Expression Of ATAD2 Research Articles

Targeting USP7 to induce ubiquitination degradation of oncogenic ATAD2 and effects on cholangiocarcinoma growth.

e16215 Background: Cholangiocarcinoma (CCA) is a highly fatal disease due to limited therapeutic targets. Deubiquitinase USP7 is an oncogene and has been suggested to be deeply involved in regulating tumor initiation and progression, but its abundance and role in CCA still remain unclear. Methods: USP7 mRNA expression was performed using TCGA and GEO database. Survival analysis in CCA patients with high- and low-USP7 mRNA expression was performed using NODE database. CCK8, colony formation, flow cytometry and shRNA interference assay were used to evaluate the biological function of USP7-ATAD2 axis on CCA cancer cells in vitro. 4D label-free quantification proteomics was used to analyze the targets and networks regulated by USP7. The molecular mechanism of USP7 and ATAD2 in CCA was determined by conducting Western blot, co-immunoprecipitation, IHC and quantitative real-time PCR (qPCR) analyses. The CCA PDX model was used to evaluate the efficacy of targeting USP7 in the treatment of cholangiocarcinoma. Results: Here, we found USP7 is highly expressed in CAA, and is associated with poor outcomes of CCA patients. We show that USP7 depletion induces S phase arrest, and suppresses CCA tumor cells proliferation. Next, we provide a comprehensive understanding of targets and networks affected by USP7 depletion by performing a 4D label-free quantification proteomics analysis. We show that ATAD2 is a USP7 target, and USP7 interacts with and deubiquitinates ATAD2 to stabilize ATAD2 protein level. Ectopic expression of ATAD2 in USP7-depleted cells rescues the cell proliferation phenotype. Further, pharmacological blocking the activity of USP7 by P5091 inhibits tumor growth in CCA patient-derived xenograft (PDX) mouse model. Conclusions: This study uncovers the role of USP7-ATAD2 axis in regulating tumor growth, and targeting USP7 may provide a novel therapeutic strategy for CCA.

Dual Targeting Novel WDR5/ATAD2 Oncogenic Signaling through CK2/Ikaros Axis Demonstrates Synergistic Efficacy in T-ALL

Background WD repeat-containing protein 5 (WDR5) is highly expressed in human cancers and its high expression is linked with adverse prognoses. However, the oncogenic roles of WDR5 and underlying mechanisms are undefined in T-cell acute lymphoblastic leukemia (T-ALL). Casein Kinase II (CK2) is a pro-oncogene, and CK2-mediated IKAROS dysfunction plays critical roles in high-risk ALL, and CK2 inhibitor CX-4945 has the anti-leukemic effect by restoring IKAROS function. This study aims to explore the synergistic anti-leukemic efficacy of dual targeting novel WDR5/ATAD2 oncogenic signaling via the CK2/IKAROS axis in T-ALL. Methods Clinical samples from 38 T-ALL patients and 32 healthy controls were obtained from Zhongda Hospital Southeast University. The expression of WDR5 and ATAD2 were tested by q-PCR and Western blot. RNA-seq was performed after CEM cells were treated with WDR5 inhibitor (OICR-9429), CX-4945 and vehicle control for 72 hours. The human leukemia xenograft mouse model is established by intravenously injected with WDR5 knockdown CEM (CEM-shWDR5) cells and shRNA control (CEM-shNC) into NSG mice. Mice were treated for 25 days in 4 groups [CEM-shNC + vehicle, CEM-sh WDR5 + vehicle; CEM-shNC + CX-4945 (100 mg/kg via gavage), CEM-sh WDR5 + CX-4945]. Leukemia burden in mice spleen and bone marrow was analyzed by flow cytometry. The survival of mice was compared by the Kaplan-Meier method. The synergistic effect of CX-4945 and OICR-9429 was analyzed by CCK8 assay. This study was approved by the Ethics Committee of Zhongda Hospital Southeast University Results WDR5 ishighly expressed in T-ALL patients compared with normal controls( P<0.001) and is higher in the relapsed samples than paired newly-diagnosed controls ( P<0.05) (Fig. 1A-B). WDR5 knockdown in T-ALL cells significantly suppressed cell proliferation and induced cell cycle arrest in vitro ( P<0.01) (Fig. 1C), and in vivo leukemia development (Fig. 1D). Also, WDR5 inhibitor OICR-9429 induces cell proliferation and cell cycle arrest in the cells. RNA-seq revealed that ATAD2 is downregulated upon OICR-9429 treatment and indicated that ATAD2 is the downstream target of WDR5. WDR5 knockdown suppresses ATAD2 expression ( P<0.01) (Fig. 1E). In addition, ATAD2 ishighly expressed in T-ALL patients versus normal controls ( P<0.05) (Fig. 1F), and ATAD2 silencing significantly suppresses cell proliferation and induces cell cycle arrest of T-ALL cells ( P<0.01) (Fig. 1G). These data indicate WDR5/ATAD2 signaling act as a novel potential oncogenic pathway in T-ALL. Furthermore, we found that IKAROS transcriptionally suppresses WDR5 expression, and CK2-mediated hyperphosphorylation of IKAROS is one of the major mechanisms of IKAROS dysfunction in ALL. CX-4945 strongly suppresses WDR5 expression in an IKAROS-dependent manner (Fig. 1H-J). The synergistic effect of CX-4945 with WDR5 disruption on cell proliferation suppression and cell cycle arrest was observed in T-ALL both in in vitro and in vivo xenograft mouse model. The combination of CX-4945 with WDR5 disruption significantly prolonged the survival of mice compared with single CX-4945 [55(52-58) days vs. 38.5(36-41) days， P<0.001] and single WDR5 disruption control [55(52-58) days vs. 41.5(39-44) days, P<0.001] (Fig. 1K). Consistently, the combination significantly reduces the size and weight of the spleen (Fig. 1L), % human CD45+ T-ALL cells in the spleen (Fig. 1M) and bone marrow (data not shown), and the expression of WDR5 and ATAD2 in the spleen versus single treatment controls ( P<0.01) (Fig. 1N-P). Finally, a synergistic effect of CX-4945 with OICR-9429 on cell proliferation suppression and down-regulation of WDR5 and ATAD2 was observed in the primary leukemic cells from T-ALL patients (Fig. 2A-D). Together, these data indicated the synergistic efficacy of CX-4945 with OICR-9429 on T-ALL and the novel model was summarized in Fig.2E. Conclusion In this study, we identified a novel WDR5/ATAD2 oncogenic signaling regulated by CK2/IKAROS axis in T-ALL. Our study reveals a model that dual targeting WDR5/ATAD2 signaling through direct inhibiting oncoproteins and via CK2/IKAROS axis to transcriptionally repress the oncoprotein to achieve synergistic efficacy. Our results further highlight the combination of CX-4945 with WDR5 inhibition is a potential option for the therapy of T-ALL patients.

