Loss of ATRX promotes aggressive features of osteosarcoma with increased NF-κB signaling and integrin binding
Suzanne Bartholf DeWitt, Sarah Hoskinson Plumlee, Hailey E. Brighton, Dharshan Sivaraj, E.J. Martz, Maryam Zand, Vardhman Kumar, Maya U. Sheth, Warren Floyd, Jacob V. Spruance, Nathan Hawkey, Shyni Varghese, Jianhua Ruan, David G. Kirsch, Jason A. Somarelli, Ben Alman, William C. Eward
Suzanne Bartholf DeWitt, Sarah Hoskinson Plumlee, Hailey E. Brighton, Dharshan Sivaraj, E.J. Martz, Maryam Zand, Vardhman Kumar, Maya U. Sheth, Warren Floyd, Jacob V. Spruance, Nathan Hawkey, Shyni Varghese, Jianhua Ruan, David G. Kirsch, Jason A. Somarelli, Ben Alman, William C. Eward
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Research Article Oncology

Loss of ATRX promotes aggressive features of osteosarcoma with increased NF-κB signaling and integrin binding

Abstract

Osteosarcoma (OS) is a lethal disease with few known targeted therapies. Here, we show that decreased ATRX expression is associated with more aggressive tumor cell phenotypes, including increased growth, migration, invasion, and metastasis. These phenotypic changes correspond with activation of NF-κB signaling, extracellular matrix remodeling, increased integrin αvβ3 expression, and ETS family transcription factor binding. Here, we characterize these changes in vitro, in vivo, and in a data set of human OS patients. This increased aggression substantially sensitizes ATRX-deficient OS cells to integrin signaling inhibition. Thus, ATRX plays an important tumor-suppression role in OS, and loss of function of this gene may underlie new therapeutic vulnerabilities. The relationship between ATRX expression and integrin binding, NF-κB activation, and ETS family transcription factor binding has not been described in previous studies and may impact the pathophysiology of other diseases with ATRX loss, including other cancers and the ATR-X α thalassemia intellectual disability syndrome.

Authors

Suzanne Bartholf DeWitt, Sarah Hoskinson Plumlee, Hailey E. Brighton, Dharshan Sivaraj, E.J. Martz, Maryam Zand, Vardhman Kumar, Maya U. Sheth, Warren Floyd, Jacob V. Spruance, Nathan Hawkey, Shyni Varghese, Jianhua Ruan, David G. Kirsch, Jason A. Somarelli, Ben Alman, William C. Eward

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Figure 8

ATRX KO sensitizes cells to pharmacological inhibition of integrin signaling in vivo, and integrin inhibition is sufficient to reverse aggressive phenotypes seen with ATRX loss.

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ATRX KO sensitizes cells to pharmacological inhibition of integrin sign...
(A and B) Treatment with the integrin inhibitor SB273005 significantly reduced tumor growth in ATRX-null U-2OS cells (1-way repeated-measures ANOVA, P < 0.0001 for volume change over time; 1-tailed Student’s t test, P = 0.02 for final tumor volume change; vehicle-treated: n = 4 females, 5 males; integrin inhibitor-treated: n = 5 females, 5 males). (C) The integrin inhibitor reversed the increased rate of migration conferred by loss of ATRX in the MG-63 cell line (repeated-measures ANOVA, P < 0.0001; WT vehicle, n = 19 replicates; WT drug, n = 26 replicates; KO vehicle, n = 18 replicates; KO drug, n = 25 replicates). (D and E) The integrin inhibitor SB273005 reversed the increased migration and invasion observed with the MG-63 ATRX-KO cells in both uncoated transwell assays (D) and Matrigel-coated transwell assays (E). Scale bars: 100 μm. (Tukey’s multiple-comparison test for uncoated wells: ATRX-KO vehicle versus WT Vehicle P = 0.028, KO Vehicle versus WT Drug P = 0.035, KO Vehicle versus KO Drug P = 0.0117, n = 12 wells, 2 experiments. Tukey’s multiple-comparison test for Matrigel-coated wells: ATRX-KO vehicle versus WT vehicle, P < 0.0001; KO vehicle versus WT drug, P < 0.0001; KO vehicle versus KO drug, P < 0.0001; n = 12 wells; 2 experiments). (F) Integrin inhibitor treatment partially reversed the upregulation of NF-κB transcription factors p65 and RelB in the ATRX-KO cells (Tukey’s multiple-comparison tests for p65: ATRX-KO vehicle versus WT vehicle, P < 0.0001; KO vehicle versus WT drug, P < 0.0001; KO vehicle versus KO drug, P = 0.028; n = 3. Tukey’s multiple-comparison tests for RelB: ATRX-KO vehicle versus WT vehicle, P < 0.0001; KO vehicle versus WT drug, P < 0.0001; KO vehicle versus KO drug, P = 0.0002; n = 3).