Apoptosis signaling molecules as treatment targets in head and neck squamous cell carcinoma

Thomas J. Ow; Carlos Thomas; Cory D. Fulcher; Jianhong Chen; Andrea López; Denis E. Reyna; Michael B. Prystowsky; Richard V. Smith; Bradley A. Schiff; Gregory Rosenblatt; Thomas J. Belbin; Thomas M. Harris; Geoffrey C. Childs; Nicole Kawachi; Nicolas F. Schlecht; Evripidis Gavathiotis

doi:10.1002/lary.28441

Apoptosis signaling molecules as treatment targets in head and neck squamous cell carcinoma

Thomas J. Ow, Carlos Thomas, Cory D. Fulcher, Jianhong Chen, Andrea López, Denis E. Reyna, Michael B. Prystowsky, Richard V. Smith, Bradley A. Schiff, Gregory Rosenblatt, Thomas J. Belbin, Thomas M. Harris, Geoffrey C. Childs, Nicole Kawachi, Nicolas F. Schlecht, Evripidis Gavathiotis

Research output: Contribution to journal › Article › peer-review

15 Scopus citations

Abstract

Objectives: To evaluate BCL-2 family signaling molecules in head and neck squamous cell carcinoma (HNSCC) and examine the ability of therapeutic agents with variable mechanisms of action to induce apoptosis in HNSCC cells. Methods: messenger ribonculeic acid (mRNA) expression of BAK, BAX, B-cell lymphoma (Bcl-2), BCL2 Like 1 (BCL2L1), and MCL1 were measured in The Cancer Genome Atlas (TCGA) head and neck cancer dataset, as well as in a dataset from a cohort at Montefiore Medical Center (MMC). Protein expression was similarly evaluated in a panel of HNSCC cell lines (HN30, HN31, HN5, MDA686LN, UMSCC47). Cell viability and Annexin V assays were used to assess the efficacy and apoptotic potential of a variety of agents (ABT-263 [navitoclax], A-1210477, and bortezomib. Results: Expression of BAK, BAX, BCL2L1, and MCL1 were each significantly higher than expression of BCL2 in the TCGA and MMC datasets. Protein expression demonstrated the same pattern of expression when examined in HNSCC cell lines. Treatment with combined ABT-263 (navitoclax)/A-1210477 or with bortezomib demonstrated apoptosis responses that approached or exceeded treatment with staurospaurine control. Conclusion: HNSCC cells rely on inhibition of apoptosis via BCL-xL and MCL-1 overexpression, and induction of apoptosis remains a potential therapeutic option as long as strategies overcome redundant anti-apoptotic signals. Level of Evidence: NA Laryngoscope, 130:2643–2649, 2020.

Original language	English (US)
Pages (from-to)	2643-2649
Number of pages	7
Journal	Laryngoscope
Volume	130
Issue number	11
DOIs	https://doi.org/10.1002/lary.28441
State	Published - Nov 1 2020

Keywords

A-1210477
ABT-263
Head and neck squamous carcinoma
apoptosis
bortezomib

ASJC Scopus subject areas

Otorhinolaryngology

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

Access to Document

10.1002/lary.28441

Cite this

Ow, T. J., Thomas, C., Fulcher, C. D., Chen, J., López, A., Reyna, D. E., Prystowsky, M. B., Smith, R. V., Schiff, B. A., Rosenblatt, G., Belbin, T. J., Harris, T. M., Childs, G. C., Kawachi, N., Schlecht, N. F., & Gavathiotis, E. (2020). Apoptosis signaling molecules as treatment targets in head and neck squamous cell carcinoma. Laryngoscope, 130(11), 2643-2649. https://doi.org/10.1002/lary.28441

Ow, TJ, Thomas, C, Fulcher, CD, Chen, J, López, A, Reyna, DE, Prystowsky, MB , Smith, RV , Schiff, BA, Rosenblatt, G, Belbin, TJ, Harris, TM, Childs, GC, Kawachi, N, Schlecht, NF & Gavathiotis, E 2020, 'Apoptosis signaling molecules as treatment targets in head and neck squamous cell carcinoma', Laryngoscope, vol. 130, no. 11, pp. 2643-2649. https://doi.org/10.1002/lary.28441

