Nucleus Accumbens-associated Protein Research Articles

Nacc1 Mutation in Mice Models Rare Neurodevelopmental Disorder with Underlying Synaptic Dysfunction.

A missense mutation in the transcription repressor Nucleus accumbens-associated 1 (NACC1) gene at c.892C>T (p.Arg298Trp) on chromosome 19 causes severe neurodevelopmental delay ( Schoch et al., 2017). To model this disorder, we engineered the first mouse model with the homologous mutation (Nacc1+/R284W ) and examined mice from E17.5 to 8 months. Both genders had delayed weight gain, epileptiform discharges and altered power spectral distribution in cortical electroencephalogram, behavioral seizures, and marked hindlimb clasping; females displayed thigmotaxis in an open field. In the cortex, NACC1 long isoform, which harbors the mutation, increased from 3 to 6 months, whereas the short isoform, which is not present in humans and lacks aaR284 in mice, rose steadily from postnatal day (P) 7. Nuclear NACC1 immunoreactivity increased in cortical pyramidal neurons and parvalbumin containing interneurons but not in nuclei of astrocytes or oligodendroglia. Glial fibrillary acidic protein staining in astrocytic processes was diminished. RNA-seq of P14 mutant mice cortex revealed over 1,000 differentially expressed genes (DEGs). Glial transcripts were downregulated and synaptic genes upregulated. Top gene ontology terms from upregulated DEGs relate to postsynapse and ion channel function, while downregulated DEGs enriched for terms relating to metabolic function, mitochondria, and ribosomes. Levels of synaptic proteins were changed, but number and length of synaptic contacts were unaltered at 3 months. Homozygosity worsened some phenotypes including postnatal survival, weight gain delay, and increase in nuclear NACC1. This mouse model simulates a rare form of autism and will be indispensable for assessing pathophysiology and targets for therapeutic intervention.

Critical domains for NACC2-NTRK2 fusion protein activation.

Neurotrophic receptor tyrosine kinases (NTRKs) belong to the receptor tyrosine kinase (RTK) family. NTRKs are responsible for the activation of multiple downstream signaling pathways that regulate cell growth, proliferation, differentiation, and apoptosis. NTRK-associated mutations often result in oncogenesis and lead to aberrant activation of downstream signaling pathways including MAPK, JAK/STAT, and PLCγ1. This study characterizes the NACC2-NTRK2 oncogenic fusion protein that leads to pilocytic astrocytoma and pediatric glioblastoma. This fusion joins the BTB domain (Broad-complex, Tramtrack, and Bric-a-brac) domain of NACC2 (Nucleus Accumbens-associated protein 2) with the transmembrane helix and tyrosine kinase domain of NTRK2. We focus on identifying critical domains for the biological activity of the fusion protein. Mutations were introduced in the charged pocket of the BTB domain or in the monomer core, based on a structural comparison of the NACC2 BTB domain with that of PLZF, another BTB-containing protein. Mutations were also introduced into the NTRK2-derived portion to allow comparison of two different breakpoints that have been clinically reported. We show that activation of the NTRK2 kinase domain relies on multimerization of the BTB domain in NACC2-NTRK2. Mutations which disrupt BTB-mediated multimerization significantly reduce kinase activity and downstream signaling. The ability of these mutations to abrogate biological activity suggests that BTB domain inhibition could be a potential treatment for NACC2-NTRK2-induced cancers. Removal of the transmembrane helix leads to enhanced stability of the fusion protein and increased activity of the NACC2-NTRK2 fusion, suggesting a mechanism for the oncogenicity of a distinct NACC2-NTRK2 isoform observed in pediatric glioblastoma.

Identification of a NACC1-Regulated Gene Signature Implicated in the Features of Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC), characterized by a deficiency in estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor2 (HER2), is among the most lethal subtypes of breast cancer (BC). Nevertheless, the molecular determinants that contribute to its malignant phenotypes such as tumor heterogeneity and therapy resistance, remain elusive. In this study, we sought to identify the stemness-associated genes involved in TNBC progression. Using bioinformatics approaches, we found 55 up- and 9 downregulated genes in TNBC. Out of the 55 upregulated genes, a 5 gene-signature (CDK1, EZH2, CCNB1, CCNA2, and AURKA) involved in cell regeneration was positively correlated with the status of tumor hypoxia and clustered with stemness-associated genes, as recognized by Parametric Gene Set Enrichment Analysis (PGSEA). Enhanced infiltration of immunosuppressive cells was also positively correlated with the expression of these five genes. Moreover, our experiments showed that depletion of the transcriptional co-factor nucleus accumbens-associated protein 1 (NAC1), which is highly expressed in TNBC, reduced the expression of these genes. Thus, the five genes signature identified by this study warrants further exploration as a potential new biomarker of TNBC heterogeneity/stemness characterized by high hypoxia, stemness enrichment, and immune-suppressive tumor microenvironment.

