levels. The log-rank test was used to compare survival curves for high and low MCL1 (A, D), high and low NOXA (B, E), and high or low MCL1 in correlation with low or high NOXA (C, F). The initial numbers of patients at risk in each group are indicated in the key.

Article Snippet: Reagents and antibodies Entinostat (SNDX-275) was provided by Syndax Pharmaceuticals, Inc. Pimasertib (AS703026) was provided by EMD Serono, Inc. We obtained anti-NOXA (EMD Millipore, Billerica, MA), anti-MCL1 (R&D Systems, Minneapolis, MN, or Thermo Fisher Scientific, Waltham, MA), anti-PUMA, anti-BIM, anti-BAK, anti-BAX, anti-cleaved caspase 3, anti-cleaved caspase 9, and anti-Ki67 (Cell Signaling Technology, Beverly, MA), anti-α-tubulin (clone B-5-1-2; Sigma-Aldrich), and anti-horseradish peroxidase-conjugated antibodies (Thermo Scientific, Rockford, IL).

Techniques:

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Histone deacetylase inhibitor enhances the efficacy of MEK inhibitor through NOXA-mediated MCL1 degradation in triple-negative and inflammatory breast cancer

doi: 10.1158/1078-0432.CCR-16-2622

Figure Lengend Snippet: SUM190PT, SUM149PT, and HCC1806 cells were treated with clinically achievable (≤ 1 μmol/L) doses, representative data shown for entinostat (1 μM) and pimasertib (1 μM) for 48–72 hours. The IC50 values of entinostat were determined for SUM190PT, SUM149PT, and HCC1806 cell lines to be 0.6 μM, 0.3 μM, and 0.9 μM, respectively; the IC50 values of pimasertib were 1.9 μM, 0.6 μM, and 2.5 μM, respectively. Cell proliferation and apoptosis were measured by SRB staining (A) and Annexin V-PE staining (B), respectively. Data were pooled from three independent experiments and presented as mean ± SEM. *, P < 0.05; **, P < 0.001; ***, P < 0.0001. MCL1, NOXA, PUMA, and mitochondrial apoptosis-related proteins BIM, BAX, BAK, and cleaved caspase-9 were examined through immunoblotting analysis (C). D, NOXA/MCL1 binding on SUM190PT and SUM149PT cells was determined after entinostat (1 μM) and pimasertib (1 μM) individual and combination treatment by immunoprecipitation (IP) using anti-MCL1 antibody and immunoblotting with anti-NOXA antibody. After-IP samples were also blotted with NOXA antibody as an IP control. Pixel density of proteins was quantified for each condition, and the ratios of protein/tubulin or treatment/control are shown next to the blots; tubulin expression was used as a protein loading control.

Article Snippet: Reagents and antibodies Entinostat (SNDX-275) was provided by Syndax Pharmaceuticals, Inc. Pimasertib (AS703026) was provided by EMD Serono, Inc. We obtained anti-NOXA (EMD Millipore, Billerica, MA), anti-MCL1 (R&D Systems, Minneapolis, MN, or Thermo Fisher Scientific, Waltham, MA), anti-PUMA, anti-BIM, anti-BAK, anti-BAX, anti-cleaved caspase 3, anti-cleaved caspase 9, and anti-Ki67 (Cell Signaling Technology, Beverly, MA), anti-α-tubulin (clone B-5-1-2; Sigma-Aldrich), and anti-horseradish peroxidase-conjugated antibodies (Thermo Scientific, Rockford, IL).

Techniques: Staining, Western Blot, Binding Assay, Immunoprecipitation, Expressing

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Histone deacetylase inhibitor enhances the efficacy of MEK inhibitor through NOXA-mediated MCL1 degradation in triple-negative and inflammatory breast cancer

doi: 10.1158/1078-0432.CCR-16-2622

Figure Lengend Snippet: SUM190PT and SUM149PT cells were transfected with NOXA (siNOXA) or Scrambled (siSCR) siRNA through electroporation. Knockdown of NOXA mRNA and induction of apoptosis as measured by cleaved caspase-3 after siRNA inhibition were confirmed by quantitative RT-PCR and immunoblotting analysis (A), respectively, after entinostat treatment for 24 and 72 hours. Cell proliferation after siRNA and entinostat treatment was measured by SRB staining after 72 hours (B). SUM190PT, SUM149PT, and HCC1806 cells were transfected with either a NOXA-expressing vector or empty control vector by electroporation. Expression of NOXA, as well as MCL1, was analyzed by immunoblotting analysis 72 hours after transfection (C). Pixel density of MCL1 was quantified for each condition, and the ratios of MCL1/tubulin are shown above the blots; tubulin expression was used as a protein loading control. Proliferation of cells with NOXA overexpression in response to treatment with pimasertib (2.5 μM) was determined by SRB staining after 72 hours (D). Data were pooled from three independent experiments and presented as mean ± SEM. *, P < 0.05; **, P < 0.005; ***, P < 0.0001.

