Wei-Lin Liu

Sonic Hedgehog inhibition as a strategy to augment radiosensitivity of hepatocellular carcinoma

Sonic Hedgehog (SHH) is a regulator in tumorigenesis of hepatocellular carcinoma (HCC). This study aimed to determine whether radiation‐induced SHH signaling occurs in HCC and whether SHH inhibitor acts as a radiosensitizer.

Radiosensitization by combining an aurora kinase inhibitor with radiotherapy in hepatocellular carcinoma through cell cycle interruption.

Radiotherapy has been integrated into the multimodal treatment of hepatocellular carcinoma (HCC), especially of localized hepatic tumor(s) refractory to conventional treatment. However, tumor control remains unsatisfactory mainly because of insufficient dose, and sublethally irradiated tumor may associate with metastasis. Our aim was to assess the effect of combining a molecularly targeted Aurora kinase inhibitor, VE‐465, with radiotherapy in in vitro and in vivo models of human HCC. Human HCC cell lines (Huh7 and PLC‐5) were used to evaluate the in vitro synergism of combining VE‐465 with irradiation. Flow cytometry analyzed the cell cycle changes, while western blot investigated the protein expressions after the combined treatment. Severe combined immunodeficient (SCID) mice bearing ectopic and orthotopic HCC xenografts were treated with VE‐465 and/or radiotherapy for the in vivo response. VE‐465 significantly enhanced radiation‐induced death in HCC cells by a mechanism involving the enhanced inhibition of histone H3 phosphorylation and interruption of cell cycle change. In SCID, mice bearing ectopic HCC xenografts, pretreatment with VE‐465 (20 mg/kg/day × 9 days) significantly enhanced the tumor‐suppressive effect of radiotherapy (5 Gy/day × 5 days) by 54.0%. A similar combinatorial effect of VE‐465 and radiotherapy was observed in an orthotopic model of Huh7 tumor growth by 17.2%. In the orthotopic Huh7 xenografts, VE‐465 significantly enhanced radiation‐induced tumor growth suppression by a mechanism involving the increased apoptosis. VE‐465 is a potent inhibitor of Aurora kinase with therapeutic value as a radiosensitizer of HCC.

Synergistic Blockade of EGFR and HER2 by New-Generation EGFR Tyrosine Kinase Inhibitor Enhances Radiation Effect in Bladder Cancer Cells

Blockade of EGFR has been proved useful in enhancing the effect of radiotherapy, but the advantages of new-generation EGFR tyrosine kinase inhibitors (TKI) in radiosensitization are not well known. We used two human bladder cancer cells with wild-type EGFR to study the synergism between irradiation and afatinib (an EGFR/HER2 dual kinase inhibitor) or erlotinib (an EGFR kinase inhibitor). Here, we showed that afatinib has better radiosensitizing effect than erlotinib in increasing cancer cell killing, the percentage of apoptotic cells, and DNA damage. Afatinib is also superior to erlotinib in combining radiation to decrease tumor size, inhibit glucose metabolism, and enhance apoptotic proteins in vivo. Finally, erlotinib suppressed cell growth and induced more DNA damage in bladder cancer cells transfected with HER2 shRNA, but not in control vector-treated cells. In conclusion, concomitant blockade of radiation-activated EGFR and HER2 signaling by a new-generation EGFR TKI better inhibits the growth of bladder cancer cells both in vitro and in vivo. The absence of radiosensitization by EGFR inhibition alone and the greater radiosensitizing effect of EGFR inhibitor in HER2 knocked down cells suggest the synergism between HER2 and EGFR in determining radiosensitivity. The regained radiosensitizing activity of erlotinib implies that with proper HER2 inhibition, EGFR tyrosine kinase is still a potential target to enhance radiotherapy effect in these seemingly unresponsive bladder cancer cells. Mol Cancer Ther; 14(3); 810–20. ©2015 AACR.

