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Purpose: Here, we aim to investigate the molecular mechanism of regorafenib and verify the potential druggable target for the treatment of hepatocellular carcinoma (HCC). Experimental Design: HCC cell lines (PLC5, HepG2, Hep3B, SK-Hep1, and HA59T) were used to investigate the in vitro effect of regorafenib. Phosphatase activity was analyzed in HCC cells and purified SHP-1 proteins. PLC5-bearing mice were used to test the therapeutic efficiency of 20 and 40 mg/kg/d treatment with regorafenib (n \ge 8 mice). The clinical relevance of STAT3 signaling was investigated with 142 tumor samples from different patients with HCC. Descriptive statistical analysis was used to compare the baseline characteristics of patients and the expression of p-STAT3. Results: Regorafenib inhibited STAT3-related signaling in a dose-dependent manner and was a more potent inhibitor of STAT3 than sorafenib. Regorafenib increased SHP-1 phosphatase activity in purified SHP-1 protein directly. N-SH2 domain deletion and D61A mutants mimicking open-form SHP-1 partially abolished regorafenib-induced STAT3 inhibition and apoptosis. Importantly, a higher level of expression of STAT3 was found in patients with advanced clinical stages (P = 0.009) and poorly differentiated tumors (P = 0.035). Conclusions: Regorafenib induced significant tumor inhibition by relieving the autoinhibited N-SH2 domain of SHP-1 directly and inhibiting p-STAT3 signals. STAT3 may be suitable as a prognostic marker of HCC development, and may be a druggable target for HCC-targeted therapy using regorafenib. Clin Cancer Res; 20(22); 5768–76. ©2014 AACR.

Piwi-like RNA-mediated gene silencing 4 (PIWIL4) is expressed in somatic tissues and is proposed to silence the transposition of repetitive elements like long interspersed nuclear elements (LINEs) through DNA methylation and maintain the genome stability. Through the knockout of PIWIL4 gene by CRISPR/Cas9 system in mesenchymal stem cells (MSCs), MDA-MB-231 (breast cancer cell line), HT29 (colon cancer cell line) and AGS (gastric cancer cell line), the piRNA biogenesis was decreased and so was the methylation within LINE1 target loci and global methylation. The recruitment of SUV39H1, a histone H3 methyl-transferase, into nucleus was blocked, leading to the reduction of silencing tri-methylation of histone H3 at lysine 9. Concurrently, the active H3K4me3 mark increased after knockout. Also, global CTCF bindings were distorted after the knockout of PIWIL4 especially at the bindings franking the piRNA clusters. This distortion implied the disorganization of genomic conformation which was validated by karyotyping. Through chromosomal G-banding, aneuploidy was observed which indicated the loss of genomic stability. The knockout of PIWIL4 also results in disorganized cytoskeleton, therefore, changed cell stiffness as measured by atomic force microscopy. Neural and hepatic induction of MSCs were also blocked by the knockout of PIWIL4. In correlation, tissue arrays detected abnormal PIWIL4 expression in gastric (n=45), colon (n=30) and breast cancers (n=30). In conclusion, we found that PIWIL4-piRNA interaction helps to maintain somatic DNA methylome, genome stability and stiffness in somatic stem cells. The loss of PIWIL4 expression might then a candidate for cancer biomarker. (Supported by: MOST-105-2314-B-371 -008, MOST-105-2320-B-194 -004, MOST Taiwan) Citation Format: Syuan-Cih Lin, Chia-Chen Chiu, Shou-Tung Chen, Yao-Li Chen, Ping-Yi Lin, Shu-Huei Hsiao, Yu-Wei Leu. Loss of PIWIL4 and piRNAs attenuate somatic methylome and genome stability [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 1398. doi:10.1158/1538-7445.AM2017-1398

