Tsung-Hao Liu

Sorafenib Overcomes Trail Resistance of Hepatocellular Carcinoma Cells through the Inhibition of Stat3

Purpose: Recombinant tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) is a promising antitumor agent. However, many hepatocellular carcinoma (HCC) cells show resistance to TRAIL-induced apoptosis. Here, we report that sorafenib improves the antitumor effect of TRAIL-related agents in resistant HCC. Experimental Design: HCC cell lines (PLC5, Huh-7, Hep3B, and Sk-Hep1) were treated with sorafenib and/or TRAIL-related agents (TRAIL or LBY135) and analyzed in terms of apoptosis and signal transduction. In vivo efficacy was determined in nude mice with PLC5 xenografts. Results: Sorafenib, the only approved drug for HCC, sensitizes resistant HCC cells to an agonistic DR5 antibody (LBY135) and TRAIL-induced apoptosis in TRAIL-resistant HCC cells. We found that STAT3 played a significant role in mediating TRAIL sensitization. Our data showed that sorafenib downregulated phospho-STAT3 (pSTAT3) and subsequently reduced the expression levels of STAT3-related proteins (Mcl-1, survivin, and cyclin D1) in a dose- and time-dependent manner in TRAIL-treated HCC cells. Knockdown of STAT3 by RNA interference overcame apoptotic resistance to TRAIL in HCC cells, and ectopic expression of STAT3 in HCC cells abolished the TRAIL-sensitizing effect of sorafenib. Moreover, SHP-1 inhibitor reversed downregulation of pSTAT3 and apoptosis induced by sorafenib, and silencing of SHP-1 by RNA interference abolished the effects of sorafenib on pSTAT3. Notably, sorafenib increased SHP-1 activity in PLC5 cells. Finally, sorafenib plus LBY135 significantly suppressed PLC5 xenograft tumor growth. Conclusions: Sorafenib sensitizes resistant HCC cells to TRAIL-induced apoptosis at clinical achievable concentrations, and this effect is mediated via the inhibition of STAT3. Clin Cancer Res; 16(21); 5189–99. ©2010 AACR.

Direct comparisons of peripheral T-cell lymphoma with diffuse B-cell lymphoma of comparable histological grades--should peripheral T-cell lymphoma be considered separately?

Peripheral T-cell lymphoma (PTCL) forms a morphologically heterogeneous group of non-Hodgkins lymphomas (NHL) with distinct immunophenotypes of mature T cells. Progress has been slow in defining specific clinicopathological entities to this particular group of NHL. In order to elucidate the specific characteristics of PTCL, a direct comparison of PTCL with a group of diffuse B-cell lymphomas (DBCL) was performed. Between June 1983 and December 1987, we studied 114 adults with NHL, using a battery of immunophenotyping markers. Adult T-cell leukemia/lymphoma, lymphoblastic lymphoma, mycosis fungoides/Sézary syndrome, follicular lymphoma, well-differentiated lymphocytic lymphoma, and true histiocytic lymphoma were excluded from this study since these are distinct clinicopathologic entities with well-recognized immunophenotypes. Of the remaining 75 patients, 70 who had adequate clinical information were analyzed, and of these, 34 were PTCL and 36 were DBCL. Classified according to the National Cancer Institute (NCI) Working Formulation (WF), 68% of PTCL and 31% of DBCL were high-grade lymphomas. Clinical and laboratory features were similar, except PTCL had a characteristic skin involvement and tended to present in more advanced stages with more constitutional symptoms. Induction chemotherapy was homogeneous in both groups, and complete remission rates were 62% for PTCL and 67% for DBCL. Patients with DBCL had a better overall survival than patients with PTCL, but the survival benefit disappeared after patients were stratified according to intermediate- or high-grade lymphoma. A subgroup of PTCL patients who had received less intensive induction chemotherapy was found to have a very unfavorable outcome. We conclude that (1) PTCL follows the general grading concept proposed in WF classification; (2) within a given intermediate or high grade, PTCL and DBCL respond comparably to treatment; (3) the intensity of induction chemotherapy has a crucial impact on the outcome of PTCL patients; and (4) with a few exceptions, the clinical and laboratory features of PTCL and DBCL are comparable.