ATAD2 promotes glycolysis and tumor progression in clear cell renal cell carcinoma by regulating the transcriptional activity of c-Myc

Clear cell renal cell carcinoma (ccRCC) is a common malignant tumor of the urogenital tract. Given that ccRCC is often resistant to radiotherapy and traditional chemotherapy, the clinical treatment of patients with ccRCC remains a challenge. The present study found that ATAD2 was significantly upregulated in ccRCC tissues. In vitro and in vivo experiments showed that the inhibition of ATAD2 expression mitigated the aggressive phenotype of ccRCC. ATAD2 was also associated with glycolysis in ccRCC. Interestingly, we found that ATAD2 could physically interact with c-Myc and promote the expression of its downstream target gene, thereby enhancing the Warburg effect of ccRCC. Overall, our study emphasizes the role of ATAD2 in ccRCC. The targeted expression or functional regulation of ATAD2 could be a promising method to reduce the proliferation and progression of ccRCC.

Abstract 5680: Hypoxia-responsive and HIF1α-regulated AAA+ ATPase ATAD2 shows high oncogenic potential in stomach cancer

Abstract The burden of stomach cancer incidence and death is a global challenge. Treating stomach cancer remains immensely difficult due to the lack of available markers to detect the disease at the early stages. Therefore, there is an immense demand for finding out an early detection biomarker for stomach cancer. Recently, ATAD2 is found to be overexpressed in stomach cancer with high prognostic significance and recognised as a promising biomarker for stomach cancer. ATAD2 is a unique cancer/testis antigen (CTA) that belongs to both AAA+ ATPase and bromodomain family proteins. However, nothing much is known about the mechanism of ATAD2-mediated stomach carcinogenesis. Using bioinformatics analyses as well as studies with stomach cancer cells and stomach biopsy tissue samples, we address the regulation and function of ATAD2 expression in stomach cancer. We show that ATAD2 is overexpressed in the most common intestinal type of stomach adenocarcinoma, and enhanced expression of ATAD2 is observed in all stages and grades of stomach cancer. Our immunofluorescence microscopy study with stomach biopsy tissues confirms the high expression of ATAD2 both in stomach adenocarcinoma and metastatic stomach tissue samples. Survival analysis indicates that upregulated ATAD2 expression drastically affects the survival of stomach cancer patients. Since H. pylori infection, and hypoxia are the major contributing factors for stomach carcinogenesis; we further study their role in ATAD2-mediated stomach malignancy. An enhanced expression of ATAD2 is observed in H. pylori-infected stomach cancer cells. We identify ATAD2 as a hypoxia-responsive and HIF1α-regulated gene and elucidate that upregulated expression of ATAD2 enhances proliferation and migration of hypoxic stomach cancer cells. We further recognize the protein-protein interaction (PPI) network of ATAD2 with the top-ranked partners like ESR1 (or ERα), NCOA3 (or ACTR/SRC3/AIB1), SUMO2, HDA11 (HDAC11), SPTN2 (SPTBN2), and MYC. Among them, ESR1, MYC, NCOA3, and HDA11 are oncoproteins. Most strikingly, we report the participation of two druggable targets of ATAD2, i.e., AAA+ ATPase and bromodomain in the ATAD2-PPI network; ESR1, SUMO2, SPTN2, and MYC show a preference for bromodomain, whereas NCOA3 and HDA11 prefer ATPase domain of ATAD2. The importance of such findings in terms of targeting bromodomain-PPI and/or ATPase-PPI interfaces to curtail stomach cancer is elucidated. Citation Format: Anasuya Roychowdhury, Aditi Nayak, Sugandh Kumar, Anshuman Dixit, Asima Bhattacharyya. Hypoxia-responsive and HIF1α-regulated AAA+ ATPase ATAD2 shows high oncogenic potential in stomach cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5680.

