Sleeping Beauty Insertional Mutagenesis in Mice Identifies Drivers of Steatosis-Associated Hepatic Tumors.

Hepatic steatosis is a strong risk factor for the development of hepatocellular carcinoma (HCC), yet little is known about the molecular pathology associated with this factor. In this study, we performed a forward genetic screen using Sleeping Beauty (SB) transposon insertional mutagenesis in mice treated to induce hepatic steatosis and compared the results to human HCC data. In humans, we determined that steatosis increased the proportion of female HCC patients, a pattern also reflected in mice. Our genetic screen identified 203 candidate steatosis-associated HCC genes, many of which are altered in human HCC and are members of established HCC-driving signaling pathways. The protein kinase A/cyclic AMP signaling pathway was altered frequently in mouse and human steatosis-associated HCC. We found that activated PKA expression drove steatosis-specific liver tumorigenesis in a mouse model. Another candidate HCC driver, the N-acetyltransferase NAT10, which we found to be overexpressed in human steatosis-associated HCC and associated with decreased survival in human HCC, also drove liver tumorigenesis in a steatotic mouse model. This study identifies genes and pathways promoting HCC that may represent novel targets for prevention and treatment in the context of hepatic steatosis, an area of rapidly growing clinical significance. Cancer Res; 77(23); 6576-88. ©2017 AACR.

Different subcellular localizations and functions of human ARD1 variants.

ARD1 is present in various species and has several variants derived from alternative splicing of mRNA. Previously, we reported differential biological functions and cellular distributions of mouse ARD1 (mARD1) variants. However, in comparison to mARD1 variants, human ARD1 (hARD1) variants have been rarely studied. In this study, we characterized a hARD1 variant, hARD1(131) and investigated its cellular activities. hARD1(131) mRNA was isolated from HeLa cells and sequenced. Sequence alignment revealed that, compared to hARD1(235), the most common form of hARD1, the mRNA sequence encoding hARD1131 possesses an altered reading frame due to a 46-bp deletion. Thus, hARD1(131) and hARD1(235) differ in their C-terminal regions with a partially deleted acetyltransferase domain at the C-terminus of hARD1(131). Moreover, hARD1(131) and hARD1(235) showed different subcellular localizations and biological functions. hARD1(131) was mostly localized in the cell nucleus, whereas hARD1(235) was primarily localized in the cytoplasm. In addition, hARD1(235) stimulated cell proliferation by upregulation of cyclin D1, however hARD1(131) had no influence on cyclin D1 expression and cell growth. Because hARD1(235) enhances cell proliferation by its autoacetylation activity, we examined the autoacetylation activity of hARD1(131) and observed that this function was absent in hARD1(131). These results suggest that human ARD1 variants have different effects on cell prolifer-ation, which may result from distinct subcellular localizations and autoacetylation activities.

High expression of N-acetyltransferase 10: a novel independent prognostic marker of worse outcome in patients with hepatocellular carcinoma.

N-acetyltransferase 10 (NAT10) is a nucleolar protein involved in histone acetylation, telomerase activity regulation, DNA damage response and cytokinesis. The expression of NAT10 was found to be enhanced in several types of tumors, suggesting its correlation with tumor development. However, the specific role of NAT10 in hepatocellular carcinoma (HCC) is still unclear. The aim of this study was to investigate the expression of NAT10 in HCC patients and to assess the relationship of NAT10 expression with clinicopathological characteristics and tumor prognosis. We selected 17 pairs of HCC samples and adjacent non-neoplastic tissue for mRNA expression analysis. We also performed immunohistochemistry in 186 HCC samples to evaluate the NAT10 protein expression. Cox regression and Kaplan-Meier analysis was used to study the diagnostic and prognostic value of NAT10. The results showed that NAT10 expression was mainly localized in the nuclei/nucleoli and was significantly higher in HCC tissues than peritumoral tissues (P < 0.01). High NAT10 expression was positively correlated with histological differentiation (P < 0.01) and TNM classification (P < 0.01). Cox regression univariate and multivariable analysis revealed that expression of NAT10 in HCC was an independent prognostic factor for patient survival time. Our data suggested that NAT10 might be a promising prognostic marker and potential therapeutic target in HCC.