Synergistic interactions between sorafenib and bortezomib in hepatocellular carcinoma involve PP2A-dependent Akt inactivation

BACKGROUND & AIMS Previously we reported that Akt inactivation determines the sensitivity of hepatocellular carcinoma (HCC) cells to bortezomib. Here we report that combined treatment with sorafenib and bortezomib shows synergistic effects in HCC. METHODS HCC cell lines (PLC/PRF/5, Huh-7, and Hep3B) were treated with sorafenib and/or bortezomib and analyzed in terms of apoptosis signal transduction. In vivo efficacy was determined in nude mice with PLC/PRF/5 xenografts. RESULTS Pretreatment with sorafenib enhanced bortezomib-induced apoptotic cell death by restoring bortezomibs ability to inactivate Akt in PLC/PRF/5 cells. Knocking down Akt1 by RNA-interference overcame apoptotic resistance to bortezomib in PLC/PRF/5 cells and ectopic expression of active Akt in HCC cells abolished the bortezomib sensitizing effect of sorafenib, indicating Akt inactivation plays a key role in mediating the combinational effects. Moreover, okadaic acid, a protein phosphatase 2A (PP2A) inhibitor, reversed down-regulation of phospho-Akt (P-Akt) expression induced by co-treatment with sorafenib and bortezomib, and 1, 9 di-deoxy-forskolin, a PP2A agonist, restored bortezomibs effect on P-Akt and apoptosis. Importantly, silencing of PP2A by RNA-interference reduced the apoptotic effect induced by sorafenib-bortezomib co-treatment, indicating that PP2A is indispensable for mediating the effects of these drugs. Notably, sorafenib with bortezomib increased PP2A activity in PLC/PRF/5 cells without altering protein levels of PP2A complex or the interaction between PP2A and Akt proteins. Finally, sorafenib plus bortezomib significantly suppressed PLC/PRF/5 xenograft tumor growth, down-regulated P-Akt expression, and up-regulated PP2A activity. CONCLUSIONS The combination of sorafenib and bortezomib shows synergy in HCC through PP2A-dependent Akt inactivation.

Modified CLIP with objective liver reserve assessment retains prognosis prediction for patients with advanced hepatocellular carcinoma.

The Cancer of the Liver Italian Program (CLIP) score is a commonly used staging system for hepatocellular carcinoma (HCC) helpful with predicting prognosis of advanced HCC. CLIP uses the Child‐Turcotte‐Pugh (CTP) score to evaluate liver reserve. A new scoring system, the albumin–bilirubin (ALBI) grade, has been proposed as they objectively evaluate liver reserve. We examined whether the modification of CLIP with ALBI retained its prognosis prediction for patients with advanced HCC.

Successful Hepatic Arterial Infusion of Chemotherapy in a Patient with Advanced Hepatocellular Carcinoma and Impending Liver Failure

Dear Editor, We read with great interest the study of Moriguchi et al. [1] regarding the efficacy of hepatic arterial infusion of chemotherapy (HAIC) in treating advanced hepatocellular carcinoma (HCC) with portal vein tumor thrombosis (PVTT). HAIC has been proposed to be beneficial for patients with advanced HCC owing to a numerically higher response rate than with sorafenib. We would like to share a unique case of a patient with advanced HCC and Child-Pugh (CP) class C liver reserve to endorse the findings of Moriguchi et al. [1]. A 59-year-old male with no prior history of systemic disease, including chronic viral hepatitis or alcoholic liver disease, was found to have an infiltrative mass occupying the majority of the right liver, along with main PVTT and dilated left intrahepatic bile ducts (Fig. 1A, B) identified using computed tomography (CT). Laboratory examination revealed severe hyperbilirubinemia (total/direct bilirubin 36.2/23.6 mg/ dL) and hypoalbuminemia (2.2 g/dL), prothrombin time was 13.3 s (international normalized ratio of 1.26), and the alpha-fetoprotein level was 712.9 ng/mL. Liver reserve was determined to be CP class C. A biopsy of the liver tumor confirmed the diagnosis of HCC. Percutaneous transhepatic cholangiography revealed obstructed left intrahepatic bile ducts for which a biliary drainage tube was inserted. The total bilirubin level decreased to 17.6 mg/dL but failed to decrease further despite adequate drainage. Because of the patient’s poor liver reserve, sorafenib was not recommended. The patient consented to receive HAIC and was given 500 mg/m2/day of 5-fluorouracil on days 1–3 and 60 mg/m2 of cisplatin on day 2, which was repeated every 3 weeks [2]. The total bilirubin level decreased to 5.3 mg/dL before the second HAIC cycle and then further improved to 1.5 mg/dL before the third cycle (Fig. 2). The alpha-fetoprotein level also decreased to 183.4 ng/mL. After 3 cycles of HAIC, a CT scan showed right liver tumor shrinkage and resolution of the main and left PVTT (Fig. 1C, D). Percutaneous transhepatic cholangiogPublished online: April 20, 2018