MicroRNA-217 modulates pancreatic cancer progression via targeting ATAD2

AimsPancreatic cancer is a fatal disease across the world with 5 years survival rate less than 10%. ATAD2, a valid cancer drug-target, is overexpressed in pancreatic malignancy with high oncogenic potential. However, the mechanism of the upregulated expression of ATAD2 in pancreatic cancer is unknown. Since microRNAs (miRNAs) could potentially control target mRNA expressions, and are involved in cancer as tumor-suppressors, oncomiR or both, we examine the possibility of miRNA-mediated regulation of ATAD2 in pancreatic cancer cells (PCCs). Main methodsOur in-silico approach first identifies hsa-miR-217 as a candidate regulator for ATAD2 expression. For further validation, luciferase reporter assay is performed. We overexpress hsa-miRNA-217 and assess cellular viability, migration, apoptosis and cell cycle progression in three different PCCs (BxPC3, PANC1, and MiaPaCa2). Key findingsWe find hsa-miRNA-217 has potential binding site at the 3′UTR of ATAD2. Luciferase assay confirms that ATAD2 is a direct target of hsa-miR-217. Overexpression of hsa-miR-217 drastically downregulates ATAD2 expression in PCCs, thus, corroborating binding studies. The elevated expression of hsa-miRNA-217 diminishes cell proliferation and migration as well as induces apoptosis and cell cycle arrest in PCCs. Finally, siRNA mediated ATAD2 knockdown or overexpression of hsa-miRNA-217 in PCCs showed inactivation of the AKT signaling pathway. Therefore, hsa-miR-217 abrogates pancreatic cancer progression through inactivation of the AKT signaling pathway and this might be partly due to miR-217 mediated suppression of ATAD2 expression. SignificanceThe application of hsa-miR-217 mimic could be a promising therapeutic strategy for the treatment of pancreatic cancer patients in near future.

Oncogenic potential of ATAD2 in stomach cancer and insights into the protein-protein interactions at its AAA + ATPase domain and bromodomain

ATAD2 has recently been shown to promote stomach cancer. However, nothing is known about the functional network of ATAD2 in stomach carcinogenesis. This study illustrates the oncogenic potential of ATAD2 and the participation of its ATPase and bromodomain in stomach malignancy. Expression of ATAD2 in stomach cancer is analyzed by in silico and in vitro techniques including western blot and immunofluorescence microscopy of stomach cancer cells (SCCs) and tissues. The oncogenic potential of ATAD2 is examined thoroughly using genetic alterations, driver gene prediction, survival analysis, identification of interacting partners, and analysis of canonical pathways. To understand the protein-protein interactions (PPI) at residue level, molecular docking and molecular dynamics simulations (1200 ns) are performed. Enhanced expression of ATAD2 is observed in H. pylori-infected SCCs, patient biopsy tissues, and all stages and grades of stomach cancer. High expression of ATAD2 is found to be negatively correlated with the survival of stomach cancer patients. ATAD2 is a cancer driver gene with 37 mutational sites and a predictable factor for stomach cancer prognosis with high accuracy. The top canonical pathways of ATAD2 indicate its participation in stomach malignancy. The ATAD2-PPI in stomach cancer identify top-ranked partners; ESR1, SUMO2, SPTN2, and MYC show preference for the bromodomain whereas NCOA3 and HDA11 have preference for the ATPase domain of ATAD2. The oncogenic characterization of ATAD2 provides strong evidence to consider ATAD2 as a stomach cancer biomarker. These studies offer an insight for the first time into the ATAD2-PPI interface presenting a novel target for cancer therapeutics. Communicated by Ramaswamy H. Sarma

HIF1α-dependent upregulation of ATAD2 promotes proliferation and migration of stomach cancer cells in response to hypoxia

Stomach cancer is a difficult-to-treat disease. Lack of detection markers and limited understanding of the disease mechanisms contribute to the aggressive nature of stomach cancer cells (SCCs). Recently, an ATPase, ATAD2 has been found to be highly expressed in stomach cancer contributing to increased malignancy. However, nothing is known about the mechanism of ATAD2 upregulation and its involvement in stomach carcinogenesis. Since hypoxic microenvironment plays a crucial role in the progression of solid tumors like stomach cancer; we have examined the regulation and function of ATAD2 expression in hypoxic SCCs. ATAD2 is induced in hypoxia-treated SCCs. Stomach adenocarcinoma and metastatic tissues with high HIF1α level also show enhanced ATAD2 expression. In the absence of hypoxia-inducible factor HIF1α, ATAD2 protein level is found to be less indicating towards a potential correlation between them. We identify the presence of HIF1α-binding site (HBS) and HIF1α ancillary site (HAS) in the ATAD2 promoter. Using both in vitro and in vivo binding studies, we confirm that HIF1α binds with the ATAD2 promoter in hypoxic condition. ATAD2 upregulation promotes proliferation and migration of SCCs exposed to hypoxia. Thus, we identify ATAD2 as a hypoxia-responsive and HIF1α-regulated gene and elucidate that upregulated expression of ATAD2 enhances tumor-promoting functions in hypoxic SCCs. Therefore, we propose ATAD2 as a promising therapeutic target for stomach cancer.

Knockdown of ATAD2 Inhibits Proliferation and Tumorigenicity Through the Rb-E2F1 Pathway and Serves as a Novel Prognostic Indicator in Gastric Cancer.