@article{1bc7b196a95b4dfdbce90b75e0837bb9,

title = "Apoptosis signaling molecules as treatment targets in head and neck squamous cell carcinoma",

abstract = "Objectives: To evaluate BCL-2 family signaling molecules in head and neck squamous cell carcinoma (HNSCC) and examine the ability of therapeutic agents with variable mechanisms of action to induce apoptosis in HNSCC cells. Methods: messenger ribonculeic acid (mRNA) expression of BAK, BAX, B-cell lymphoma (Bcl-2), BCL2 Like 1 (BCL2L1), and MCL1 were measured in The Cancer Genome Atlas (TCGA) head and neck cancer dataset, as well as in a dataset from a cohort at Montefiore Medical Center (MMC). Protein expression was similarly evaluated in a panel of HNSCC cell lines (HN30, HN31, HN5, MDA686LN, UMSCC47). Cell viability and Annexin V assays were used to assess the efficacy and apoptotic potential of a variety of agents (ABT-263 [navitoclax], A-1210477, and bortezomib. Results: Expression of BAK, BAX, BCL2L1, and MCL1 were each significantly higher than expression of BCL2 in the TCGA and MMC datasets. Protein expression demonstrated the same pattern of expression when examined in HNSCC cell lines. Treatment with combined ABT-263 (navitoclax)/A-1210477 or with bortezomib demonstrated apoptosis responses that approached or exceeded treatment with staurospaurine control. Conclusion: HNSCC cells rely on inhibition of apoptosis via BCL-xL and MCL-1 overexpression, and induction of apoptosis remains a potential therapeutic option as long as strategies overcome redundant anti-apoptotic signals. Level of Evidence: NA Laryngoscope, 130:2643–2649, 2020.",

keywords = "A-1210477, ABT-263, Head and neck squamous carcinoma, apoptosis, bortezomib",

author = "Ow, {Thomas J.} and Carlos Thomas and Fulcher, {Cory D.} and Jianhong Chen and Andrea L{\'o}pez and Reyna, {Denis E.} and Prystowsky, {Michael B.} and Smith, {Richard V.} and Schiff, {Bradley A.} and Gregory Rosenblatt and Belbin, {Thomas J.} and Harris, {Thomas M.} and Childs, {Geoffrey C.} and Nicole Kawachi and Schlecht, {Nicolas F.} and Evripidis Gavathiotis",