Expression of NAC1 Restrains the Memory Formation of CD8+ T Cells during Viral Infection.

Nucleus accumbens-associated protein 1 (NAC1) is a transcription co-factor that has been shown to possess multiple roles in stem cell and cancer biology. However, little is known about its roles in regulation of the immune system. In the current study, we observed that expression of NAC1 impacted the survival of CD8+ T cells in vitro. NAC1−/− CD8+ T cells displayed lower metabolism, including reduced glycolysis and oxidative phosphorylation. In vivo, compared with wild-type (WT) mice, NAC1−/− mice produced a lower response to vaccinia virus (VACV) infection, and viral antigen (Ag)-specific CD8+ T cells decreased more slowly. Additionally, we observed that the NAC1−/− mice demonstrated a stronger memory formation of viral Ag-specific CD8+ T cells post-viral infection. Mechanically, we identified that compared with WT CD8+ T cells, the Interferon Regulatory Factor 4 (IRF4), a key transcription factor in T cell development, was highly expressed in NAC1−/− CD8+ T cells, insinuating that IRF4 could be a critical regulatory target of NAC1 in the memory formation of CD8+ T cells. Our results indicate that NAC1 restrains the memory formation of CD8+ T cells by modulating IRF4, and targeting NAC1 may be exploited as a new approach to boosting CD8+ T cell memory.

Activating transcription factor 3-activated long noncoding RNA forkhead box P4-antisense RNA 1 aggravates colorectal cancer progression by regulating microRNA-423-5p/nucleus accumbens associated 1 axis

ABSTRACT Long noncoding RNAs (lncRNAs) have vital roles in the progression of colorectal cancer (CRC). Forkhead box P4-antisense RNA 1 (FOXP4-AS1) showed a potential unfavorable prognostic factor for CRC, while its underlying mechanism remains elusive. Thus, the goal of this research is to determine mechanism of FOXP4-AS1 in CRC occurrence and development. Herein, a Dual-luciferase reporter assay was performed to assess the regulation of miR-423-5p to nucleus accumbens-associated protein 1 (NACC1) and activating transcription factor 3 (ATF3) to FOXP4-AS1 promoter. Hematoxylin-eosin (H&E) staining was performed to detect the pathological changes of tumor tissues. Flow cytometry, cell counting kit 8, Transwell, and wound healing assays were conducted to assess apoptosis, proliferation, migration, and invasion of CRC cells, respectively. The results showed that FOXP4-AS1 was highly expressed in CRC cell lines and tissues. CRC progression was promoted by the overexpression of FOXP4-AS1 in HTC116 cells and animal models. Furthermore, FOXP4-AS1 served as a molecular sponge for miR-423-5p, and NACC1 is a direct target of miR-423-5p. MiR-423-5p silencing or overexpression of NACC1 increased proliferation, migration, and invasion of HCT116 cells while suppressing apoptosis. We also found that the upregulation of FOXP4-AS1 was activated by ATF3 in CRC cells. Collectively, our results demonstrated that ATF3-activated FOXP4-AS1 aggravates CRC progression by regulating miR-423-5p/NACC1 axis, indicating a new target for CRC treatment.

Silencing of NACC1 inhibits the proliferation, migration and invasion of nasopharyngeal carcinoma cells via regulating the AKT/mTOR signaling pathway.

