Abstract P5-17-04: Combined PI3K and NOS inhibition enhances efficacy of taxane-based chemotherapy in metaplastic breast cancer
Authors: Reddy Tejaswini P , Mahboubi Bijan , Rosato Roberto R. , Guzman-Rojas Liliana , Qian Wei , Zhou Jianying , Kim Baek , Moulder Stacy , Piwnica-Worms Helen , Chang Jenny C. 
Published: March 2022

Abstract
Background: Metaplastic breast cancer (MpBC) is a therapeutically chemoresistant, aggressive, and heterogeneous breast cancer variant accounting for &lt;5% of all breast cancers. Most MpBCs harbor a triple-negative breast cancer (TNBC) phenotype, yet have a worse prognosis and decreased survival compared to TNBC. Despite its chemorefractory nature, the current mainstay of treatment for MpBC is surgery and systemic chemotherapy. Common molecular alterations found in MpBC associated with poor prognosis and worse overall survival include 1) hyperactivation of the phosphoinositide 3-kinase (PI3K) signaling pathway and 2) enhanced production of nitric oxide via inducible nitric oxide synthase (iNOS). In MpBC, both the PI3K and NOS signaling pathways may synergistically work together to enhance chemoresistance. We propose that combined inhibition of PI3K and iNOS will enhance the efficacy of taxane-based chemotherapy in MpBC. Methods: For in vitro and in vivo studies, we used MpBC cell lines (Hs578T and BT549) and TNBC/MpBC Patient-Derived Xenograft (PDX) models, respectively. For all studies, we used pan-NOS inhibitor NG-monomethyl-l-arginine (L-NMMA, L), PI3K inhibitor alpelisib (A), and docetaxel (D). Immunohistochemistry (IHC), Western Blotting (WB), Cell Proliferation Assays, Flow Cytometry (to evaluate cell death and cell cycle distribution analysis), and HIV reverse transcriptase-based dNTP assay to quantify dNTPs were performed. For in vivo studies, five MpBC PDX models were implanted into the mammary fat pad of NSG mice and they received single therapy (vehicle control, L, A, D), dual therapy (D+A, D+L), or triple combination therapy (D+A+L). Tumor volumes were recorded twice weekly. Results: 66% (4/6) MpBC and 33% (7/21) TNBC PDX models had double-positive IHC staining of both iNOS and p-Akt (Ser473), supporting the concept that both signaling pathways are typically activated in MpBC tumors, relative to non-metaplastic TNBC tumors. Apoptosis and cell proliferation analysis found that MpBC cell lines treated with triple-combination (D+A+L) had an increased number of apoptotic cells and decreased cell proliferation relative to MpBC cells treated with dual combination (L+A), or single treatment (vehicle, D, A, or L). Cell cycle distribution analysis of treated MpBC cells found that in a time-dependent manner, there was a substantial decrease in the % of MpBC cells in S-phase and an increase in the % cells in G2/M cell cycle arrest due to dual and triple combination. This result was supported by dNTP quantification analysis revealing that combined PI3K and NOS inhibition induced greater nucleotide depletion within 8 hours of treatment relative to single treatment in MpBC cells. WB analysis revealed that dual/triple combination therapy in MpBC cells resulted in an enhanced DNA damage response signaling relative to single treatment, as indicated with increased expression of γ-H2AX, p-Chk1, p-Chk2, p-P53 (Ser15 and 20), and p21. Pro-survival PI3K signaling pathway was activated in response to docetaxel treatment alone in MpBC cells, but its activation was significantly reduced when docetaxel was coupled with PI3K and NOS inhibition. In vivo studies revealed that triple combination therapy significantly reduced tumor volume and improved survival proportions compared to dual/single therapy and vehicle control. Conclusions: The present data suggest that combined PI3K and NOS inhibition enhances docetaxel-mediated DNA damage by depleting nucleotide pools, leading to enhanced DNA damage response, growth arrest, and apoptosis. Ongoing studies are investigating how docetaxel coupled with PI3K and NOS inhibition influences DNA repair signaling and MpBC metastatic capacity. The addition of PI3K and NOS inhibitors to taxane-based chemotherapy may be a novel therapeutic strategy for aggressive MpBCs.
Citation Format: Tejaswini P Reddy, Bijan Mahboubi, Roberto R. Rosato, Liliana Guzman-Rojas, Wei Qian, Jianying Zhou, Baek Kim, Stacy Moulder, Helen Piwnica-Worms, Jenny C. Chang. Combined PI3K and NOS inhibition enhances efficacy of taxane-based chemotherapy in metaplastic breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-17-04.