note = "Funding Information: t.j.o : supported in part by the Triological Society Career Development Award, the National Institutes of Health (NIH)/National Cancer Institute (NCI) grant 2K12 CA132783‐06, and the National Institutes of Health (NIH)/The National Institute of Dental and Craniofacial Research (NIDCR) grant 1 K23 DE027425‐01. e.g. and d.e.r . supported from the National Institutes of Health (NIH)/National Cancer Institute (NCI) grant CA178394 and an award from the Pershing Square Sohn Cancer Research Alliance. a.l .: supported from a T32 Institutional Training Program grant T32 AG 23475. The authors also acknowledge support from the Albert Einstein Cancer Center the National Institutes of Health (NIH)/National Cancer Institute (NCI) grant P30CA013330, and specifically the flow cytometry core facility, part of the Albert Einstein shared resources, as well as Roswell Park Comprehensive Cancer Center grant CA016056. The authors have no other funding, financial relationships, or conflicts of interest to disclose. Funding Information: Data from a total of 496 HNSCC patients in The Cancer Genome Atlas (TCGA) cohort include level 3 ribonucleic acid (RNA) sequencing data (log2 transformed RSEM value, as reads per kilobase of transcript per million mapped reads) and clinical data (human papillomavirus [HPV] status, tumor stage, and primary tumor site) downloaded from National Cancer Institute Genomic Data Commons (GDC, https://portal.gdc.cancer.gov/). Relative gene expression levels of targeted genes were presented as box plots using GraphPad Prism version 8 (GraphPad Software, Inc., San Diego, CA). In parallel, the relative gene expression levels were examined from the targeted genes within tumor samples obtained and evaluated at Montefiore Medical Center (MMC). RNA from HNSCC tumors were hybridized to the Illumina HumanHT-12-v3 Expression BeadChip (Illumina, San Diego, CA), and data were normalized and evaluated, as described in previous reports. For this analysis, evaluation was restricted to tumors from patients with histologically proven HNSCC, without previous treatment, and without a history of a previous HNSCC primary tumor. Gene expression data from 131 HNSCC patients was log2 transformed, analyzed, and presented as box plots using GraphPad Prism version 8 (GraphPad Software, Inc.). Pairwise Pearson correlation analysis was conducted with relative gene expression levels of BAX, BAK1, BCL2, BCL2L1, and MCL1 within TCGA and MMC cohorts using log2 transformed data. A correlation coefficient matrix was produced by combining pairwise correlation coefficients from TCGA and MMC data, respectively. The matrix was used for unsupervised hierarchical clustering using the pheatmap package applied in R statistical software. Data from a total of 496 HNSCC patients in The Cancer Genome Atlas (TCGA) cohort include level 3 ribonucleic acid (RNA) sequencing data (log2 transformed RSEM value, as reads per kilobase of transcript per million mapped reads) and clinical data (human papillomavirus [HPV] status, tumor stage, and primary tumor site) downloaded from National Cancer Institute Genomic Data Commons (GDC, https://portal.gdc.cancer.gov/). Relative gene expression levels of targeted genes were presented as box plots using GraphPad Prism version 8 (GraphPad Software, Inc., San Diego, CA). In parallel, the relative gene expression levels were examined from the targeted genes within tumor samples obtained and evaluated at Montefiore Medical Center (MMC). RNA from HNSCC tumors were hybridized to the Illumina HumanHT-12-v3 Expression BeadChip (Illumina, San Diego, CA), and data were normalized and evaluated, as described in previous reports. For this analysis, evaluation was restricted to tumors from patients with histologically proven HNSCC, without previous treatment, and without a history of a previous HNSCC primary tumor. Gene expression data from 131 HNSCC patients was log2 transformed, analyzed, and presented as box plots using GraphPad Prism version 8 (GraphPad Software, Inc.). Pairwise Pearson correlation analysis was conducted with relative gene expression levels of BAX, BAK1, BCL2, BCL2L1, and MCL1 within TCGA and MMC cohorts using log2 transformed data. A correlation coefficient matrix was produced by combining pairwise correlation coefficients from TCGA and MMC data, respectively. The matrix was used for unsupervised hierarchical clustering using the pheatmap package applied in R statistical software. Cell lines HN30, HN31, HN5, MDA686LN, and UMSCC47 were graciously provided by Dr. Jeffrey N. Myers, MD, PhD at the University of Texas, MD Anderson Cancer Center, Houston, Texas. Transfer agreements required the following permissions (HN30, HN31?John Ensley, MD, Wayne State University, Michigan, MI; MDA686LN?Peter