Nucleus accumbens-associated protein 1 (NACC1) has been reported to serve as an oncogenic role in several types of cancer; however, its role in nasopharyngeal carcinoma (NPC) remains to be determined. The present study aimed to investigate the role of NACC1 in NPC and elucidate the underlying mechanisms. Therefore, NACC1 expression in the normal nasopharyngeal epithelial cell line, NP69, and various NPC cell lines was determined by reverse transcription-quantitative PCR and western blot analyses. NACC1 expression was silenced in the NPC SUNE-1 cell line by transfection with a short hairpin RNA. Cell viability, proliferation, migration, invasion and epithelial-mesenchymal transition (EMT) were then evaluated using MTT, colony formation, wound healing, Transwell and western blot assays, respectively. SC79 was employed to activate AKT expression in NACC1-silenced SUNE-1 cells, and the aforementioned cellular processes were observed. The results revealed that NACC1 expression was upregulated in NPC cell lines. NACC1-knocdown inhibited SUNE-1 cell proliferation, migration, invasion and EMT. Moreover, the levels of phosphorylated AKT and mTOR were decreased upon NACC1 silencing. Mechanistically, the presence of SC79 significantly blocked all the effects of NACC1-knockdown on SUNE-1 cells. The findings of the present study demonstrated that NACC1-knockdown effectively suppressed NPC cell proliferation, migration and invasion by inhibiting the activation of the AKT/mTOR signaling pathway. NACC1 may thus serve as a potential target for the diagnosis and therapy of NPC.

Nucleus Accumbens-Associated Protein 1 Binds DNA Directly through the BEN Domain in a Sequence-Specific Manner.

Nucleus accumbens-associated protein 1 (NAC1) is a nuclear protein that harbors an amino-terminal BTB domain and a carboxyl-terminal BEN domain. NAC1 appears to play significant and diverse functions in cancer and stem cell biology. Here we demonstrated that the BEN domain of NAC1 is a sequence-specific DNA-binding domain. We selected the palindromic 6 bp motif ACATGT as a target sequence by using a PCR-assisted random oligonucleotide selection approach. The interaction between NAC1 and target DNA was characterized by gel shift assays, pull-down assays, isothermal titration calorimetry (ITC), chromatin-immunoprecipitation assays, and NMR chemical shifts perturbation (CSP). The solution NMR structure revealed that the BEN domain of human NAC-1 is composed of five conserved α helices and two short β sheets, with an additional hitherto unknown N-terminal α helix. In particular, ITC clarified that there are two sequential events in the titration of the BEN domain of NAC1 into the target DNA. The ITC results were further supported by CSP data and structure analyses. Furthermore, live cell photobleaching analyses revealed that the BEN domain of NAC1 alone was unable to interact with chromatin/other proteins in cells.

Circular RNA hsa_circ_0001588 promotes the malignant progression of lung adenocarcinoma by modulating miR-524-3p/NACC1 signaling

AimsPrevious studies have demonstrated that circular RNAs play significant roles in several tumors, including lung adenocarcinoma; however, specific biological functions and molecular mechanisms underlying this process remain unclear. Materials and methodsHere, we conducted real-time quantitative PCR (qRT-PCR) to measure hsa_circ_0001588 expression levels in 60 paired lung adenocarcinoma tissues and cell lines. Furthermore, the association between hsa_circ_0001588 and clinical features of lung adenocarcinoma was analyzed. Functional experiments were conducted to assess the influence of hsa_circ_0001588 on proliferation, migration, and invasion in lung adenocarcinoma cells. We detected possible downstream targets of hsa_circ_0001588 using bioinformatics analysis. Luciferase reporter assays, qRT-PCR, and western blotting assays were performed to verify the molecular mechanism underlying hsa_circ_0001588 functions. Key findingsWe found that hsa_circ_0001588 was prominently upregulated in lung adenocarcinoma tissues and cell lines; elevated expression of hsa_circ_0001588 was positively correlated with poor clinicopathological features of lung adenocarcinoma. Functional experiments revealed that hsa_circ_0001588 acts as an oncogene to promote the proliferation, migration, and invasion of lung adenocarcinoma in vitro. Mechanistically, hsa_circ_0001588 promoted the proliferation, migration, and metastasis of lung adenocarcinoma by binding to miR-524-3p to promote nucleus accumbens-associated protein 1(NACC1) expression.Significance: Together, our results revealed that hsa_circ_0001588 upregulated the expression of NACC1 by combining with miR-524-3p to promote the proliferation, migration, and invasion of lung adenocarcinoma cells, suggesting that hsa_circ_0001588 may be an underlying therapeutic target for lung adenocarcinoma.

NAC1 attenuates BCL6 negative autoregulation and functions as a BCL6 coactivator of FOXQ1 transcription in cancer cells.

