Chi Hang Wong

Enhanced Antitumor Activity with Combining Effect of mTOR Inhibition and Microtubule Stabilization in Hepatocellular Carcinoma

Mammalian target of rapamycin (mTOR) and the microtubules are shown to be potential targets for treating hepatocellular carcinoma (HCC). PI3K/Akt/mTOR activation is associated with resistance to microtubule inhibitors. Here, we evaluated the antitumor activity by cotargeting of the mTOR (using allosteric mTOR inhibitor everolimus) and the microtubules (using novel microtubule-stabilizing agent patupilone) in HCC models. In vitro studies showed that either targeting mTOR signaling with everolimus or targeting microtubules with patupilone was able to suppress HCC cell growth in a dose-dependent manner. Cotargeting of the mTOR (by everolimus) and the microtubules (by patupilone, at low nM) resulted in enhanced growth inhibition in HCC cells (achieving maximal growth inhibition of 60–87%), demonstrating potent antitumor activity of this combination. In vivo studies showed that everolimus treatment alone for two weeks was able to inhibit the growth of Hep3B xenografts. Strikingly, the everolimus/patupilone combination induced a more significant antitumor activity. Mechanistic study demonstrated that this enhanced antitumor effect was accompanied by marked cell apoptosis induction and antiangiogenic activity, which were more significant than single-agent treatments. Our findings demonstrated that the everolimus/patupilone combination, which had potent antitumor activity, was a potential therapeutic strategy for HCC.

Targeting Angiogenic Genes as a Therapeutic Approach for Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is a complex liver disease with limited treatment options and often resulting in a poor prognosis. The development of HCC depends on the formation of new blood vessels and it demonstrates hypervascularity and invasive property to the surrounding vasculature clinically. A complex network of growth factors acting on both tumor cells and endothelial cells mediates the angiogenesis in HCC. It is an attractive approach to inhibit the angiogenic processes as the treatment of HCC and therefore, anti-angiogenic TKIs were developed to inhibit the vessel formation in the tumors. However, it is currently perceived that the efficacy of these anti-angiogenic TKIs has reached plateau, and it is necessary to develop novel agents with non-TKI mechanism to inhibit the angiogenic targets. With the better understanding of molecular mechanisms that govern angiogenesis, as well as the advancement in biomedical engineering, new approaches of gene therapy have brought hopes for therapeutic intervention in HCC. Gene therapy is based on the transfer of genetic material to the patients with the aim to modify or correct the malignancy from its molecular basis. In this article, we will discuss the conventional anti-angiogenic therapies and the gene therapy approaches in HCC. The therapeutic potential of gene therapy for HCC treatment has been demonstrated and further development of anti-angiogenic may result in new treatment option for HCC patients.

Abstract 2558: The mechanistic study on the effect of platinum-based chemotherapy efficacy imposed by EGFR-TKI regulated ERCC1 in non-small cell lung cancer (NSCLC)