Sacks, MD, New York University, New York, NY/University of Texas MD Anderson Cancer Center, Houston, TX; UMSCC47?Thomas Carey, University of Michigan, Ann Arbor, MI). All cells were grown in Dulbecco's Modified Eagle's Medium (DMEM) combined with 10% fetal bovine serum, nonessential amino acids, sodium pyruvate, and 1% antibiotic: penicillin/strepotmycin. Incubation was maintained at 37?C, 5% CO2. Stocks of each line used were authenticated with short tandem repeat genotyping. Whole cell lysates from HSNCC cells were prepared in radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitor cocktail (Pierce, Thermo Scientific, Rockford, IL). Protein concentrations were measured using DC protein assay (Bio-Rad, Hercules, CA). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was run at 80 V and membrane immunoblotted overnight. Primary antibodies were incubated in 1% bovine serum albumin/Tris-buffered saline with 0.1% polysorbate-20 at concentrations varying from 1:500 to 1:5000 (anti-?-actin?8H10D10,1:5000; anti-Bcl-2 (124) 15071S, 1:500; anti-Mcl-1 (D35A5) 5453S; anti-BAX 2772S, 1:500; anti-Bcl-xL (54H6) 2764s, Cell Signaling, 1:500; each from Cell Signaling, Danvers, MA; and anti-BAK,1:500,NT, EMD Millipore Corp., Burlington, MA. Fluorescent secondary antibodies against mouse or rabbit were sourced from LI-COR (LI- COR Biosciences, Lincoln, NE). Immunolabeled proteins were visualized with the LI-COR Odyssey FC Imaging System. Protein quantification was determined from 3 independent experiments using Image Studio software, Image Studio Lite 4.0 (LI-COR Biosciences). A pipetting robot was utilized to plate cells and test agents in a rapid, high throughput fashion. HNSCC cells (1,500 cells/well) were seeded in 96-well white plates. Cells were incubated with serial dilutions of bortezomib. Control (untreated) wells were maintained in parallel dilutions of dissolvent dimethylsulfoxide. Cell Titer-Glo (Promega, Madison, WI) was used to assess cell viability according to the manufacturer's protocol. The Infinite? M200 microplate reader (Tecan, M?nnedorf, Switzerland) was used to measure luminescence. Viability assays were performed in duplicates and the data normalized to vehicle-treated control wells. IC50 values were determined by nonlinear regression analysis using Prism software, version 8 (GraphPad Software, Inc.). HNSCC cell lines were plated on 96-well plates at a density of 1,500 cells/well. Cells were allowed to incubate for 24 hours before addition of bortezomib, with doses ranging from 0.31 nM to 160 nM. After 48-hour drug incubation, 10 ?L of MTT (VWR Chemicals, Solon, OH) at 5 mg/mL was added until visualization of formazan crystals by 150 minutes. Cell viability was determined by spectrophotometry at 570 nM. IC50 values were calculated by nonlinear regression analysis of the recorded absorbances with respect to vehicle control wells using GraphPad Prism version 8 (GraphPad Software, Inc.). HNSCC cells were plated onto 10 cm plates and allowed to grow for 24 hours. The following day, DMEM containing 5 nM or 10 nM bortezomib (EMD Millipore Corp.) was added and incubated for 24 hours at 37?C with 5% CO2. Whole cell lysate was extracted in RIPA buffer at the end of incubation. One hundred twenty-five thousand HNSCC cells were seeded in each well of six well plates and incubated for 24 hours. After 24 hours, HNSCCs were treated with apoptosis-inducing agents at the following concentrations: 1 ?M staurosporine (Adipogen Life Sciences, San Diego, CA), 5 ?M ABT-263 (navitoclax) (Selleck Chemicals, Houston, TX), 5 ?M A-1210477 (Selleck Chemicals, Houston, Tx), and 5?M ABT-263 (navitoclax)/5 ?M A-1210477 combination, 5 nM bortezomib, and 10 nM bortezomib. Cells were incubated for another 48 hours and harvested with Accutase? (Sigma Aldrich, St. Louis, MO), washed in phosphate-buffered saline and stained with PE Annexin V Apoptosis Detection Kit I (BD Biosciences, San Diego, CA). Analysis was conducted by flow analyzer (BD LSRII Yellow). Flow cytometer data was analyzed, and figures were produced by FlowJo version 10 software (FlowJo, LLC, Ashland, OR). Mean averages were calculated for each transcript assessed from gene expression data in TCGA and MMC datasets, and transcripts were compared against each other using two-tailed Student t tests. Median or mean averages were calculated where appropriate for Annexin V data and for protein quantification, and box plots were created to represent data where described. For statistical analyses, a P value of 0.05 was accepted as the threshold for significance. Statistical analyses for specific assays are included in more detail in individual sections above. Publisher Copyright: {\textcopyright} 2020 The American Laryngological, Rhinological and Otological Society, Inc.",