Background: Nucleus accumbens-associated protein 1 (NAC1) has multifaceted roles in cancer pathogenesis and progression, including the development of drug resistance, promotion of cytokinesis, and maintenance of “stem cell-like” phenotypes. NAC1 is a transcriptional co-regulator belonging to the bric-a-brac tramtrack broad (BTB) family of proteins, although it lacks the characteristic DNA binding motif of the BTB family. The formation of higher-order transcription complexes likely depends on its interaction with other DNA-binding co-factors.Results: NAC1 interacts with BCL6 via its C-terminal BEN domain and forms a complex that binds the promoter region and activates transcription of the NAC1 target gene, FOXQ1. NAC1 and BCL6 were coordinately upregulated. Our analysis also identified a novel function of NAC1 in attenuating BCL6 auto-downregulation in ovarian cancer. Lastly, we found a significant overlap among NAC1- and BCL6-regulated genes in tumor cells, suggesting that NAC1 and BCL6 coordinately control transcription in cancer.Conclusions: The results of this study provide a novel mechanistic insight into the oncogenic roles of NAC1 and underline the importance of developing the NAC1/BCL6-targeted cancer therapy.Methods: Using the Cistrome database and Chromatin Immunoprecipitation (ChIP) analyses, we identified BCL6 as a potential NAC1- interacting molecule. Co-immunoprecipitation (Co-IP), luciferase reporter assay, immunohistochemistry and microarray analysis were performed to analyze the interaction between NAC1 and BCL6 and the mechanisms by which they regulate the downstream genes including FOXQ1.

NACC1, as a Target of MicroRNA-331-3p, Regulates Cell Proliferation in Urothelial Carcinoma Cells.

The nucleus accumbens-associated protein 1 (NACC1) is a transcription factor constitutively expressed in the urothelium, where it regulates cell growth, senescence, autophagy, and epithelial-mesenchymal transition. microRNA (miRNA) constitutes a class of small non-coding RNAs which are involved in cell proliferation, differentiation, and progression of tumors. miRNAs and their target molecules are utilized for molecular diagnosis of urothelial carcinoma. NACC1 is one of several putative target molecules of miR-331-3p, and is associated with cell proliferation in cancers such as prostate and cervical cancer. Functional experiments involving miR-331-3p and its target molecule NACC1 were conducted using the urothelial carcinoma (UC) cell lines, T24, UMUC6, and KU7. Furthermore, quantitative reverse transcription polymerase chain reaction and immunostaining were performed to evaluate the expression of NACC1 in UC derived from transurethral resection of bladder tumor (TUR-Bt) specimens. The methane thiosulfonate (MTS) assay revealed that cell proliferation was significantly reduced after transient transfection of miR-331-3p precursor and/or NACC1 siRNA in UC cells. Cell senescence via cell cycle arrest at the G1 phase was induced by NACC1 inhibition. On the other hand, suppression of NACC1 induced cell migration and invasion abilities. Immunohistochemical analysis of TUR-Bt specimens revealed that over 70% of UC cells presented strongly positive results for NACC1. In contrast, normal urothelial cells were weakly positive for NACC1. It was also found that NACC1 expression was lower in invasive UC cells than in non-invasive UC cells. Loss of NACC1 induced vessel invasion in invasive UC tissues. The present results indicate that NACC1 regulated by miR-331-3p contributes to cell proliferation, and is involved in cell migration and invasion. This suggests that NACC1 can serve as a potential target molecule for the prediction and prognosis of UC, and can contribute to effective treatment strategies.

NAC1 promotes the migration of prostate cancer cells and participates in osteoclastogenesis by negatively regulating IFNβ.

Nucleus accumbens-associated protein 1 (NAC1), a transcriptional co-regulator, is overexpressed in advanced prostate cancer. However, the NAC1-regulated transcriptome has not been completely explored. In the present study, the functional silencing of NAC1 blocked the migration of prostate cancer cells and suppress osteoclastogenesis. The present study also determined that NAC1 was overexpressed in the highly aggressive prostate cancer cell lines PC-3, DU-145 and LNCaP. NAC1 small interfering RNA treatment of DU-145 cells decreased cell migration, but interestingly had no significant effects on cell proliferation. Furthermore, microarray analysis showed that a group of genes may be associated with the development of prostate cancer after NAC1 knockdown, including interferon-β (IFNβ), which is reported to be involved in osteoclastogenesis, an important factor affecting bone metastasis. The mechanisms of NAC1 function were further explored by co-culture studies using PC-3 and RAW264.7 osteoclast precursor cells, which demonstrated that silencing NAC1 downregulated the genes associated with the activation of osteoclasts. Furthermore, it was revealed that NAC1 had the ability to affect the release of IFNβ into the extracellular environment. Together, these findings indicated that NAC1 promoted cell migration, and that NAC1 may have a key role in osteoclastogenesis.