IntroductionThe aim of the present study was to examine the expression of ATAD2 in gastric cancer (GC) specimens and to evaluate its correlation with clinicopathologic features, including survival of GC patients. The potential roles of ATAD2 in the GC cell proliferation, apoptosis, and tumour growth were further explored.Materials and MethodsQuantitative reverse transcription-polymerase chain reaction (qRT-PCR), Western blotting and immunohistochemistry (IHC) were applied to determine the mRNA and protein expression of ATAD2 in GC and corresponding adjacent non-tumourous specimens. The relationship between ATAD2 expression and clinicopathological features of GC patients was analysed. Kaplan-Meier analysis was performed to assess the prognostic value of ATAD2 expression levels. The proliferation, colony formation, apoptosis and tumorigenesis roles of ATAD2 were measured using in vitro and in vivo experiments.ResultsThe expression of ATAD2 mRNA and protein was overexpressed in GC tissues compared with corresponding adjacent non-tumourous tissues. ATAD2 expression was significantly correlated with tumour size, tumour differentiation, and clinical tumour-node-metastasis (TNM) stage. Patients with high ATAD2 expression were likely to experience significantly shorter postoperative overall survival (OS) and disease-free survival (DFS). Multivariate Cox analysis suggested ATAD2 as an independent variable for OS and DFS. Knockdown of ATAD2 significantly suppressed cell proliferation, colony formation in vitro and tumorigenicity in vivo. Cell cycle and apoptotic assays showed that the anti-proliferative effect of pLV-ATAD2 shRNA was mediated by arresting cells in the G1 phase and inducing cell apoptosis. Silencing of ATAD2 reduced the expression of cyclinD1, ppRb, E2F1 and cyclinE and upregulated the expression of cleaved-PARP and cleaved-Caspase 3.ConclusionOur study indicated that ATAD2 plays an important role in the process of tumorigenesis and progression in GC, and it could serve as a novel prognostic biomarker and a therapeutic target for the treatment of GC patients.

ATAD2 predicts poor outcomes in patients with ovarian cancer and is a marker of proliferation.

The oncogene ATPase family AAA domain-containing protein 2 (ATAD2) has been demonstrated to promote malignancy in a number of different types of tumor; however, its expression and role in ovarian cancer (OC) remain unknown. In the present study, it was demonstrated that ATAD2 acts as both a marker and a driver of cell proliferation in OC. Immunohistochemistry (IHC) and bioinformatics analyses were used to evaluate ATAD2 expression in OC, and multi-omics integrated analyses were used to dissect which factor resulted in its upregulation. Multiplex IHC assay was used to reveal the specific expression of ATAD2 in proliferating OC cells. CRISPR-Cas9-mediated gene editing was performed to investigate the effect of ATAD2 deletion on OC proliferation. The results demonstrated that ATAD2 is elevated in primary OC tissues compared with the adjacent normal tissue and metastases from the stomach. Genetic copy number amplification is a primary cause resulting in upregulation of ATAD2, and this was most frequently observed in OC. High ATAD2 expression was associated with advanced progression and predicted an unfavorable prognosis. ATAD2 could be used to identify cases of OC with a high proliferation signature and could label proliferating cells in OC. CRISPR-Cas9-mediated ATAD2 deletion resulted in a significant decrease in both cell proliferation and colony formation ability. Mechanistically, ATAD2-knockdown resulted in deactivation of the mitogen-activated protein kinase (MAPK) pathways, particularly the JNK-MAPK pathway, resulting in suppression of proliferation. Collectively, the data from the present study demonstrated that the ATD2 gene was frequently amplified and protein expression levels were upregulated in OC. Therefore, ATAD2 may serve as an attractive diagnostic and prognostic OC marker, which may be used to identify patients with primary OC, whom are most likely to benefit from ATAD2 gene-targeted proliferation intervention therapies.

Correlations between DHT/AR and ATAD2 expression in hepatocellular carcinoma

Objective To investigate the expression of androgen receptor(AR), ATAD2 in hepatocellular carcinoma(HCC) and the correlations with clinicopathological features, and the role of DHT/AR and ATAD2 in proliferation of HCC cells. Methods The samples of 75 patients with HCC in the First Affiliated Hospital of China Medical University from February 2012 to December 2012 were collected. LM3 and Huh7 cells were divided into control group, DHT group, DHT + CDX (bicalutamide) group and CDX group; and also divided into Ri-ATAD2 group (adding interference fragments) and Ri-C group (adding control vector sequence). Immunohistochemistry was used to detect the expression of AR and ATAD2, and to analyze the correlations between clinical features and survival of patients. Real-time PCR and Western Blot were used to detect the expression of AR and ATAD2, and CCK-8 was used to detect cell proliferation. Results HCC patient samples were grouped according to AR and ATAD2 expression. Compared with low AR expression group (n=31), the ratio of tumor <5 cm in high expression group (n=44) was higher, and the ratio of TNM stage I + II was lower. Compared with low ATAD2 expression group (n=35), the ratio of metastasis and tumor differentiation grade III + IV was higher in high expression group (n=40), and the difference was statistically significant (P<0.05). The overall survival rate of patients with high expression of ATAD2 was lower than other patients, and the differences were statistically significant (P<0.05). Multivariate Cox regression analysis showed that ATAD2 expression (HR=1.935, 95% CI: 1.066~3.515) and metastasis (HR=2.212, 95% CI: 1.059~4.619) were independent predictors of poor prognosis. Compared with LO2 cells, the mRNA and protein level of AR and ATAD2 in LM3 and Huh7 cells were significantly higher, and the differences were statistically significant (P<0.05). And the proliferation rate of HCC cells increased significantly after 48 and 72 hours compared with the control group, and the differences were statistically significant (P<0.05). After adding CDX, the proliferation of LM3 and Huh7 induced by DHT was inhibited. DHT enhanced the expression of ATAD2, while CDX inhibited the expression of ATAD2. The expression of ATAD2 protein decreased when LM3 and Huh7 cells were interfered. Compared with Ri-C group, the proliferation of HCC cells in Ri-ATAD2 group decreased significantly after the DHT treatment 48 and 72 hours, and the difference was statistically significant (P<0.05). Conclusions DHT/AR promoted the proliferation of HCC cells by inducing ATAD2 expression. Modulating ATAD2 expression may be the potential mechanism of DHT/AR in HCC proliferation. Key words: Dihydrotestosterone; Androgen recceptor; ATPase family AAA domain-containing protein 2; Hepatocellular carcinoma; Proliferation