Increased Expression of Programmed Death-Ligand 1 in Infiltrating Immune Cells in Hepatocellular Carcinoma Tissues after Sorafenib Treatment

Objective: Programmed death-ligand 1 (PD-L1) expression in the tumor microenvironment (TME) has been reported to be related to prognosis in patients with hepatocellular carcinoma (HCC) after hepatectomy. The impact of sorafenib on PD-L1 expression in the TME of advanced HCC is unclear. Patients and Methods: Patients with HCC who received sorafenib for advanced disease at National Taiwan University Hospital, Taipei, Taiwan, and who had paired HCC tissues obtained before and after sorafenib treatment were included in the study group. HCC patients not treated with sorafenib who had paired primary and recurrent or metastatic tissues were identified as the reference group. The membrane PD-L1 staining, detected by immunohistochemistry (IHC) using SP142 antibody, was semiquantitatively scored in tumor cells (TCs) or tumor-infiltrating immune cells (ICs). Additional IHC assays were employed to characterize the PD-L1-expressing ICs. Results: Twenty-three advanced HCC patients with pre- and post-sorafenib paired HCC tissues were included in the study group. The median duration of sorafenib treatment was 4.3 months (range: 1.3–18.7). PD-L1 expression in ICs was significantly higher in post-sorafenib HCC tissues than in pre-sorafenib HCC tissues (pre-sorafenib vs. post-sorafenib IHC 0/1/2/3: 11/5/5/2 vs. 5/5/2/11, p = 0.016). However, PD-L1 expression in TCs was not significantly different between pre- and post-sorafenib tissues (IHC 0/1/2/3: 19/2/0/2 vs. 14/5/0/4, p = 0.094). In the reference group of 44 patients not treated with sorafenib, PD-L1 expression in ICs and TCs was not significantly different between the paired primary and metastatic HCC tissues. By performing IHC double staining with PD-L1 and CD68, we found the PD-L1-expressing ICs were mainly CD68-positive macrophages. PD-L1 expression levels of pre- and post-sorafenib tissues were not associated with patients’ overall survival or duration of sorafenib treatment. Conclusions: PD-L1 expression in ICs was significantly increased in post-sorafenib HCC tissues. The mechanisms and clinical significance of this observation warrants further investigation.

Abstract 1636: Increased expression of programmed death-ligand 1 (PD-L1) on infiltrating immune cells of hepatocellular carcinoma (HCC) tissues after sorafenib treatment