NAC1 Regulates Somatic Cell Reprogramming by Controlling Zeb1 and E-cadherin Expression.

SummaryReprogramming somatic cells to induced pluripotent stem cells (iPSCs) is a long and inefficient process. A thorough understanding of the molecular mechanisms underlying reprogramming is paramount for efficient generation and safe application of iPSCs in medicine. While intensive efforts have been devoted to identifying reprogramming facilitators and barriers, a full repertoire of such factors, as well as their mechanistic actions, is poorly defined. Here, we report that NAC1, a pluripotency-associated factor and NANOG partner, is required for establishment of pluripotency during reprogramming. Mechanistically, NAC1 is essential for proper expression of E-cadherin by a dual regulatory mechanism: it facilitates NANOG binding to the E-cadherin promoter and fine-tunes its expression; most importantly, it downregulates the E-cadherin repressor ZEB1 directly via transcriptional repression and indirectly via post-transcriptional activation of the miR-200 miRNAs. Our study thus uncovers a previously unappreciated role for the pluripotency regulator NAC1 in promoting efficient somatic cell reprogramming.

1602P - Syndecan-1 up-regulates microRNA-331-3p and mediates epithelial-to-mesenchymal transition in prostate cancer

Syndecan-1 is an important regulator, and may contribute to various mechanisms in the progression of prostate cancer, such as cell growth, and epithelial-to-mesenchymal transition (EMT), through regulation of microRNAs (miRNAs). We recently found that miR-331-3p is down-regulated by syndecan-1 silencing in PC3 cells, based on an miRNA expression array and quantitative RT-PCR assay. The aim of the present study was to investigate whether syndecan-1-regulated miR-331-3p expression positively affects EMT. The expression of miR-331-3p, E-cadherin, and vimentin was detected in twenty-three prostate cancer tissues with in situ hybridization and immunohistochemistry. For functional analysis of miR-331-3p and syndecan-1, miR-331-3p precursor or syndecan-1 siRNA was transfected into PC3 cell lines. Cell proliferation and migration were evaluated with MTS assay and wound healing assay, respectively. For bioinformatics prediction, quantitative RT-PCR and luciferase assay were used to identify the target of the miRNA. In situ hybridization and immunohistochemistry of radical prostatectomy samples revealed miR-331-3p expression in cancer cells with high Gleason patterns, and EMT was demonstrated by decreased E-cadherin and increased vimentin staining. Overexpression of miR-331-3p up-regulated mesenchymal markers such as vimentin, N-cadherin, and Snail, and down-regulated epithelial markers such as E-cadherin and desmoplakin in the prostate cancer cell line PC3. We identified Neuropilin 2 (NRP2) and nucleus accumbens-associated protein 1 (NACC1) as putative target molecules in silico, as they were closely associated with the expression of miR-331-3p and TGF-&bgr;/Smad 4 signals. Syndecan-1 gene silencing decreased the levels of Dicer, which is involved in miRNA maturation. miR-331-3p can suppress tumor growth and the migration of prostate cancer cells by targeting NRP2 and NACC1, which may provide a potential therapeutic target for prostate cancer treatment. Moreover, syndecan-1-mediated miRNA maturation by Dicer and miR-331-3p-mediated EMT via effects on TGF-&bgr;/Smad 4 signaling are essential for the development of prostate cancer.

Protein complex formation and intranuclear dynamics of NAC1 in cancer cells

Nucleus accumbens-associated protein 1 (NAC1) is a cancer-related transcription regulator protein that is also involved in the pluripotency and differentiation of embryonic stem cells. NAC1 is overexpressed in various carcinomas including ovarian, cervical, breast, and pancreatic carcinomas. NAC1 knock-down was previously shown to result in the apoptosis of ovarian cancer cell lines and to rescue their sensitivity to chemotherapy, suggesting that NAC1 may be a potential therapeutic target, but protein complex formation and the dynamics of intranuclear NAC1 in cancer cells remain poorly understood. In this study, analysis of HeLa cell lysates by fast protein liquid chromatography (FPLC) on a sizing column showed that the NAC1 peak corresponded to an apparent molecular mass of 300–500 kDa, which is larger than the estimated molecular mass (58 kDa) of the protein. Furthermore, live cell photobleaching analyses with green fluorescent protein (GFP)-fused NAC1 proteins revealed the intranuclear dynamics of NAC1. Collectively our results demonstrate that NAC1 forms a protein complex to function as a transcriptional regulator in cancer cells.

