Pharmacological Inhibition of NOS Activates ASK1/JNK Pathway Augmenting Docetaxel-Mediated Apoptosis in Triple-Negative Breast Cancer

• Published in: Clinical cancer research 2018-03, Vol.24 (5), p.1152-1162
• Main Authors: Dávila-González, Daniel, Choi, Dong Soon, Rosato, Roberto R, Granados-Principal, Sergio M, Kuhn, John G, Li, Wen-Feng, Qian, Wei, Chen, Wen, Kozielski, Anthony J, Wong, Helen, Dave, Bhuvanesh, Chang, Jenny C
• Format: Article
• Language: English
• Subjects: Animal models; Animals; Antineoplastic Combined Chemotherapy Protocols - pharmacology; Antineoplastic Combined Chemotherapy Protocols - therapeutic use; Apoptosis; Apoptosis - drug effects; Breast cancer; Cancer; Cell Line, Tumor; Cellular stress response; Chemoresistance; Chemotherapy; Cytometry; Docetaxel - pharmacology; Docetaxel - therapeutic use; Drug Synergism; Endoplasmic reticulum; Experimental design; Female; Flow cytometry; Fuel consumption; Humans; Inhibition; Iodides; JNK Mitogen-Activated Protein Kinases - metabolism; JNK protein; MAP kinase; MAP Kinase Kinase Kinase 5 - metabolism; MAP Kinase Signaling System - drug effects; Mass spectrometry; Mass spectroscopy; Medical prognosis; Mice; Mice, SCID; Nitric oxide; Nitric Oxide Synthase Type II - antagonists & inhibitors; Nitric-oxide synthase; omega-N-Methylarginine - pharmacology; omega-N-Methylarginine - therapeutic use; Pharmacology; Propidium iodide; Rodents; Stress; Stress response; Stresses; Survival; Triple Negative Breast Neoplasms - drug therapy; Triple Negative Breast Neoplasms - pathology; Xenograft Model Antitumor Assays; Xenografts; Xenotransplantation
• ISSN: 1078-0432; 1557-3265
• DOI: 10.1158/1078-0432.ccr-17-1437

Chemoresistance in triple-negative breast cancer (TNBC) is associated with the activation of a survival mechanism orchestrated by the endoplasmic reticulum (EnR) stress response and by inducible nitric oxide synthase (iNOS). Our aim was to determine the effects of pharmacologic NOS inhibition on TNBC. TNBC cell lines, SUM-159PT, MDA-MB-436, and MDA-MB-468, were treated with docetaxel and NOS inhibitor (L-NMMA) for 24, 48, and 72 hours. Apoptosis was assessed by flow cytometry using Annexin-V and propidium iodide. Western blot was used to assess ER stress and apoptosis, and rtPCR was used to evaluate s-XBP1. TNBC patient-derived xenografts (PDX) were treated either with vehicle, docetaxel, or combination therapy (NOS inhibition + docetaxel). Mouse weight and tumor volumes were recorded twice weekly. Docetaxel concentration was determined using mass spectrometry. To quantify proliferation and apoptosis, PDX tumor samples were stained using Ki67 and TUNEL assay. L-NMMA ameliorated the iNOS upregulation associated with docetaxel. Apoptosis increased when TNBC cells were treated with combination therapy. In TNBC PDXs, combination therapy significantly reduced tumor volume growth and increased survival proportions. In the BCM-5998 PDX model, intratumoral docetaxel concentration was higher in mice receiving combination therapy. Coupling docetaxel with NOS inhibition increased EnR-stress response via coactivation of ATF4 and CHOP, which triggered the pASK1/JNK proapoptotic pathway, promoting cleavage of caspases 3 and 9. iNOS is a critical target for docetaxel resistance in TNBC. Pharmacologic inhibition of NOS enhanced chemotherapy response in TNBC PDX models. Combination therapy may improve prognosis and prevent relapse in TNBC patients who have failed conventional chemotherapy. .