Targeting histone deacetylase 4/ubiquitin‐conjugating enzyme 9 impairs DNA repair for radiosensitization of hepatocellular carcinoma cells in mice

Several strategies to improve the efficacy of radiation therapy against hepatocellular carcinoma (HCC) have been investigated. One approach is to develop radiosensitizing compounds. Because histone deacetylase 4 (HDAC4) is highly expressed in liver cancer and known to regulate oncogenesis through chromatin structure remodeling and controlling protein access to DNA, we postulated that HDAC4 inhibition might enhance radiations effect on HCC cells. HCC cell lines (Huh7 and PLC5) and an ectopic xenograft were pretreated with HDAC inhibitor or short hairpin RNA to knock down expression of HDAC4 and then irradiated (2.5‐10.0 Gy). We evaluated cell survival by a clonogenic assay; apoptosis by Annexin V immunofluorescence; γH2AX, Rad51, and HDAC4 by immunofluorescence staining; HDAC4, Rad51, and ubiquitin‐conjugating enzyme 9 (Ubc9) in HCC cell nuclei by cell fractionation and confocal microscopy; physical interaction between HDAC4/Rad51/Ubc9 by immunoprecipitation; and the downstream targets of HDAC4 knockdown by immunoblotting. Both HDAC4 knockdown and HDAC inhibitor enhanced radiation‐induced cell death and reduced homologous recombination repair of DNA double‐strand breaks and protein kinase B activation, leading to increased apoptosis. HDAC4 knockdown with or without an HDAC inhibitor significantly delayed tumor growth in a radiation‐treated xenograft model. Radiation stimulated nuclear translocation of Rad51 in an HDAC4‐dependent manner and the binding of Ubc9 directly to HDAC4, which led to Ubc9 acetylation. Moreover, these effects were accompanied by HDAC4/Ubc9/Rad51 complex dissociation through inhibiting nuclear translocation. Conclusion: HDAC4 signaling blockade enhances radiation‐induced lethality in HCC cells and xenografts. These findings raise the possibility that HDAC4/Ubc9/Rad51 complex in DNA repair may be a target for radiosensitization of HCC. (Hepatology 2018;67:586‐599).

Targeting Histone deacetylase 4/Ubc9 impairs DNA repair for radiosensitization of hepatocellular carcinoma cells

Several strategies to improve the efficacy of radiation therapy against hepatocellular carcinoma (HCC) have been investigated. One approach is to develop radiosensitizing compounds. Because histone deacetylase 4 (HDAC4) is highly expressed in liver cancer and known to regulate oncogenesis through chromatin structure remodeling and controlling protein access to DNA, we postulated that HDAC4 inhibition might enhance radiations effect on HCC cells. HCC cell lines (Huh7 and PLC5) and an ectopic xenograft were pretreated with HDAC inhibitor or short hairpin RNA to knock down expression of HDAC4 and then irradiated (2.5‐10.0 Gy). We evaluated cell survival by a clonogenic assay; apoptosis by Annexin V immunofluorescence; γH2AX, Rad51, and HDAC4 by immunofluorescence staining; HDAC4, Rad51, and ubiquitin‐conjugating enzyme 9 (Ubc9) in HCC cell nuclei by cell fractionation and confocal microscopy; physical interaction between HDAC4/Rad51/Ubc9 by immunoprecipitation; and the downstream targets of HDAC4 knockdown by immunoblotting. Both HDAC4 knockdown and HDAC inhibitor enhanced radiation‐induced cell death and reduced homologous recombination repair of DNA double‐strand breaks and protein kinase B activation, leading to increased apoptosis. HDAC4 knockdown with or without an HDAC inhibitor significantly delayed tumor growth in a radiation‐treated xenograft model. Radiation stimulated nuclear translocation of Rad51 in an HDAC4‐dependent manner and the binding of Ubc9 directly to HDAC4, which led to Ubc9 acetylation. Moreover, these effects were accompanied by HDAC4/Ubc9/Rad51 complex dissociation through inhibiting nuclear translocation. Conclusion: HDAC4 signaling blockade enhances radiation‐induced lethality in HCC cells and xenografts. These findings raise the possibility that HDAC4/Ubc9/Rad51 complex in DNA repair may be a target for radiosensitization of HCC. (Hepatology 2018;67:586‐599).