Nucleus accumbens-associated protein 1 is a feasible marker for distinguishing oral epithelial dysplasia from squamous cell carcinoma

Background: Oral epithelial dysplasia (OED) and carcinoma in situ (CIS) are defined as dysplastic epithelial cells in the epithelium. Accurate diagnosis of such lesions is difficult even histopathologically. Over one-third of cases of oral squamous cell carcinoma (OSCC) are associated with OED. Nucleus accumbens-associated protein 1 (NAC1) belongs to the Poxvirus and Zinc finger/Bric-a-brac Tramtrack Broad complex family of proteins that mediates several cellular functions, including proliferation, apoptosis, and transcription.

Syndecan-1 up-regulates microRNA-331-3p and mediates epithelial-to-mesenchymal transition in prostate cancer.

MicroRNAs (miRNAs) are small noncoding RNAs with a length of approximately 19-24 nucleotides that regulate gene expression through translational inhibition and contribute to the progression of various tumors including prostate cancer. Aberrant expression of miRNAs has been implicated in the progression and metastasis of prostate cancer. The present study aimed to investigate whether miR-331-3p controlled by syndecan-1 positively affects the epithelial-to-mesenchymal transition (EMT). Overexpression of miR-331-3p upregulated mesenchymal markers such as vimentin, N-cadherin, and snail and downregulated epithelial markers such as E-cadherin and desmoplakin in the prostate cancer cell line PC3. We identified Neuropilin 2 and nucleus accumbens-associated protein 1 as putative target molecules in silico, as they were closely associated with the expression of miR-331-3p and TGF-β/Smad 4 signals. In situ hybridization and immunohistochemistry of radical prostatectomy samples revealed miR-331-3p in cancer cells with high Gleason patterns, in which EMT was demonstrated by decreased E-cadherin, and increased vimentin staining. Syndecan-1 gene silencing decreased levels of Dicer, which is involved in miRNA maturation. MiR-331-3p-mediated miRNA maturation and enhanced EMT via effects on TGF-β/Smad 4 and Dicer are essential for the development of prostate cancer mediated by syndecan-1. © 2015 Wiley Periodicals, Inc.

Nucleus Accumbens-Associated Protein 1 Expression Has Potential as a Marker for Distinguishing Oral Epithelial Dysplasia and Squamous Cell Carcinoma.

BackgroundOral epithelial dysplasia (OED) and carcinoma in situ (CIS) are defined by dysplastic cells in the epithelium. Over a third of oral squamous cell carcinoma (OSCC) patients present with associated OED. However, accurate histopathological diagnosis of such lesions is difficult. Nucleus accumbens-associated protein 1 (NAC1) is a member of the Pox virus and Zinc finger/Bric-a-brac Tramtrack Broad complex family of proteins, and is overexpressed in OSCC. This study aimed to determine whether NAC1 has the potential to be used as a marker to distinguish OED and OSCC.Methods and FindingsThe study included 114 patients (64 men, 50 women). There were 67, 10, and 37 patients with OED, CIS, and OSCC, respectively. NAC1 labeling indices (LIs) and immunoreactivity intensities (IRI) were evaluated. The patients’ pathological classification was significantly associated with age, sex, NAC1 LIs, and NAC1 IRI (p = 0.025, p = 0.022, p < 0.001, and p < 0.001, respectively). As a result of multivariate analysis, a predictive model was made; this identified the NAC1 LIs (OR [95% CI] 1.18 [1.11–1.28], p < 0.001) and NAC1 IRI (0.78 [0.68–0.86], p < 0.001) as predictive factors for CIS/OSCC. The NAC1 LIs/IRI cut-off values which discriminated between OED and CIS/OSCC were 50%/124 pixels. For NAC1 LIs with > 50% positivity the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were 0.766, 0.910, 0.857, and 0.847, respectively. For NAC1 IRI with ≤ 124 positive pixels, the sensitivity, specificity, PPV, and NPV were 0.787, 0.866, 0.804, and 0.853, respectively. Though there are several potential limitations to this study and the results were obtained from a retrospective analysis of a single site cohort, the data suggest that the NAC1 LIs/IRI is a strong predictor of CIS/OSCC.ConclusionsNAC1 has potential as a marker for distinguishing OED from CIS/OSCC.