Abstract 5084: Potent and isoform-selective ATAD2 bromodomain inhibitor with unprecedented chemical structure and mode of action

Abstract ATAD2 (ATPase family AAA-domain containing protein 2, also called ANCCA) is an epigenetic regulator that binds to chromatin through its bromodomain (BD), a motif specialized for acetyl-lysine recognition. ATAD2 directly associates with multiple transcription factors such as ERα, AR, E2F, and Myc; hence, ATAD2 has been proposed to act as a co-factor for oncogenic transcription factors. Furthermore, we have recently reported a novel role for ATAD2 during DNA replication, uncovering interactions between ATAD2 and histone acetylation marks on newly synthesized histone H4. High expression of ATAD2 strongly correlates with poor patient prognosis in multiple tumor types, including gastric, endometrial, hepatocellular, ovarian, breast and lung cancers. However, the exact function of ATAD2 in these tumor types remains unclear. A more thorough validation of ATAD2 as a therapeutic target is hampered by the lack of isoform-selective, potent and cellularly active ATAD2 inhibitors. A systematic assessment of crystal structures of BD-containing protein family predicted that development of selective inhibitors of ATAD2 would be challenging. In line with this prediction, only limited progress in developing lead compounds targeting ATAD2 has been reported so far. A few notable exceptions relied on fragments as starting points, however, their weak potency, insufficient selectivity against other BDs, permeability limitations or modest cellular activity have curbed their further development towards drug candidates. Here we embarked on a novel strategy to identify ATAD2 inhibitors: 11 different DNA-encoded libraries adding up to 67 billion unique encoded compounds were combined and incubated with ATAD2 BD followed by two rounds of affinity-mediated selection. This approach provided with several series of binders, for which specific target engagement of their SMOL moiety upon off-DNA synthesis was confirmed in biochemical and biophysical assays. Several rounds of potency optimization led to the identification of BAY-850, a highly potent and ATAD2 (isoform A) mono-selective inhibitor, which holds an amine substituted 3-(2-furyl)benzamide core. This compound shows - as revealed by size exclusion chromatography and native mass spectrometry - a novel mode of action for a BD inhibitor based on specific target dimerization. In a cellular fluorescence recovery after photobleaching (FRAP) assay BAY-850 displaced wild-type ATAD2 from the chromatin to the same extent as the genetic mutagenesis of ATAD2 BD. In contrast, chemically very similar inactive control compounds showed no major effects on ATAD2 association with the chromatin. These results qualify BAY-850 as the first biologically active ATAD2 isoform A-specific chemical probe, which will enable further elucidation of the cancer biology of this intriguing protein. Citation Format: Amaury E. Fernández-Montalván, Markus Berger, Benno Kuropka, Seong Joo Koo, Volker Badock, Joerg Weiske, Simon J. Holton, Apirat Chaikuad, Laura Díaz-Sáez, James Bennett, Oleg Federov, Kilian Huber, Paolo Centrella, Matthew A. Clark, Christoph E. Dumelin, Eric A. Sigel, Holly S. Soutter, Dawn M. Troast, Ying Zhang, John W. Cuozzo, Anthony D. Keefe, Didier Roche, Vincent Rodeschini, Jan Hübner, Hilmar Weinmann, Ingo V. Hartung, Matyas Gorjanacz. Potent and isoform-selective ATAD2 bromodomain inhibitor with unprecedented chemical structure and mode of action [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5084. doi:10.1158/1538-7445.AM2017-5084

Expression of ATAD2 in different liver lesions and its clinical significance

Objective: To examine the expression of ATAD2 in different liver lesions and its clinical significance. Methods: ATAD2 expression in 60 hepatocellular carcinoma (HCC) surgical specimens (49 of which have concurrent liver cirrhosis), 43 HCC biopsy specimens, 2 high-grade liver dysplastic nodule specimens, 3 low-grade liver dysplastic nodule specimens, 50 liver cirrhosis tissue samples, and 20 normal liver tissue samples were measured using immunohistochemistry. The F-test, q-test, t-test, and chi-square test were used for statistical analysis of data. Results: ATAD2 was expressed in 56 HCC surgical specimens (93.33%), 35 HCC biopsy specimens (81.40%), and 2 high-grade liver dysplastic nodule specimens (2/2), but not in the low-grade liver dysplastic nodule, liver cirrhosis tissue, and normal liver tissue samples. The mean expression of ATAD2 was significantly higher in HCC tissues than in high-grade and low-grade liver dysplastic nodule tissues, liver cirrhosis tissue, and normal liver tissue (F = 22.96, q = 3.138, 3.972, 12.272, and 9.101, respectively, all P < 0.01). There were no significant differences in the mean expression and positive expression rate of ATAD2 between HCC surgical and biopsy specimens (t = 1.40, P > 0.05; χ² = 3.47, P >0.05). Of the 35 HCC biopsy specimens that expressed ATAD2, the mean ATAD2 expression was ≥1% in 35 specimens (100%), ≥3% in 27 specimens (77.14%), and ≥5 % in 23 specimens (65.71%). In addition, among the pathological grade I-II HCC biopsy specimens, the mean ATAD2 expression was ≥1% in 28 specimens (100%), ≥3% in 22 specimens (62.86%), and ≥5% in 19 specimens (54.29%). Moreover, ATAD2 expression in HCC was associated with serum alpha-fetoprotein level, presence of hepatitis B virus surface antigen (HBsAg), and presence of concurrent liver cirrhosis (t = 2.09, 2.30, and 2.18, respectively, all P < 0.05). Conclusion: ATAD2 may play an important role in HCC tumorigenesis, and may be involved in malignant transformation of cells. ATAD2 expression can be a valuable marker for differentiating the nature of lesions in liver biopsy tissues during clinical practice.

