Abstract A80: NAC1 attenuates BCL6 negative autoregulation and functions as a BCL6 coactivator of FOXQ1 transcription in ovarian cancer (OVCA).
Authors: Herlinger Alice Laschuk , Gao Min , Wu Ren-Chin , Wang Tian-Li , Rangel Leticia B A , Shih Ie-Ming 
Published: May 2016

Nucleus accumbens-associated protein 1 (NAC1) is a transcriptional co-regulator that lacks the DNA binding domain, thus relying on other co-factors to form higher-order transcription complexes. The present study aimed to elucidate the mechanism by which NAC1 modulates gene expression in OVCA. Nacc1-/- mice were euthanized, and spleens were collected. Promoter sequences were retrieved using Biostrings and BSgenome packages of Bioconductor 2.12. The position frequency matrix of BCL6 motif was obtained from Cistrome. BCL6 binding motifs on targeted promoters were identified by matchPWM, and log-odd scores calculated by PWMscoreStartingAt. For chromatin immunoprecipitation (ChIP), cells were fixed with 1% (w/v) paraformaldehyde prior to cross-linking with DTBP. Anti-BCL6, anti-NAC1 or rabbit IgG isotype were used for IP, and the genes of interest were amplified by qPCR. HEK293T cells were used for co-immunoprecipitaion (co-IP), and proteins were analyzed by Western blot. Luciferase reporter assays were performed in HeLa cells transfected with siNAC1 and/or siBCL6. Immunohistochemistry was done on tissue microarray (TMA) constituted of 51 primary ovarian high-grade serous carcinoma samples (HGSOC), using anti-NAC1 or anti-BCL6 antibodies. For microarray experiments, OVCAR5 cells were transfected with siNAC1 or siBCL6. Two-tailed t-test was used analyze data (means ± SD; p&lt;0.05). BCL6 consensus binding motifs were mapped to the promoters of the NAC1 targeted genes FOXQ1 (positions A -1000, B -800, and C -150). NAC1 binds to BCL6 motifs as proved by ChIP and qPCR assays conducted in HeLa and MCF7 cells (p&lt; 0.01). Reciprocal co-IP experiments done in HEK293T cells co-transfected with BCL6-FLAG and full-length NAC1-V5 indicated that NAC1 interacts with BCL6 via its C-terminal domain (NAC1-C186). Functional NAC1 is required to form putative NAC1/BCL6 transcription complex, and to modulate FOXQ1 expression in HeLa N130 tTA cells (p&lt; 0.001). Luciferase reporter assays run in HeLa cells transfected with siNAC1 and siBCL6 proved that the genes interact and collaborate to regulate FOXQ1 transcriptional activity (p&lt; 0.01). NAC1 and BCL6 transcriptional and protein expressions correlate in OVCA cell lines (R2=0.7025; p=0.0185), and in HGSOC TMA (R2=0.31; p=0.027), respectively. Furthermore, NAC1 modulates the expression of BCL6 as shown in OSE-NAC1 transfected cells (p=0.041), and in OVCAR5- (p=0.016), OVCAR3- (p=0.0089), SKOV3- (p=0.0216), and taxol-resistant SKOV3TR-siRNA-NAC1 (p=0.0002) transfected cells. BCL6 transcript expression was reduced by 50% in the spleen of Nacc-/- mice (p=0.0068). Endogenous NAC1 and BCL6 ChIP on the consensus BCL6 binding sequence within the BCL6 promoter in OVCA cells showed that BCL6 binds to its own promoter, and that NAC1 was enriched in the ChIP. Competition assay performed in NAC1-C186-OVCAR-3 cells revealed that BCL6 expression is modulated by NAC1 (p&lt;0.0001), thus pointing to a direct interaction between NAC1 and BCL6 as a key phenomenon that attenuates BCL6 auto-downregulation by NAC1. Modulation of gene expression was assessed by cDNA microarray in siNAC1 or siBCL6 transfected OVCAR5 cells: 238 and 188 genes were down- whereas 139 and 113 genes were up-regulated by NAC1 and BCL6, respectively, amongst which 54 genes were down- and 25 genes were up-regulated concomitantly. In conclusion, our study establishes a new mechanism for NAC1-driven OVCA, in which it interacts with BCL6 via its C-terminal domain forming a transcription complex that modulates the expression of target genes. We have also described a novel function of NAC1 in attenuating BCL6 autoregulation in OVCA. Finally, we have found a significant overlap among NAC1- and BCL6-regulated, suggesting a transcription collaboration of NAC1 and BCL6 in OVCA cells. CORRESPONDING AUTHORS: RANGEL, LBA; SHIH, IM.

Citation Format: Alice Laschuk Herlinger, Min Gao, Ren-Chin Wu, Tian-Li Wang, Leticia B A Rangel, Ie-Ming Shih. NAC1 attenuates BCL6 negative autoregulation and functions as a BCL6 coactivator of FOXQ1 transcription in ovarian cancer (OVCA). [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A80.