Identification of the NAC1-Regulated Genes in Ovarian Cancer

Nucleus accumbens-associated protein 1 (NAC1), encoded by the NACC1 gene, is a transcription co-regulator that plays a multifaceted role in promoting tumorigenesis. However, the NAC1-regulated transcriptome has not been comprehensively defined. In this study, we compared the global gene expression profiles of NAC1-overexpressing SKOV3 ovarian cancer cells and NAC1-knockdown SKOV3 cells. We found that NAC1 knockdown was associated with up-regulation of apoptotic genes and down-regulation of genes involved in cell movement, proliferation, Notch signaling, and epithelial-mesenchymal transition. Among NAC1-regulated genes, FOXQ1 was further characterized because it is involved in cell motility and epithelial-mesenchymal transition. NAC1 knockdown decreased FOXQ1 expression and promoter activity. Similarly, inactivation of NAC1 by expression of a dominant-negative construct of NAC1 suppressed FOXQ1 expression. Ectopic expression of NAC1 in NACC1 null cells induced FOXQ1 expression. NAC1 knockdown resulted in decreased cell motility and invasion, whereas constitutive expression of FOXQ1 rescued motility in cells after NAC1 silencing. Moreover, in silico analysis revealed a significant co-up-regulation of NAC1 and FOXQ1 in ovarian carcinoma tissues. On the basis of transcription profiling, we report a group of NAC1-regulated genes that may participate in multiple cancer-related pathways. We further demonstrate that NAC1 is essential and sufficient for activation of FOXQ1 transcription and that the role of NAC1 in cell motility is mediated, at least in part, by FOXQ1.

Low expression of nucleus accumbens‐associated protein 1 predicts poor prognosis for patients with pancreatic ductal adenocarcinoma

Nucleus accumbens-associated protein 1 (NAC1) is overexpressed in various carcinomas including ovarian, cervical, breast, and pancreatic carcinomas. High expression of NAC1 is considered to have adverse effects on prognosis through negative regulation of growth arrest and DNA-damage-inducible 45-γ interacting protein 1 (GADD45GIP1) in ovarian and cervical carcinomas. In the present study, the expression of NAC1 in pancreatic ductal adenocarcinoma (PDA) was measured using immunohistochemistry and computer-assisted image analysis in order to investigate its correlation with various clinicopathological parameters and prognosis. Patients with low-NAC1 PDA had worse overall survival (P = 0.0010) and a shorter disease-free survival (P = 0.0036) than patients with high-NAC1 PDA. This was a clinical effect opposite to that reported in ovarian and cervical carcinomas. Furthermore, knockdown of NAC1 in pancreatic carcinoma cell lines did not increase expression of the GADD45GIP1 protein. These results indicate that the gene(s) regulated by NAC1 vary depending on the types of carcinoma or originating tissue, and that low expression of NAC1 predicts poor prognosis for patients with PDA.

Nuclear localization signal in a cancer-related transcriptional regulator protein NAC1

Nucleus accumbens-associated protein 1 (NAC1) might have potential oncogenic properties and participate in regulatory networks for pluripotency. Although NAC1 is described as a transcriptional regulator, the nuclear import machinery of NAC1 remains unclear. We found, using a point mutant, that dimer formation was not committed to the nuclear localization of NAC1 and, using deletion mutants, that the amino-terminal half of NAC1 harbored a potential nuclear localization signal (NLS). Wild type, but not mutants of this region, alone was sufficient to drive the importation of green fluorescent protein (GFP) into the nucleus. Bimax1, a synthetic peptide that blocks the importin α/β pathway, impaired nuclear localization of NAC1 in cells. We also used the binding properties of importin to demonstrate that this region is an NLS. Furthermore, the transcriptional regulator function of NAC1 was dependent on its nuclear localization activity in cells. Taken together, these results show that the region with a bipartite motif constitutes a functional nuclear import sequence in NAC1 that is independent of NAC1 dimer formation. The identification of an NAC1 NLS thus clarifies the mechanism through which NAC1 translocates to the nucleus to regulate the transcription of genes involved in oncogenicity and pluripotency.