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Platinum-based chemotherapy is conventionally the first line treatment for EGFR wild type patients while EGFR-TKI is the standard for patients with mutation. Subgroup biomarker studies conducted in FASTACT-2 (Wu et al Lancet Oncology 2013) indicated that patients with positive ERCC1 attained longer overall survival and progression free survival under intercalated chemotherapy and EGFR-TKI, in comparison with solely chemotherapy. EGFR-TKI is postulated to down-regulate ERCC1 expression in EGFR wild type NSCLC cells, hence enhancing the Chemo efficacy. The study aims to investigate the missing link between EGFR and ERCC1 in the perspective of mechanistic means. In vitro and in vivo studies were devised to examine the impact imposed on ERCC1 by EGFR-TKI. H1993 cells underwent a 72-hour EGFR-TKI treatment and transient siRNA transfection for ERCC1 suppression. Cell viability assay was employed for detecting the cisplatin sensitivity of the transfected cells. Xenograft tumor models were established with a 2-week treatment of oral erlotinib on animals, using western blot and IHC to study the ERCC1 expression. Human EGFR Phosphorylation Membrane Array was used to study the EGFR phosphorylation pattern involved in ERCC1 level alterations and its downstream pathways were further analyzed with western blot. ERCC1 expression level is not correlated with EGFR-TKI sensitivity. Nonetheless, ERCC1 level gradually decreased along the 72-hour exposure of EGFR-TKI. The cells’ sensitivity towards cisplatin was raised upon ERCC1 knock-down with its IC50 reduced from 14.67μM to 0.16μM. Furthermore, TKI showed a significant reduction in ERCC1 level with IHC staining after treatment yet no sequel on the tumor volume of the xenografts. On the mechanistic side, 6 out of 17 EGFR phosphorylation sites were found compelling in respond to variations on ERCC1 level. We discovered that the MAPK/ERK and JNK pathways were the potential candidates that regulated EGFR associated ERCC1 expression. In summary, the data manifested that EGFR-TKI may reduce ERCC1 expression and latently enhance sensitivity towards cisplatin. MAPK/ERK and JNK pathways may contribute in ERCC1 regulation. With the preliminary screening results, it warrants further investigation to validate the underlying mechanism for the regulation of ERCC1 expression level. Citation Format: Hio Teng Cheong, Connie Wun Chun Hui, Fei Xu, Tony Shu Kam Mok, Chi Hang Wong. The mechanistic study on the effect of platinum-based chemotherapy efficacy imposed by EGFR-TKI regulated ERCC1 in non-small cell lung cancer (NSCLC). [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 2558. doi:10.1158/1538-7445.AM2015-2558

Infer related genes from large gene expression dataset with embedding

Artificial neural networks (ANNs) have been utilized for classification and prediction task with remarkable accuracy. However, its implications for unsupervised data mining using molecular data is under-explored. We adopted a method of unsupervised ANN, namely word embedding, to extract biologically relevant information from TCGA gene expression dataset. Ground truth relationship, such as cancer types of the input sample and semantic meaning of genes, were showed to retain in the resulting entity matrices. We also demonstrated the interpretability and usage of these matrices in shortlisting candidates from a long gene list. This method is feasible to mine big volume of biological data, and would be a valuable tool to discover novel knowledge from omics data. The resulting embedding matrices mined from TCGA gene expression data are interactively explorable online (http://bit.ly/tcga-embedding-cancer) and could serve as an informative reference.

Upregulation of Bcl2 in NSCLC with acquired resistance to EGFR‑TKI

Lung cancer has the highest incidence and mortality rate worldwide among all malignancy-associated mortalities, of which non-small cell lung cancer accounts for 80% of all cases. Resistance against epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) develops following 8–12 months of disease progression, and is a critical issue. HCC827 cell lines with resistance to EGFR-TKIs were successfully screened. The half maximal inhibitory concentration values were 1,000-fold higher than the values for the parental HCC827 cell line, thereby demonstrating cross-resistance against the same family of TKIs. The expression of B-cell lymphoma 2 (Bcl2) was markedly increased in the resistant clones, as well as in the patient biopsies. The phosphatase and tensin homolog phosphoinositide 3-kinase signaling axis is a potential mechanism for acquiring resistance, and therefore targeting Bcl2 may be a useful strategy for further investigations.

Abstract 5059: Preclinical study on the efficacy of Panobinostat in hepatocellular carcinoma

