Abstract P3-03-02: Inhibition of NOS promotes ER stress response and augments docetaxel-mediated apoptosis in TNBC
Authors: Davila-Gonzalez D , Choi DS , Kuhn J , Granados SM , Rosato RR , Dave B , Chang JC 
Published: March 2017

Introduction: Chemoresistance in triple negative breast cancer (TNBC) is related to an activation of a survival response orchestrated by endoplasmic reticulum (ER) stress. We hypothesize that attenuation of nitric oxide (NO) signaling pathway can overcome treatment resistance, preventing relapse, ultimately improving survival of TNBC patients. Here, we aimed to investigate the effects of pharmacological iNOS (inducible nitric oxide synthase) inhibition by L-NMMA on docetaxel-meditated ER stress response and to determine whether the therapeutic NOS inhibition may improve chemotherapy-based response.

Methods: BT-549, SUM-149, MDA-MB-436, and MDA-MD-468 TNBC cell lines were treated with docetaxel (D; 5 nm)/ L-NMMA (L; 4mM)/ amlodipine (A; 5 µm) daily for 48 and 72 hours. Cell death and proliferation were assayed by Annexin V and ATP quantification, respectively. Western Blot (WB) was used to measure ER stress markers. In vivo regimen treatment followed three 2-weekscycles of D (20 mg/kg intraperitoneal [IP] on day 1) and L (200 mg/kg oral gavage on day 2-6); A (10 mg/kg IP on day 2-6) A was administered together with L to counteract the well-known effects of L on blood pressure (hypertension). TNBC Patient derived xenograft (PDX) models #2147, #5998, #3107 and #4664 were transplanted into the mammary fat pad of SCID Beige mice. PDX #2147 received either, single drug (vehicle, L, A, D), double (L+A, D+L, D+A), or triple drug combination (L+A+D). Models #4664, #3107 and #5998 received only vehicle, D or D+ L+A. Mice weight and tumor volumes were recorded twice weekly. D concentration was measured by mass spectrometry.

Results: Studies on SUM-159 cell line showed that, when compared to the docetaxel-treated group, D+L+A increased cell death significantly, as indicated by a rise in annexin V/propidium iodide-positive cells. Increase in cell death by D+L+A was further demonstrated by accumulation of mitochondrial cleaved BAX. The enhanced apoptotic effects of D+L+A in MDA MD 468, BT 549 and MDA MD TNBC cell lines were confirmed by a decrease in ATP levels compared to D alone. WB revealed a survival stress response activated by docetaxel. When it was coupled with NOS inhibition, ER stress response showed higher expression of ATF4 and CHOP, triggering a proapoptotic response by pASK1/JNK pathway and cleaved caspases (CC3 and CC9). PDX #2147 showed that L, A and L+A treatment groups had similar tumor volume growth as the untreated group. However, combination therapy, D+L+A, significantly reduced the tumor volume and increased survival proportions compared with vehicle and docetaxel. Combination therapy also dramatically reduced tumor size on TNBC #4664 and #3107, and significantly improved response on #5998 compared with docetaxel alone. Intratumoral docetaxel concentration was 5.3-fold higher in mice receiving D+L+A than in those receiving docetaxel alone (#5998). In both groups, docetaxel was not detected in the plasma one week after injection.

Conclusion: The present data suggest that iNOS may be a critical target for docetaxel resistance in TNBC. iNOS inhibition enhanced chemotherapy response in TNBC PDX models indicating that addition of iNOS inhibitor may improve prognosis and prevent relapse in TNBC patients who have failed conventional chemotherapy.

Citation Format: Davila-Gonzalez D, Choi DS, Kuhn J, Granados SM, Rosato RR, Dave B, Chang JC. Inhibition of NOS promotes ER stress response and augments docetaxel-mediated apoptosis in TNBC [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-03-02.