The role of miR-372 in ovarian carcinoma cell proliferation

ObjectivesMicroRNA-372 has been shown to be associated with multiple tumors' development and progression, by regulating the expression of proteins involved in cell cycle and apoptosis. However, the specific mechanism and function of miR-372 in ovarian carcinoma are not clear. Our study explored the role of miR-372 in ovarian carcinoma cell cycle and proliferation. Materials and methodsMiR-372 expression was quantified in normal ovarian tissue, benign tumors, primary ovarian carcinomas and metastatic omentum by qRT-PCR. MTT assay and plate clone formation assay were performed to evaluate the cell viability and proliferation. EDU assay and cell apoptosis assay were also used to determine cell growth. We used Western Blot to analysis expression of the known miR-372 targets. ResultsWe found that miR-372 expression was significantly lower in ovarian carcinoma than normal ovarian tissues and benign tumors. Moreover, miR-372 overexpression showed significant inhibition of cell proliferation and promoted cell apoptosis. Western Blot revealed that miR-372 downregulated the expression of ATAD2, LATS2, P62, DKK1 and cyclinA1 to inhibit the proliferation of cells. ConclusionsOur findings indicate that miR-372 has a prominent role in inhibiting tumor growth and it is a valuable target for ovarian cancer therapy.

Long noncoding RNA PCAT-14 induces proliferation and invasion by hepatocellular carcinoma cells by inducing methylation of miR-372.

Long non-coding RNAs (lncRNAs) regulate oncogenesis by inducing methylation of CpG islands to silence target genes. Here we show that the lncRNA PCAT-14 is overexpressed in patients with hepatocellular carcinoma (HCC), and is associated with a poor prognosis after surgery. Our results demonstrate that PCAT-14 promotes proliferation, invasion, and cell cycle arrest in HCC cells. In addition, PCAT-14 inhibits miR-372 expression by inducing methylation of the miR-372 promoter. Simultaneously, miR-372 eliminates the effects of PCAT-14 on proliferation, invasion, and cell cycle in HCC cells. Moreover, PCAT-14 regulates expression of ATAD2 and activation of the Hedgehog pathway via miR-372. These findings indicate that PCAT-14 plays an important role in HCC, and may serve as a novel prognostic factor and therapeutic target.

Expression and correlation of ATAD2 and E-cadherin in hepatocellular carcinoma

Objective To investigate the correlation and clinicopathological features of ATAD2 and E-cadherin expression in human hepatocellular carcinoma (HCC), and role of ATAD2 in HCC cells invasion and metastases. Methods The expressions of ATAD2 and E-cadherin in 90 HCC specimens and 16 specimens collected from peritumoral tissue were detected by immunohistochemistry (IHC) test. The correlations between ATAD2 and E-cadherin expression and clinicopathological features were analyzed. The lentiviral vector of sh-ATAD2 RNA was used to interfere with ATAD2 expression. Western blot was performed to evaluate the changes of E-cadherin and Vimentin expression in HCC cells after ATAD2 expression interference. The role of ATAD2 in HCC cells invasion and metastasis was assessed by Transwell analysis. Results ATAD2 protein expression was obviously upregulated in HCC tissue compared to peritumoral tissue (56.7% vs. 31.3%, P<0.05). Meanwhile, E-cadherin expression was significantly downregulated in HCC tissue (43.3% vs. 68.7%, P<0.05). ATAD2 and E-cadherin expressions were both correlated with HCC metastasis (P<0.05). Correlation analysis demonstrated that the ATAD2 expression was negatively correlated with E-cadherin (r=-0.263, P<0.05). After interfering ATAD2 expression, cell migration and invasion of HCCLM3 cells were suppressed (migration, 42.5±2.6 vs. 78.1±3.8, P<0.05; invasion, 33.0±4.7 vs. 72.7±5.6, P<0.05). Meanwhile, the E-cadherin expression was remarkably upregulated (P<0.05). However, the Vimentin expression downregulated (P<0.05). Conclusions ATAD2 expression was upregulated in HCC, and silencing ATAD2 could upregulate E-cadherin expression and downregulate Vimentin expression, and inhibit the invasion and metastasis of HCC cells. Modulating E-cadherin expression may be the potential mechanism of ATAD2 in HCC invasion and metastasis. Key words: ATPase family, ATAD2; E-cadherin; Hepatocellular carcinoma; Invasion; Metastasis