DNA methylation is a dominant silencing epigenetic modification that is inheritable in somatic cells. The recruitment of DNA methylation was proven to be able to reshape the cell physiology and cause tumorigenesis. The reversible nature of the methylation modification also makes the DNA methylation a therapeutic target and the monitoring of the methylation/demethylation dynamics in vivo is thus important for the development of demethylation agents. A targeted DNA methylation method and a two-component reporter system were combined to monitor the increase and demolishment of DNA methylation in vivo. The two constructs of the reporter systems are: target gene promoter (like HIC1 and ENSA) linked with the Tet repressor gene; the other is the Tet operator linked with the reporter like EGFP (in cell), IFP and iRFP. Both constructs were co-transfected into cells like mesenchymal stem cells, MDA-MB-231 and HCT116, and stable clones with both constructs were isolated and validated. Targeted DNA methylation method was used to transfect the isolated cell and, as the increase of DNA methylation, the reporters expressed. The increased methylation was then quenched after the adding of demethylation agents. To test this system in vivo, cells with both constructs were injected into the immune-deficient mice and the tumors grew afterward. Targeted DNA methylation was performed in vivo and the increase of methylation/expression of reporter genes were observed by fluorescence-based tomography. This combined system is also effective to monitor the demethylation effects of the demethylation agents. (Supported by: NSC- 102-2320-B-194-003-MY3 and NSC-102-2314-B-371- 003-MY3, MOST Taiwan) Citation Format: Yao-Li Chen, Shou-Tung Chen, Chun-Chun Chung, Chia-Chen Hsu, Ping-Yi Lin, Wan-Ling Chuang, Yi-Ju Chu, Yu-Wei Leu, Shu-Huei Hsiao. In vivo visualization of the targeted DNA methylation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-153. doi:10.1158/1538-7445.AM2015-LB-153

Purpose. The aim of this study is to investigate the relationship between SHP-1 phosphatase activity and clinicopathological characteristics in hepatocellular carcinoma (HCC). Methods. Patients. Between July 2011 and August 2012, 86 patients who underwent surgical hepatectomy resection at Changhua Christian Hospital were enrolled retrospectively. This study was approved by the Institutional of Review Board. For each patient, the diagnosis of hepatocellular carcinoma was confirmed by pathology reports. The clinical data of all patients were obtained through chart review. The data included hepatitis B or C status and the levels of alpha-fetoprotein (AFP). Histological differentiation and pathological staging were studied regarding the 7th American Joint Committee on Cancer (AJCC) cancer staging manual. Further analysis with standard immunohistochemistry and tissue microarray, which used 86 paraffin-embedded paired specimens of HCC. Immunopositivity was assessed by two pathologists who were blinded to the clinical information. SHP-1 immunoreactivity was scored as negative (0), weak (1), moderate (2), and strong expression (3), respectively. A final immunohistochemical score was calculated by the percentages of stained cells and the intensities of the staining. Statistical Analysis. The patients were classified into two subgroups based on the expression of SHP-1. Categorical variables were compared using the χ2 test or Fisher exact test when appropriate. For continuous variables, Mann-Whitney U tests were used to examine the differences between two subgroups in terms of the clinicopathological characteristics of patients with HCC. P values less than 0.05 were considered to be statistically significant. All statistical analysis was performed with SPSS for Windows Version 16.0 (SPSS Inc; Chicago, IL, USA). Preliminary Results. A total of 86 patients were enrolled in this study. The mean age among patients was 63 years (SD, 11 years) and most of them were male (69.8%). Regarding the clinicopathological characteristics of the two subgroups based on their SHP-1 gene expression status. There were no statistical differences between the two subgroups regarding age, gender, tumor size, HbsAg, HCV and TNM stage. However, the variables of AFP levels and histological grade were found to be statistically significant. Conclusions. In the present study, we performed immunohistochemical staining of SHP-1 in a tissue array of 86 paired specimens from HCC patients. SHP-1 was found overexpressed in the tissues. Addtionally, there were no statistical differences in clinicopathological data except AFP levels and histological grade. However, the role of SHP-1 in tumorigenesis is still unknown. With this preliminary finding, we suggest that it is essential to conduct a further study to clarify the tumorigenesis pathways of SHP-1 and to follow up the prognostic outcomes among these HCC patients. Citation Format: Ping-Yi Lin, Yao-Li Chen, Pei-Yi Chu, Kuen-Feng Chen. Src homology region 2 domain-containing phosphatase-1 (SHP-1) is overexpressed in hepatocellular carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1799. doi:10.1158/1538-7445.AM2013-1799