Article Snippet: Reagents and antibodies Entinostat (SNDX-275) was provided by Syndax Pharmaceuticals, Inc. Pimasertib (AS703026) was provided by EMD Serono, Inc. We obtained anti-NOXA (EMD Millipore, Billerica, MA), anti-MCL1 (R&D Systems, Minneapolis, MN, or Thermo Fisher Scientific, Waltham, MA), anti-PUMA, anti-BIM, anti-BAK, anti-BAX, anti-cleaved caspase 3, anti-cleaved caspase 9, and anti-Ki67 (Cell Signaling Technology, Beverly, MA), anti-α-tubulin (clone B-5-1-2; Sigma-Aldrich), and anti-horseradish peroxidase-conjugated antibodies (Thermo Scientific, Rockford, IL).

Techniques: Transfection, Electroporation, Inhibition, Quantitative RT-PCR, Western Blot, Staining, Expressing, Plasmid Preparation, Over Expression

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Histone deacetylase inhibitor enhances the efficacy of MEK inhibitor through NOXA-mediated MCL1 degradation in triple-negative and inflammatory breast cancer

doi: 10.1158/1078-0432.CCR-16-2622

Figure Lengend Snippet: SUM190PT and SUM149PT cells were transfected with either MCL1-expressing or empty control vectors by electroporation (A, B). Induced expression of MCL1 protein was confirmed by immunoblotting analysis. The ability of MCL1 overexpression to induce cell proliferation after entinostat (5 μM) and pimasertib (5 μM) single and combination treatments was measured by SRB staining after 72 hours. Cell proliferation (C) and apoptosis (D) were determined by SRB staining and Annexin V-PE staining, respectively, in SUM190PT and SUM149PT cells after inhibition of MCL1 by the small molecule inhibitor UMI-77 (0.3 μM and 5 μM, respectively) in combination with pimasertib (1 μM). Data were pooled from three independent experiments and presented as mean ± SEM. Tubulin expression was used as a protein loading control. *, P < 0.05; **, P < 0.001; ***, P < 0.0001.

Article Snippet: Reagents and antibodies Entinostat (SNDX-275) was provided by Syndax Pharmaceuticals, Inc. Pimasertib (AS703026) was provided by EMD Serono, Inc. We obtained anti-NOXA (EMD Millipore, Billerica, MA), anti-MCL1 (R&D Systems, Minneapolis, MN, or Thermo Fisher Scientific, Waltham, MA), anti-PUMA, anti-BIM, anti-BAK, anti-BAX, anti-cleaved caspase 3, anti-cleaved caspase 9, and anti-Ki67 (Cell Signaling Technology, Beverly, MA), anti-α-tubulin (clone B-5-1-2; Sigma-Aldrich), and anti-horseradish peroxidase-conjugated antibodies (Thermo Scientific, Rockford, IL).

Techniques: Transfection, Expressing, Electroporation, Western Blot, Over Expression, Staining, Inhibition

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Histone deacetylase inhibitor enhances the efficacy of MEK inhibitor through NOXA-mediated MCL1 degradation in triple-negative and inflammatory breast cancer

doi: 10.1158/1078-0432.CCR-16-2622

Figure Lengend Snippet: SUM190PT and SUM149PT cell lines were treated with entinostat (0.01 and 0.05 μM, respectively) and/or pimasertib (0.01 and 0.05 μM, respectively) and allowed to grow in an anchorage-independent environment for 2–3 weeks; clonal growth was measured at the treatment endpoint by colony formation (A). Data were pooled from three independent experiments and presented as mean ± SEM. Tumor volume measurements for SUM190PT and SUM149PT tumor xenograft-bearing mice (n=12/group and 10/group, respectively) treated via oral gavage daily for up to 2 months with vehicle, entinostat (20 or 5 mg/kg), and/or pimasertib (30 or 0.5 mg/kg) (B). C, Immunohistochemistry (IHC) staining from SUM190PT and SUM149PT tumor xenografts treated with vehicle or the indicated drugs. Paraformaldehyde-fixed paraffin sections were incubated with anti-Ki-67 antibody, and TUNEL staining was performed. Representative images of 5 IHC staining experiments are illustrated. Magnification, 20x. The images were converted by ImageJ software to accomplish quantification of Ki-67 and TUNEL expression. Quantification of IHC staining is represented as mean ± SEM. D–E, Protein expression (represented as mean ± SEM) relative to loading control after immunoblotting analysis of NOXA, MCL1, and cleaved caspase-9 expression in protein lysates of five representative tumor samples from each treatment group of mice bearing SUM190PT or SUM149PT tumors. Tubulin expression was used as a protein loading control. Pixel density of protein bands was quantified for each condition using ImageJ software. *, P < 0.05; **, P < 0.001; ***, P < 0.0001.

Article Snippet: Reagents and antibodies Entinostat (SNDX-275) was provided by Syndax Pharmaceuticals, Inc. Pimasertib (AS703026) was provided by EMD Serono, Inc. We obtained anti-NOXA (EMD Millipore, Billerica, MA), anti-MCL1 (R&D Systems, Minneapolis, MN, or Thermo Fisher Scientific, Waltham, MA), anti-PUMA, anti-BIM, anti-BAK, anti-BAX, anti-cleaved caspase 3, anti-cleaved caspase 9, and anti-Ki67 (Cell Signaling Technology, Beverly, MA), anti-α-tubulin (clone B-5-1-2; Sigma-Aldrich), and anti-horseradish peroxidase-conjugated antibodies (Thermo Scientific, Rockford, IL).

Techniques: Immunohistochemistry, Incubation, TUNEL Assay, Staining, Software, Expressing, Western Blot