year = "2020",

month = nov,

day = "1",

doi = "10.1002/lary.28441",

language = "English (US)",

volume = "130",

pages = "2643--2649",

journal = "Laryngoscope",

issn = "0023-852X",

publisher = "John Wiley and Sons Inc.",

number = "11",

}

TY - JOUR

T1 - Apoptosis signaling molecules as treatment targets in head and neck squamous cell carcinoma

AU - Ow, Thomas J.

AU - Thomas, Carlos

AU - Fulcher, Cory D.

AU - Chen, Jianhong

AU - López, Andrea

AU - Reyna, Denis E.

AU - Prystowsky, Michael B.

AU - Smith, Richard V.

AU - Schiff, Bradley A.

AU - Rosenblatt, Gregory

AU - Belbin, Thomas J.

AU - Harris, Thomas M.

AU - Childs, Geoffrey C.

AU - Kawachi, Nicole

AU - Schlecht, Nicolas F.

AU - Gavathiotis, Evripidis

N1 - Funding Information: t.j.o : supported in part by the Triological Society Career Development Award, the National Institutes of Health (NIH)/National Cancer Institute (NCI) grant 2K12 CA132783‐06, and the National Institutes of Health (NIH)/The National Institute of Dental and Craniofacial Research (NIDCR) grant 1 K23 DE027425‐01. e.g. and d.e.r . supported from the National Institutes of Health (NIH)/National Cancer Institute (NCI) grant CA178394 and an award from the Pershing Square Sohn Cancer Research Alliance. a.l .: supported from a T32 Institutional Training Program grant T32 AG 23475. The authors also acknowledge support from the Albert Einstein Cancer Center the National Institutes of Health (NIH)/National Cancer Institute (NCI) grant P30CA013330, and specifically the flow cytometry core facility, part of the Albert Einstein shared resources, as well as Roswell Park Comprehensive Cancer Center grant CA016056. The authors have no other funding, financial relationships, or conflicts of interest to disclose. Funding Information: Data from a total of 496 HNSCC patients in The Cancer Genome Atlas (TCGA) cohort include level 3 ribonucleic acid (RNA) sequencing data (log2 transformed RSEM value, as reads per kilobase of transcript per million mapped reads) and clinical data (human papillomavirus [HPV] status, tumor stage, and primary tumor site) downloaded from National Cancer Institute Genomic Data Commons (GDC, https://portal.gdc.cancer.gov/). Relative gene expression levels of targeted genes were presented as box plots using GraphPad Prism version 8 (GraphPad Software, Inc., San Diego, CA). In parallel, the relative gene expression levels were examined from the targeted genes within tumor samples obtained and evaluated at Montefiore Medical Center (MMC). RNA from HNSCC tumors were hybridized to the Illumina HumanHT-12-v3 Expression BeadChip (Illumina, San Diego, CA), and data were normalized and evaluated, as described in previous reports. For this analysis, evaluation was restricted to tumors from patients with histologically proven HNSCC, without previous treatment, and without a history of a previous HNSCC primary tumor. Gene expression data from 131 HNSCC patients was log2 transformed, analyzed, and presented as box plots using GraphPad Prism version 8 (GraphPad Software, Inc.). Pairwise Pearson correlation analysis was conducted with relative gene expression levels of BAX, BAK1, BCL2, BCL2L1, and MCL1 within TCGA and MMC cohorts using log2 transformed data. A correlation coefficient matrix was produced by combining pairwise correlation coefficients from TCGA and MMC data, respectively. The matrix was used for unsupervised hierarchical clustering using the pheatmap package applied in R statistical software. Data from a total of 496 HNSCC patients in The Cancer Genome Atlas (TCGA) cohort include level 3 ribonucleic acid (RNA) sequencing data (log2 transformed RSEM value, as reads per kilobase of transcript per million mapped reads) and clinical data (human papillomavirus [HPV] status, tumor stage, and primary tumor site) downloaded from National Cancer Institute Genomic Data Commons (GDC, https://portal.gdc.cancer.gov/). Relative gene expression levels of targeted genes were presented as box plots using GraphPad Prism version 8 (GraphPad Software, Inc., San Diego, CA). In parallel, the relative gene expression levels were examined from the targeted genes within tumor samples obtained and evaluated at Montefiore Medical Center (MMC). RNA from HNSCC tumors were hybridized to the Illumina HumanHT-12-v3 Expression BeadChip (Illumina, San Diego, CA), and data were normalized and evaluated, as described in previous reports. For this analysis, evaluation was restricted to tumors from patients with histologically proven HNSCC, without previous treatment, and without a history of a previous HNSCC primary tumor. Gene expression data from 131 HNSCC patients was log2 transformed, analyzed, and presented as box plots using GraphPad Prism version 8 (GraphPad Software, Inc.). Pairwise Pearson correlation analysis was conducted with relative gene expression levels of BAX, BAK1, BCL2, BCL2L1, and MCL1 within TCGA and MMC cohorts using log2 transformed data. A correlation coefficient matrix was produced by combining pairwise correlation coefficients from TCGA and MMC data, respectively. The matrix was used for unsupervised hierarchical clustering