GSK-3ß-regulated N-acetyltransferase 10 is involved in colorectal cancer invasion.

PURPOSE: NAT10 (N-acetyltransferase 10) is a nucleolar protein, but may show subcellular redistribution in colorectal carcinoma. In this study, we evaluated membranous staining of NAT10 in colorectal carcinoma and its clinical implications, and explored the mechanism of regulation of NAT10 redistribution. EXPERIMENTAL DESIGN: The expression and subcellular redistribution of NAT10, ß-catenin, E-cadherin, and GSK-3ß were evaluated by immunohistochemistry in 222 cases of colorectal carcinoma. Regulation of NAT10 and its influence on cell motility were analyzed with inhibitors of GSK-3ß, transfection of wild-type or kinase-inactivated GSK-3ß, or expression of various domains of NAT10, and evaluated with immunofluorescence, Western blotting, and Transwell assays. RESULTS: NAT10 localized mainly in the nucleoli of normal tissues, and was redistributed to the membrane in cancer cells, particularly at the invasive "leading edge" of the tumor. This correlated well with nuclear accumulation of ß-catenin (P<0.001; χ2=68.213). In addition, NAT10 membrane staining reflected the depth of invasion and tendency to metastasize (all P values<0.001), and was associated with a poorer prognosis (P=0.023; χ2=5.161). Evaluation of the mechanism involved demonstrated that subcellular redistribution of NAT10 may result from its increased stability and nuclear export, which is brought about by inhibition of GSK-3ß. Moreover, redistribution of NAT10 induces alteration of cytoskeletal dynamics and increases cancer cell motility. CONCLUSION: The subcellular redistribution of NAT10 can be induced by decreases in GSK-3ß activity. This redistribution increases cancer cell motility, and is, thus, correlated with invasive potential and poorer clinical outcome. This finding suggests that NAT10 may be a useful prognostic marker and potential therapeutic target in colorectal carcinoma.

Inverse correlation between Naa10p and MMP-9 expression and the combined prognostic value in breast cancer patients.

To analyze the expression profiles of N-a-acetyltransferase 10 protein (Naa10p/ARD1) and matrix metalloproteinase 9 (MMP-9) in human breast cancer and evaluate their possible prognostic values in breast cancer patients. Quantitative RT-PCR was used to evaluate mRNA expression of Naa10p and MMP-9 in 55 cases of fresh breast cancer tissues, and immunohistochemistry was performed for detecting Naa10p and MMP-9 proteins on breast cancer with tissue microarray containing 80 specimens. Furthermore, Naa10p and MMP-9 were measured in 253 breast cancer tissues from patients with up to 15-year follow-up. Survival curves were generated using the Kaplan-Meier method. Multivariate analysis was performed by using the Cox proportional hazard regression model to assess the prognostic values of Naa10p and MMP-9. Both Naa10p and MMP-9 expression in breast cancer tissues were significantly higher than those in the matched non-cancerous tissues (p = 0.000 for both). There was an inverse correlation between Naa10p and MMP-9 expression at mRNA and protein levels (p = 0.000 for both). Patients with MMP-9- positive expression had a poorer overall survival (OS) and disease-free survival (DFS) than those with MMP-9-negative expression (p = 0.001 for both). However, patients with Naa10p-positive expression had better OS and DFS (p = 0.000 for both). In addition, Naa10p-positive/MMP-9- negative patients had the best OS and DFS (p = 0.000 for both). In multivariate survival analysis, TNM stage, Naa10p expression, MMP-9 expression, and combined expression status of Naa10p/MMP-9 were independent prognostic factors related to OS (p = 0.000, 0.007, 0.012, and 0.000, respectively) and DFS (p = 0.000, 0.002, 0.014, and 0.000, respectively).The expression level of Naa10p was inversely correlated with that of MMP-9 in human breast cancer samples. Combined analysis of Naa10p and MMP-9 had a significantly increased value for determining the prognosis of breast cancer patients.