Abstract 1731: Dual inhibition of EGFR and HER2 in sublethally irradiated Lewis lung carcinoma cells suppresses MMP-9 production and cell invasiveness

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: Radiotherapy (RT) is an essential component in multi-modality cancer management. While RT yields effective local disease control, distant failures remain great challenges that hinder cancer cure. Tumor cells that survive sublethal irradiation become invasive and metastasize, through up-regulation of matrix metalloproteinase-9 (MMP-9) and remodeling of extracellular matrix. Studies have shown that RT induces MMP-9 production in tumor cells by activating NF-κB through PI3K/Akt and MAPK/ERK pathways. However, clinical applications of inhibitors targeting these pathways are lacking due to toxicity and low efficacy. EGFR and HER2 are canonical upstream signaling molecules that activate these pathways. Additionally, EGFR and HER2, when stimulated with ligands, have been found to induce MMP-9 expression. In this study, we aim to determine the roles of EGFR and HER2 in RT-induced MMP-9 expression, tumor cell invasiveness and metastasis using EGFR inhibitor, erlotinib and EGFR/HER2 inhibitor, afatinib. Methods and Materials: We used western blot analysis to determine EGFR, HER2, and Akt phosphorylations in irradiated Lewis lung carcinoma (LLC) cells (7.5 Gy) pretreated with erlotinib or afatinib. We then used RT-PCR, western blot assay, ELISA, and gelatin zymography to evaluate MMP-9 expression in irradiated LLC cells pretreated with erlotinib or afatinib. Clonogenic assays estimated irradiated LLC cell survival treated with erlotinib or afatinib. Matrigel-coated Boyden chamber assay was used to evaluate invasion capability of irradiated LLC cells. Lastly, tissue western blot analyses of EGFR/HER2/Akt phosphorylations and MMP-9 were performed in LLC tumor grown on the thigh of C57BL/6 mice treated with combined RT and either drug. Results: RT induced EGFR, HER2, and Akt phosphorylations as well as increased MMP-9 expression in LLC cells. Pretreatment with afatinib suppressed RT-induced MMP-9 mRNA and protein expression more significantly than erlotinib. Afatinib inhibited RT-induced invasiveness of LLC cells more than erlotinib did. Tissue western blot analysis of irradiated LLC ectopic xenografts showed more inhibition of RT-induced in vivo MMP-9 expression by afatinib than erlotinib. Afatinib was a stronger inhibitor than erlotinib for RT-induced MMP-9 up-regulation and cell invasiveness in LLC cells and ectopic xenografts. However, clonogenic assay of LLC cells showed that neither erlotinib nor afatanib sensitized LLC cells to RT in survival. Conclusion: Our results suggest that sublethal irradiation induces MMP-9 expression through EGFR and HER2 activation. Dual inhibition of EGFR and HER2 kinase better suppress RT-induced MMP-9 expression and abolish tumor cell invasiveness. Afatinib may serve as an orally bioavailable agent that ameliorates distant metastasis from inadequately irradiated primary lung tumor. Citation Format: Wei-Lin Liu, Tommy Wei-Hsien Hou, Jason Chia-Hsien Cheng. Dual inhibition of EGFR and HER2 in sublethally irradiated Lewis lung carcinoma cells suppresses MMP-9 production and cell invasiveness. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1731. doi:10.1158/1538-7445.AM2014-1731

Abstract A285: Inhibition of histone deacetylase enhances radiosensitivity in hepatocellular carcinoma by inhibiting nuclear translocation of HDAC4 and DNA repair.