using the pheatmap package applied in R statistical software. Cell lines HN30, HN31, HN5, MDA686LN, and UMSCC47 were graciously provided by Dr. Jeffrey N. Myers, MD, PhD at the University of Texas, MD Anderson Cancer Center, Houston, Texas. Transfer agreements required the following permissions (HN30, HN31?John Ensley, MD, Wayne State University, Michigan, MI; MDA686LN?Peter Sacks, MD, New York University, New York, NY/University of Texas MD Anderson Cancer Center, Houston, TX; UMSCC47?Thomas Carey, University of Michigan, Ann Arbor, MI). All cells were grown in Dulbecco's Modified Eagle's Medium (DMEM) combined with 10% fetal bovine serum, nonessential amino acids, sodium pyruvate, and 1% antibiotic: penicillin/strepotmycin. Incubation was maintained at 37?C, 5% CO2. Stocks of each line used were authenticated with short tandem repeat genotyping. Whole cell lysates from HSNCC cells were prepared in radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitor cocktail (Pierce, Thermo Scientific, Rockford, IL). Protein concentrations were measured using DC protein assay (Bio-Rad, Hercules, CA). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was run at 80 V and membrane immunoblotted overnight. Primary antibodies were incubated in 1% bovine serum albumin/Tris-buffered saline with 0.1% polysorbate-20 at concentrations varying from 1:500 to 1:5000 (anti-?-actin?8H10D10,1:5000; anti-Bcl-2 (124) 15071S, 1:500; anti-Mcl-1 (D35A5) 5453S; anti-BAX 2772S, 1:500; anti-Bcl-xL (54H6) 2764s, Cell Signaling, 1:500; each from Cell Signaling, Danvers, MA; and anti-BAK,1:500,NT, EMD Millipore Corp., Burlington, MA. Fluorescent secondary antibodies against mouse or rabbit were sourced from LI-COR (LI- COR Biosciences, Lincoln, NE). Immunolabeled proteins were visualized with the LI-COR Odyssey FC Imaging System. Protein quantification was determined from 3 independent experiments using Image Studio software, Image Studio Lite 4.0 (LI-COR Biosciences). A pipetting robot was utilized to plate cells and test agents in a rapid, high throughput fashion. HNSCC cells (1,500 cells/well) were seeded in 96-well white plates. Cells were incubated with serial dilutions of bortezomib. Control (untreated) wells were maintained in parallel dilutions of dissolvent dimethylsulfoxide. Cell Titer-Glo (Promega, Madison, WI) was used to assess cell viability according to the manufacturer's protocol. The Infinite? M200 microplate reader (Tecan, M?nnedorf, Switzerland) was used to measure luminescence. Viability assays were performed in duplicates and the data normalized to vehicle-treated control wells. IC50 values were determined by nonlinear regression analysis using Prism software, version 8 (GraphPad Software, Inc.). HNSCC cell lines were plated on 96-well plates at a density of 1,500 cells/well. Cells were allowed to incubate for 24 hours before addition of bortezomib, with doses ranging from 0.31 nM to 160 nM. After 48-hour drug incubation, 10 ?L of MTT (VWR Chemicals, Solon, OH) at 5 mg/mL was added until visualization of formazan crystals by 150 minutes. Cell viability was determined by spectrophotometry at 570 nM. IC50 values were calculated by nonlinear regression analysis of the recorded absorbances with respect to vehicle control wells using GraphPad Prism version 8 (GraphPad Software, Inc.). HNSCC cells were plated onto 10 cm plates and allowed to grow for 24 hours. The following day, DMEM containing 5 nM or 10 nM bortezomib (EMD Millipore Corp.) was added and incubated for 24 hours at 37?C with 5% CO2. Whole cell lysate was extracted in RIPA buffer at the end of incubation. One hundred twenty-five thousand HNSCC cells were seeded in each well of six well plates and incubated for 24 hours. After 24 hours, HNSCCs were treated with apoptosis-inducing agents at the following concentrations: 1 ?M staurosporine (Adipogen Life Sciences, San Diego, CA), 5 ?M ABT-263 (navitoclax) (Selleck Chemicals, Houston, TX), 5 ?M A-1210477 (Selleck Chemicals, Houston, Tx), and 5?M ABT-263 (navitoclax)/5 ?M A-1210477 combination, 5 nM bortezomib, and 10 nM bortezomib. Cells were incubated for another 48 hours and harvested with Accutase? (Sigma Aldrich, St. Louis, MO), washed in phosphate-buffered saline and stained with PE Annexin V Apoptosis Detection Kit I (BD Biosciences, San Diego, CA). Analysis was conducted by flow analyzer (BD LSRII Yellow). Flow cytometer data was analyzed, and figures were produced by FlowJo version 10 software (FlowJo, LLC, Ashland, OR). Mean averages were calculated for each transcript assessed from gene expression data in TCGA and MMC datasets, and transcripts were compared against each other using two-tailed Student t tests. Median or mean averages were calculated where appropriate for Annexin V data and for protein quantification, and box plots were created to represent data where described. For statistical analyses, a P value of 0.05 was accepted as the threshold for significance. Statistical analyses for specific assays are included in more detail in individual sections above. Publisher Copyright: © 2020 The American Laryngological, Rhinological and Otological Society, Inc.