Background: PD-L1 expression in tumor microenvironment of HCC was reported to associate with tumor aggressiveness and recurrence. The impact of sorafenib treatment on PD-L1 expression on tumor cells (TC) or tumor-infiltrating immune cells (IC) of HCC has been unclear. Patients and Methods: We reviewed patients with HCC who had received sorafenib for advanced diseases at National Taiwan University Hospital, Taipei, Taiwan. Patients with paired HCC tissues, obtained before and after sorafenib treatment, were included. Immunohistochemistry (IHC) assay with clone SP142 antibody (Spring Bioscience, Pleasanton, CA, USA) was performed to analyze PD-L1 expression on TC and IC in paired specimens obtained before and after sorafenib. PD-L1 expression was scored as IHC 0, 1, 2, or 3 if Results: Twenty-three advanced HCC patients (Male: Female= 20: 3, median age of 64 years) with paired HCC tissues were included. All of the post-sorafenib HCC tissues were obtained after disease progression. The median duration of sorafenib treatment was 4.3 months (range: 1.3 to 18.7). The PD-L1 expression on IC was significantly increased in post-sorafenib HCC tissues, compared with tissues obtained before sorafenib (pre-sorafenib vs post-sorafenib IHC 0/1/2/3: 10/5/5/3 vs 5/5/2/11, p=0.046). However, the PD-L1 expression on TC was not significantly different between pre- and post-sorafenib tissues (IHC 0/1/2/3: 19/2/0/2 vs 14/5/0/4, p=0.065). By using IHC staining of CD68, CD66b, CD11b, and CD3 on the consecutive slides, we found the PD-L1-expressing IC were mainly CD68-positive macrophages, but not neutrophils or T lymphocytes. Neither the PD-L1 expression levels on IC of pre-sorafenib or those of post-sorafenib tissues were associated with OS or duration of sorafenib treatment. Conclusions: The PD-L1 expression on IC, especially on macrophages, in tumor microenvironment was significantly increased in post-sorafenib progression HCC tissues. Whether this increased PD-L1 expression contributes to treatment failure of sorafenib warrants further investigation (This work was supported by the grants of NTUH 105-M3232 and MOST 105-2314-B-002-180). Citation Format: Li-Chun Lu, Yi-Hsuan Lee, Chun-Jung Chang, Yu-Yun Shao, Tsung-hao Liu, Ann-Lii Cheng, Chih-Hung Hsu. Increased expression of programmed death-ligand 1 (PD-L1) on infiltrating immune cells of hepatocellular carcinoma (HCC) tissues after sorafenib treatment [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 1636. doi:10.1158/1538-7445.AM2017-1636

Abstract 355: CIP2A mediates effects of bortezomib on phospho-Akt and apoptosis in hepatocellular carcinoma cells

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Previously, we reported that Akt inactivation determines the sensitivity of hepatocellular carcinoma (HCC) cells to bortezomib (Chen et al, Cancer Res 2008). Here, we report that cancerous inhibitor of protein phosphatase 2A (CIP2A), a cellular inhibitor of protein phosphatase 2A (PP2A), mediates the apoptotic effect of bortezomib in HCC. Silencing PP2A by small interference RNA (siRNA) abolishes bortezomib-induced down-regulation of phospho-Akt and apoptosis. Bortezomib increases PP2A activity in sensitive HCC cells, including Sk-Hep1, Hep3B and Huh-7 but not in resistant PLC5 cells. Bortezomib down-regulates CIP2A in a dose- and time-dependent manner in all sensitive HCC cells whereas no alterations in CIP2A were found in resistant PLC5 cells. Knockdown of CIP2A by siRNA restored bortezomibs effects on apoptosis and PP2A activity in PLC5 cells. Moreover, over-expression of CIP2A up-regulated phospho-Akt and protected Sk-Hep1 cells from bortezomib-induced apoptosis. Importantly, ectopic expression of CIP2A decreased Akt-related PP2A activity whereas silencing CIP2A increased this activity, indicating that CIP2A negatively regulates Akt-related PP2A activity in HCC cells, Furthermore, our in vivo data showed that bortezomib down-regulates CIP2A and up-regulates PP2A activity in Huh-7 tumors but not in PLC5 tumors. In conclusion, inhibition of CIP2A determines effects of bortezomib on apoptosis and PP2A-dependent Akt inactivation in HCC. CIP2A may be a biomarker for predicting clinical response of bortezomib in HCC treatment. Supported by NTUH98P03, NTUH98FTN21, NSC97-2314-B-002-182, NSC98-2314-B-002-067-MY3, and NSC98-3112-B-002-037. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 355.