Purpose: Tumor control of hepatocellular carcinoma (HCC) by radiotherapy (RT) remains unsatisfactory. The use of an effective molecularly targeted agent in combination with RT is a potential therapeutic approach. Histone deacetylase (HDAC) inhibitors are undergoing clinical evaluation for cancer therapy. HDAC modulates chromatin structure, involves the inhibition of DNA repair, and is considered to influence radio-response. The study aim was to assess the effect of combining a hydroxamic acid derivative HDAC inhibitor, LBH589, with RT on human HCC cells. Methods and Materials: Human HCC cell lines (Huh-7 and PLC-5) were used to evaluate the in vitro synergism of combining LBH589 with irradiation. Western blot and immunofluorescence investigated the protein expressions after the combined treatment. Western blot analysis of proteins from nuclear and cytoplasmic fractions was done by using anti-HDAC4 antibody. Annexin-V immunofluorescence was used to measure apoptotic cells after the combined treatment. Results: Clonogenic survival showed a greater than additive effect with LBH589 administered before RT as compared to RT alone. LBH589 significantly enhanced the accumulation of acetyl-histone H3. Notably, radiation itself induced translocation of HDAC4 to the nucleus. In contrast, treatment with LBH589 followed by irradiation resulted in HDAC4 confinement to the cytoplasm. The findings indicated that HDAC inhibition affects the nuclear localization of HDAC4. We studied the combined effect of LBH589 and RT on DNA repair in both cell lines. γ-H2AX foci representing DNA double-strand breaks (DSBs) were detected in the nuclei at 24 hours following 10-Gy irradiation. LBH589 administered before irradiation increased the persistence of ϒ-H2AX foci beyond 24 hours. Akt phosphorylation, active caspase-3, cleaved PARP in Western blot analysis, and annexin V immunofluorescence assay revealed a synergistic effect of combining LBH589 with RT on inducing apoptosis. Conclusion: This study identifies the inhibitory nuclear translocation of HDAC4 by adding LBH589 to RT, and recognizes the effect of LBH589 on sensitizing HCC cells to radiation-induced DNA DSBs and apoptosis. Taken together, LBH589 is a potent, orally bioavailable inhibitor of HDAC with therapeutic value as a radiosensitizer of HCC. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A285. Citation Format: Wei-Lin Liu, Jason Chia-Hsien Cheng, Ming Gao, Ann-Lii Cheng. Inhibition of histone deacetylase enhances radiosensitivity in hepatocellular carcinoma by inhibiting nuclear translocation of HDAC4 and DNA repair. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A285.

Abstract 72: Targeting PI-3K/Akt signaling pathway by a PI-3K inhibitor BKM120 for radiosensitization in murine hepatocellular carcinoma.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Purpose: Tumor control of hepatocellular carcinoma (HCC) by radiotherapy remains unsatisfactory and the sub-lethal effect associates with secondary spread. Phosphatidylinositol 3-kinase (PI-3K)/Akt pathway plays a critical role in promoting cancer cell proliferation and inhibiting cell death. Elevated PI-3K/Akt activity is associated with increased cellular resistance to irradiation. Our aim was to assess if the inhibition of PI-3K/Akt activity by a PI-3K inhibitor, BKM120, contributes to the increasing sensitivity of murine liver cancer cells to irradiation. Methods and Materials: Murine HCC cell line (BNL) was used to evaluate the in vitro synergism of combining BKM120 with irradiation. Flow cytometry analyzed the cell cycle changes, whereas Western blot investigated the protein expressions after the combined treatment. Balb/c mice bearing ectopic BNL xenografts were treated with BKM120 and/or radiotherapy for the in vivo response. Results: Clonogenic cell survival decreased dose-dependently either with irradiation or BKM120 treatment. Synergisms were achieved in cells treated together with greater or equal than 0.5μM BKM120 and 2 Gy radiation. BKM120 pretreatment significantly inhibited radiation-induced Akt phosphorylation. Western blots of active caspase-3 and cleaved PARP revealed that the combined BKM120 and radiation treatment strongly increased the expression of these apoptotic markers in response to irradiation compared with radiation alone. In ectopic xenografts, pretreatment with BKM120 significantly enhanced the tumor-suppressive effect by radiotherapy. Conclusion: The apparent synergism between BKM120 and irradiation or effect of pre-treatment with BKM120 suggests an option to mitigate the activation of Akt by radiation, leading to increased cell apoptosis in murine liver cancer. These data suggest that the BKM120/radiation combination be a strategy worthy of further clinical trials. Citation Format: Wei-Lin Liu, Melissa Gao, Ann-Lii Cheng, Jason Chia-Hsien Cheng. Targeting PI-3K/Akt signaling pathway by a PI-3K inhibitor BKM120 for radiosensitization in murine 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 72. doi:10.1158/1538-7445.AM2013-72