Abstract 4539: ATAD2 mediates DNA replication in cancer

Abstract ATAD2 (ATPase family AAA domain-containing protein 2) is an epigenetic regulator which associates with chromatin through its Bromodomain specialized in Acetyl-Lys binding of histones. ATAD2 was also shown to directly associate with multiple transcription factors such as ERα, AR, E2F and MYC, and is believed to function as an oncogenic transcription factor in breast cancer. Here, we propose that ATAD2 facilitates DNA replication. ATAD2 is specifically expressed in S and G2 phase during which it co-localizes with newly synthesized DNA. We found ATAD2 on nascent chromatin together with newly synthesized histone H4 acetylated on K12 and Proliferating Cell Nuclear Antigen (PCNA), a central protein coupling replication with chromatin restoration, but not on post-replicative chromatin. In line with these observations depletion of ATAD2 by siRNA led to reduced DNA replication, perturbed loading of PCNA onto chromatin and inhibition of cell proliferation. Interestingly, a brief cycloheximide treatment of the cells to prevent the deposition of newly synthesized histones (e.g. H4K5,12diac) abrogated the recruitment of ATAD2 to nascent chromatin suggesting that ATAD2 might recognize and interact with these histone marks. Indeed, extensive biochemical and biophysical analyses involving TR-FRET, MST (MicroScale Thermophresis), Biocore, and NMR revealed that the bromodomain of ATAD2 preferentially interacts with these marks characteristic of newly synthesized histones. Consequently, overexpression of ATAD2 mutants unable to interact with these marks impaired DNA replication and recruitment of PCNA onto chromatin. Taken together, our data suggest that ATAD2 is essential for DNA replication and thus predicts that it is expressed in cells undergoing S phase. To further strengthen this hypothesis we compared the expression of ATAD2 with the proliferation marker Ki67, and the late S and G2/M marker TOP2A, in various cancer types such as colorectal, gastric, lung, prostate and breast cancers by immunohistochemistry. Indeed ATAD2 expression was restricted to Ki67 and TOP2A expressing areas of tumors, independent of cancer type. Moreover, aggressive tumors, such as triple negative breast cancer and metastatic castration-resistant prostate cancer, showed more intense and abundant expression of ATAD2 whereas slow-growing tumors showed low expression of ATAD2. This research identifies a role for ATAD2 in replication, providing mechanistic and translational support for therapeutic development in cancer. Citation Format: Seong Joo Koo, Amaury Ernesto Fernandez-Montalvan, Simon Holton, Oliver von Ahsen, Volker Badock, Sarah Vittori, Christopher J. Ott, James E. Bradner, Matyas Gorjanacz. ATAD2 mediates DNA replication in cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4539.

ATAD2 overexpression links to enrichment of B-MYB-translational signatures and development of aggressive endometrial carcinoma.

We have explored the potential for clinical implementation of ATAD2 as a biomarker for aggressive endometrial cancer by investigating to what extent immunohistochemical (IHC) staining for ATAD2 is feasible, reflects clinical phenotype and molecular subgroups of endometrial carcinomas. Increased expression of the ATAD2 gene has been implicated in cancer development and progression in a number of tissues, but few studies have investigated ATAD2 expression using IHC. Here we show that high ATAD2 protein expression is significantly associated with established clinical-pathological variables for aggressive endometrial cancer, also in the subset of estrogen receptor α (ERα) positive tumors. Protein and mRNA expression of ATAD2 were highly correlated (P < 0.001), suggesting that IHC staining may represent a more clinically applicable measure of ATAD2 level in routinely collected formalin fixed paraffin embedded specimens. Gene expression alterations in samples with high ATAD2 expression revealed upregulation of several cancer-related genes (B-MYB, CDCs, E2Fs) and gene sets that previously have been linked to aggressive disease and potential for new targeting therapies. Our results support that IHC staining for ATAD2 may be a clinically applicable biomarker reflecting clinical phenotype and targetable alterations in endometrial carcinomas to be further explored in controlled clinical trials.

Epigenetic high regulation of ATAD2 regulates the Hh pathway in human hepatocellular carcinoma.

ATAD2 is associated with many cellular progresses such as cell growth, differentiation and apoptosis. Some studies suggest ATAD2 is highly expressed in cancer cells. In our previous studies, we found that ATAD2 is highly expressed in HCC tissues, compared with adjacent normal tissues, and patients with high expression of ATAD2 had a poorer prognosis. Moreover, we found mir-372 can regulate the expression of ATAD2 in HCC cell lines. We also detected a relationship between the mRNA expression of ATAD2 and Ptch1 by gene microarray. Here, we completed the function studies of ATAD2 in vivo and in vitro, and tested whether ATAD2 could regulate the Hh pathway. ATAD2 and Hh pathway protein expressions in 80 HCC specimens were examined by immunohistochemistry (IHC). The mRNA expression of ATAD2 and Hh pathway members in paired-HCC tissues and cell lines were, respectively, analyzed using quantitative PCR. ATAD2‑RNAi was transduced into HCCLM3 and Huh7 cells, using a lentiviral vector. The effect of ATAD2 in HCC cell lines on cell cycle and apoptosis were evaluated by flow cytometry. Tumorigenicity experiments in nude mice were performed to test the function of ATAD2 in vivo. Pharmacological regulation of Hh signaling was performed to test the relation between the ATAD2 and Hh pathways and C-myc. We found that ATAD2 and Ptch1 were both highly expressed in HCC tissues, compared with paired normal hepatic tissues. In addition, we found that ATAD2 could affect the expression of the Hh pathway by PCR and western blot anaysis in HCC cell lines, by observing the outcome before and after transfection. We speculate that ATAD2 cooperates with the MYC gene to regulate the expression of SMO and Gli, activating the Hh pathway and inducing an active feedback of the Hh pathway.

