Abstract
Purpose: 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.Experimental Design: 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.Results:In vitro, 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.Conclusions: 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. Clin Cancer Res; 24(5); 1152-62. ©2018 AACR.
Highlights
40,000 women with metastatic breast cancer die in the United States every year, due to treatment failure or treatment resistance [1]
To further describe this effect, we examined the expression of inducible nitric oxide synthase (iNOS) and eNOS in the presence of docetaxel over a period of 72 hours in three different Triple-negative breast cancer (TNBC) cell lines (SUM-159PT, MDA-MD 436, and MDA-MB 468)
Docetaxel induced iNOS in a bell-shaped distribution; the highest expression was noted around 12 to 24 hours, and by 72 hours, the levels were similar to basal expression (Supplementary Fig. S1). eNOS expression in MDA-MB 436 and MDA-MB 468 remained similar over the course of the treatment; SUM-159PT showed a minimal increase at 48 hours (Supplementary Fig. S1)
Summary
40,000 women with metastatic breast cancer die in the United States every year, due to treatment failure or treatment resistance [1]. Comprises 15% of all breast cancers, and patients have higher recurrence, more distant metastases, and worse mortality rates than other breast cancer types [2]. TNBC is characterized by the lack of estrogen, progesterone, and HER2 receptor expression. TNBC is a heterogeneous disease without an FDA-approved targeted therapy. It is important to differentiate TNBC subtypes and to identify therapeutic targets when treating specific patient subpopulations [3, 4]. Most TNBCs are sensitive to systemic chemotherapies such as taxanes, anthracyclines, and platinum derivatives, yet local and systemic relapse rates are high [2, 5]
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