Abstract 4429: Comparison of afatinib and erlotinib as radiosensitizing agents in bladder cancer cells.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Erlotinib is a first-generation EGFR (epidermal growth factor receptor) tyrosine kinase inhibitor and have well-established efficacy in non-small cell lung cancer patients with activating EGFR mutations. Afatinib, on the other hand, is a second-generation EGFR tyrosine kinase inhibitor with anti-HER2 activity and was found to be of benefit to patients with advanced lung adenocarcinoma who failed previous gefetinib or erlotinib. In previous study we have demonstrated the in vitro and in vivo radiosensitising activity of afatinib in a murine bladder cancer model. However, the radiosensitizing effect of afatinib and erlotinib, has never been compared in cancer cells including bladder cancer. We performed RTK (Receptor Tyrosine Kinase) antibody arrays to investigate the relative levels of phosphorylation in T24 human bladder cancer cell line. We found that EGFR and HER2 signals were activated after radiation10 Gy. Afatinib 100 nM suppressed both irradiation-induced EGFR and HER2 signals but erlotinib 100 nM only suppressed irradiation-induced EGFR signal. Clonogenic assay of T24 and NTUB1 human bladder cancer cell lines was then examined. Afatinib (100-500 nM) showed better radiosensitizing effect (radiation dose: 2-10 Gy) than erlotinib (300-1500 nM) in both T24 and NTUB1 cells by significantly decreasing the numbers of colonies 7 days after treatment. The effect is more prominent in high doses of afatinib. Flow cytometry was used to determine the distribution of cells among various cell cycle phases. T24 and NTUB1 cells were treated with vehicle, radiation 2.5 Gy, afatinib 200 nM, erlotinib 200 nM or the combination of radiation and afatinib or erlotinib. When compared with radiation or drug alone, treatment combining radiation and afatinib in T24 cells resulted in a significant increase of cells in sub-G1 phase but the phenomenon was not observed in the combination of erlotinib and radiation. A similar trend was found in NTUB1 cells. The results implicated that afatinib might enhance radiation effect by increasing apoptosis in bladder cancer cells. To study the DNA damage status after treatment, we checked intra-nuclear γH2AX foci by mmunofluorescence microscopy. T24 and NTUB1 cells were treated with vehicle, radiation 2.5 Gy, afatinib 100 nM, erlotinib 100 nM or the combination of radiation and afatinib or erlotinib. Radiation alone significantly increased γH2AX foci and combining radiation with afatinib further increased the foci in both T24 and NTUB1 cells. The enhancement was absent in the combination of radiation and erlotinib. Our data clearly show that afatinib, a new generation EGFR tyrosine kinase inhibitor with anti-HER2 activity, is superior to erlotinib as a radiosensitizer in the treatment of human bladder cancer cells. Citation Format: Yu-Chieh Tsai, Tsung-Fan Tuan, Pei-Yin Ho, Wei-Lin Liu, Liang-Yu Chang, Yeong-Shiau Pu, Ann-Lii Cheng, Jason Chia-Hsien Chen. Comparison of afatinib and erlotinib as radiosensitizing agents in bladder cancer cells. [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 4429. doi:10.1158/1538-7445.AM2013-4429