ATAD2 is highly expressed in ovarian carcinomas and indicates poor prognosis.

The purpose of this study was to explore the expression of ATAD2 in ovarian tumor tissue as well as its relationship with degree of malignancy. Tumor tissue from 110 cases of ovarian cancer was collected in accordance with the Declaration of Helsinki for evaluation of ATAD2 expression immunohistochemistry, quantitative PCR (qPCR) and Western blotting. The correlation between the ATAD2 expression and and the prognosis of ovarian cancer was evaluated by Cox regression model. In addition, HO-8910 and OVCAR-3 cells were transfected with two siRNAs targeting ATAD2. Cell viability was evaluated with MTT assay, and cell migration by transwell migration assay. ATAD2 was shown to be highly expressed in 65.5% (72/110) of ovarian cancer cases, both at transcriptional and protein levels. Moreover, highly expression was positively correlated with degree of malignancy. Knock-down of ATAD2 in HO-8910 and OVCAR-3 cells was found to reduce cell migration. In addition, follow-up visits of the patients demonstrated that the 5-year survival rate was lower in patients with high expression of ATAD2. Our study suggested that ovarian tumor tissue may have highly expressed ATAD2, which is associated with tumor stage, omentum-metastasis, ascites and CA-125. Increased ATAD2 may play important roles in tumor proliferation and migration. ATAD2 could serve in particular as a prognostic marker and a therapeutic target for ovarian cancer.

MP29-03 TET3, HELLS, TOP2A AND ATAD2 ARE NOVEL INDEPENDENT PROGNOSTIC MARKERS IN ADVANCED RENAL CELL CARCINOMA

You have accessJournal of UrologyKidney Cancer: Basic Research II1 Apr 2014MP29-03 TET3, HELLS, TOP2A AND ATAD2 ARE NOVEL INDEPENDENT PROGNOSTIC MARKERS IN ADVANCED RENAL CELL CARCINOMA Dong Chen, Matthias Maruschke, Rainer Riesenberg, Wolfgang Zimmermann, Christian. G. Stief, and Alexander Buchner Dong ChenDong Chen More articles by this author , Matthias MaruschkeMatthias Maruschke More articles by this author , Rainer RiesenbergRainer Riesenberg More articles by this author , Wolfgang ZimmermannWolfgang Zimmermann More articles by this author , Christian. G. StiefChristian. G. Stief More articles by this author , and Alexander BuchnerAlexander Buchner More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.745AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES A significant proportion of patients with renal cell carcinoma (RCC) will have metastases at first diagnosis or develop metastases during the course of their disease. Individual risk stratification based on reliable prognostic markers is needed to allow a personalized therapeutic strategy. The aim of our study was to screen for promising prognostic markers using a novel filtering approach, based on genes that are subsequently up-regulated in primary tumors and metastases. Therefore, we created microarray expression data from RCC patients including normal kidney tissue, primary tumors and metastases. The best candidate genes were validated using RT-PCR. METHODS Tissue samples from normal renal tissue (n=14), primary tumor (14x G1, 14x G3) and metastases (n=32) were collected from patients with clear-cell RCC. Follow-up data were available from all patients. The tissue was snap-frozen immediately after surgery, and total RNA was isolated. Expression profiles were created using oligonucleotide microarrays (GeneChip HG U133 Plus 2.0, Affymetrix). For analysis of expression data, the dChip 2010 software was used. Gene expression was compared between normal kidney, primary tumor and metastases. Furthermore, a significance test was applied. In all filtered genes, univariate survival analysis was carried out (Kaplan-Meier method, log-rank test). Significant genes were further analyzed using multivariate Cox regression models. The best candidate markers were further validated in an independent cohort of 52 primary tumors using quantitative RT-PCR and immunohistochemistry. RESULTS Expression analysis revealed that in 59 probe sets there was increased expression in primary RCC compared with normal kidney, and also in metastases compared with primary tumors. In uni- or multivariate survival analysis, 15 of these probe sets were significant. Undefined genes (C1orf216 and Hs.133294.1) and genes with very low mRNA level in RT-PCR (NDC80, GOLSYN, DTL, and BUB1B) were excluded. Eight genes (TOP2A, SFN, CENPF, AMPD3, ATAD2, AURKA, HELLS and TET3) were afterwards validated by quantitative RT-PCR. Survival analysis in an independent cohort of 52 primary RCCs showed that TOP2A (HR=4.4, p=0.004), TET3 (HR=2.9, p=0.031), HELLS (HR=3.6, p=0.007), and ATAD2 (HR=3.8, p=0.014) represent independent predictors for RCC patients. TOP2A IHC results support the RT-PCR findings. CONCLUSIONS High expression of TET3, HELLS, TOP2A, and ATAD2 are independent predictors of poor outcome in RCC patients and may be used for individual risk-adapted therapy in the future. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e305 Advertisement Copyright & Permissions© 2014MetricsAuthor Information Dong Chen More articles by this author Matthias Maruschke More articles by this author Rainer Riesenberg More articles by this author Wolfgang Zimmermann More articles by this author Christian. G. Stief More articles by this author Alexander Buchner More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...