Abstract 2327: Novel role of NAC1 in regulating tumor stem cells and their interaction with immune cells in triple-negative breast cancer
Authors: Ngule Chrispus M. , Hemati Hami , Ren Xingcong , Bachert Emily , Liu Xiaoqi , Yang Jin-Ming 
Published: April 2023

Abstract
One of the important features of triple-negative breast cancer (TNBC) is the enrichment of stem-like cells that contribute substantially to the malignant phenotype. Nucleus accumbens associated protein 1 (NAC1), a unique member of the BTB-POZ family, is implicated in tumor development and progression of various types of cancer. The objective of this study was to test the hypothesis that NAC1 plays a critical role in maintaining stemness of TNBC. We found that TNBC cell lines, MDA-MB-231 and HCC-1806, had high NAC1 expressions compared to normal. Hematoxylin-eosin staining of 450 human breast cancer tissues revealed high NAC1 expressions in different stages of breast tumor development. Depletion of NAC1 in TNBC cells decreased their mammosphere formation, migration and invasion abilities, and altered their EMT phenotype. Canonical TNBC stemness markers CD44 and ALDH1A1 were downregulated in TNBC cells subjected to NAC1 depletion. Down-regulation of NAC1 also decreased aldolase activity. RNA sequencing uncovered the enrichment of JAK/STAT3, PI3K/AKT, WNT, stemness and tumor progression-associated genes in TNBC with depletion of NAC1. IHC analysis revealed increased expression of phospho-STAT3 (Y705), β-catenin, PDK1, cyclin D1, cMYC, IL-6 and phosphorylated PTEN in TNBC cells in support of the sequencing data. Immunoprecipitation assays demonstrated the physical association of NAC1 with STAT3 and CD44 and the pulse-chase experiments showed that the proteasome-mediated turnover of STAT3 was faster in the NAC1-depleted cells than the control cells (half-life: 5hrs vs 24hrs, respectively). NAC1-depleted cells had a significantly reduced tumorigenic ability in vivo. TCGA immune cell infiltration analysis revealed that NAC1 expression had a positive correlation with myeloid-derived suppressor cells (MDSCs) infiltration. In vitro co-culture assays with Gr1/CD11b+ cells from NAC1 knockout mice with EO771 tumor cells showed increase of the stemness marker CD44 and aldolase activity, suggesting a role of NAC1 in modulating the stemness-immune interaction in tumor microenvironment. Further, we observed a significant increase of NAC1 in MDSCs from the tumor bearing mice compared to that of the tumor-free mice. The results of this study demonstrate a novel role of NAC1 in regulating tumor stem cells and their crosstalk with immune cells in the tumor microenvironment (TME) and suggest that targeting NAC1 may be exploited as a new approach to reverse immune-suppressive TME and to improve treatment for TNBC.
Citation Format: Chrispus M. Ngule, Hami Hemati, Xingcong Ren, Emily Bachert, Xiaoqi Liu, Jin-Ming Yang. Novel role of NAC1 in regulating tumor stem cells and their interaction with immune cells in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2327.