PY - 2020/11/1

Y1 - 2020/11/1

N2 - Objectives: To evaluate BCL-2 family signaling molecules in head and neck squamous cell carcinoma (HNSCC) and examine the ability of therapeutic agents with variable mechanisms of action to induce apoptosis in HNSCC cells. Methods: messenger ribonculeic acid (mRNA) expression of BAK, BAX, B-cell lymphoma (Bcl-2), BCL2 Like 1 (BCL2L1), and MCL1 were measured in The Cancer Genome Atlas (TCGA) head and neck cancer dataset, as well as in a dataset from a cohort at Montefiore Medical Center (MMC). Protein expression was similarly evaluated in a panel of HNSCC cell lines (HN30, HN31, HN5, MDA686LN, UMSCC47). Cell viability and Annexin V assays were used to assess the efficacy and apoptotic potential of a variety of agents (ABT-263 [navitoclax], A-1210477, and bortezomib. Results: Expression of BAK, BAX, BCL2L1, and MCL1 were each significantly higher than expression of BCL2 in the TCGA and MMC datasets. Protein expression demonstrated the same pattern of expression when examined in HNSCC cell lines. Treatment with combined ABT-263 (navitoclax)/A-1210477 or with bortezomib demonstrated apoptosis responses that approached or exceeded treatment with staurospaurine control. Conclusion: HNSCC cells rely on inhibition of apoptosis via BCL-xL and MCL-1 overexpression, and induction of apoptosis remains a potential therapeutic option as long as strategies overcome redundant anti-apoptotic signals. Level of Evidence: NA Laryngoscope, 130:2643–2649, 2020.

AB - Objectives: To evaluate BCL-2 family signaling molecules in head and neck squamous cell carcinoma (HNSCC) and examine the ability of therapeutic agents with variable mechanisms of action to induce apoptosis in HNSCC cells. Methods: messenger ribonculeic acid (mRNA) expression of BAK, BAX, B-cell lymphoma (Bcl-2), BCL2 Like 1 (BCL2L1), and MCL1 were measured in The Cancer Genome Atlas (TCGA) head and neck cancer dataset, as well as in a dataset from a cohort at Montefiore Medical Center (MMC). Protein expression was similarly evaluated in a panel of HNSCC cell lines (HN30, HN31, HN5, MDA686LN, UMSCC47). Cell viability and Annexin V assays were used to assess the efficacy and apoptotic potential of a variety of agents (ABT-263 [navitoclax], A-1210477, and bortezomib. Results: Expression of BAK, BAX, BCL2L1, and MCL1 were each significantly higher than expression of BCL2 in the TCGA and MMC datasets. Protein expression demonstrated the same pattern of expression when examined in HNSCC cell lines. Treatment with combined ABT-263 (navitoclax)/A-1210477 or with bortezomib demonstrated apoptosis responses that approached or exceeded treatment with staurospaurine control. Conclusion: HNSCC cells rely on inhibition of apoptosis via BCL-xL and MCL-1 overexpression, and induction of apoptosis remains a potential therapeutic option as long as strategies overcome redundant anti-apoptotic signals. Level of Evidence: NA Laryngoscope, 130:2643–2649, 2020.

KW - A-1210477

KW - ABT-263

KW - Head and neck squamous carcinoma

KW - apoptosis

KW - bortezomib

UR - http://www.scopus.com/inward/record.url?scp=85078293119&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85078293119&partnerID=8YFLogxK

U2 - 10.1002/lary.28441

DO - 10.1002/lary.28441

M3 - Article

C2 - 31894587

AN - SCOPUS:85078293119

SN - 0023-852X

VL - 130

SP - 2643

EP - 2649

JO - Laryngoscope

JF - Laryngoscope

IS - 11

ER -

Apoptosis signaling molecules as treatment targets in head and neck squamous cell carcinoma

Abstract

Keywords

ASJC Scopus subject areas

UN SDGs

Access to Document

Other files and links

Fingerprint

Cite this