Background Aberrant regulation of histone deacetylases (HDACs) is known to play a pivotal role in HCC pathogenesis as well as other human malignancies. Panobinostat (LBH589) is a pan-HDAC inhibitor covering a wide range of HDACs (Class I, II and IV) with high inhibitory activity at nanomolar concentration. It has been approved by FDA for treating multiple myeloma and has demonstrated promising anti-proliferative and cytotoxic activity in breast, prostate, colon and pancreatic cancer cell lines. This study investigated in vitro and in vivo effect of Panobinostat in HCC cell lines. Methods Basal expressions of HR23B and HDACs of 7 HCC cell lines (HepG2, PLC/PRF/5, Huh-7, Hep3B, SNU-182, SNU-398 and SNU-449) were determined by western blotting. Their corresponding IC50 for 24, 48 and 72 hours towards Panobinostat were determined by cell viability assay. Huh-7, Hep3B and SNU-449 were selected for further in vitro experiments. Their cell cycle distribution after Panobinostat treatment was evaluated by flow cytometry. Apoptosis was detected by Cell Death Detection ELISA. Huh-7 and Hep3B xenograft model were used for in vivo investigation. Cells were inoculated subcutaneously into the flanks of 3-4 week old male athymic nude mice. When tumors were established, Panobinostat was administrated intraperitoneally at 7.5mg/kg and 15mg/kg five days per week for 2 weeks. Results All cell lines were able to achieve nearly 100% growth inhibition and had displayed a dose- and time-dependent manner towards Panobinostat. Maximum growth inhibition was 20-70% at 24hr compared to over 90% at 72hr. There was significant reduction in cell viability at low nanomolar concentrations (IC50 at 48hr: HepG2=8.81±0.72nM, PLC/PRF/5=18.9±0.74nM, Huh-7=14.01±1.12nM, Hep3B=25.00±3.69nM, SNU-182=73.33±15.52nM, SNU-398=12.86±3.25nM, SNU-449=73.01±9.09nM). Flow cytometry analysis showed Panobinostat induced accumulation of cells at G0/G1 phase in Huh-7 and SNU-449. Meanwhile, an increase in sub G1 population was detected in Hep3B after exposure to 25nM Panobinostat for 48h. Apoptotic induction was further confirmed by cell death detection ELISA and western blotting. Panobinostat promoted apoptosis more remarkable in Hep3B than other 2 cell lines as evidenced by a stronger cleaved PARP expression level. Panobinostat treatment delayed tumor growth in Hep3B (p Conclusion Panobinostat has been demonstrated to inhibit in vitro and in vivo HCC cell growth. Further study on the mechanism behind Panobinostat sensitivity is warranted. The study was supported by Novartis. Citation Format: Chi Tung Choy, Wing Yu Man, Chi Hang Wong, Stephen Lam Chan. Preclinical study on the efficacy of Panobinostat in hepatocellular carcinoma [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 5059. doi:10.1158/1538-7445.AM2017-5059

Low expressions of ASS1 and OTC in glioblastoma suggest the potential clinical use of recombinant human arginase (rhArg).

cell lines. None of the six cell lines had ASS1 expression, and only M059J, M059K and U373 were weakly OTC positive (Fig. 1a). Sensitivity of GBM cell lines towards PEG-BCT-100 were then evaluated using MTT cytotoxicity assay. Cells were treated with PEG-BCT-100 for 48 h and maximum 70–80 % growth inhibition was observed. The EC50 of A172, M059J, M059K, T98G, U373 and U-87 MG were 0.4436, 0.2979, 0.5224, 0.1333, 0.3048 and 0.3795 IU/ ml (Fig. 1b), respectively. Prior studies in hepatocellular carcinoma (HCC) cell lines which were deemed sensitive to PEG-BCT-100 have shown similar EC50 values [2] and clinical trials of PEG-BCT-100 are underway. To examine the occurrence of ASS1/OTC deficiency in GBM patients, immunohistochemistry (IHC) analyses of samples from 70 GBM patients were conducted. All samples were analyzed for OTC expression, and 28 of the 70 samples were randomly chosen for analysis of ASS1 expression. The performances of antibodies were affirmed using normal mouse liver (known to be ASS1 and OTC positive) in parallel experiment. None of the examined samples were ASS1 or OTC positive (Fig. 1c–e).

Abstract 5499: Preclinical evaluation of PI3K inhibitor BYL719 as a single agent and its synergism in combination with cisplatin or MEK inhibitor in nasopharyngeal carcinoma (NPC) using 3D cell culture system

BYL719 is a novel specific inhibitor against the alpha-isoform of Class I PI3K that imposes impacts on AKT-mTOR signaling axis, thereby mediating relevant pathways such as MAPK, STAT3 and EGFR. The study aims to investigate the effects of BYL719 on nasopharyngeal carcinoma (NPC) which has a high prevalence in Southeast Asia, and its synergism on combination with cisplatin or MEK inhibitor. Six NPC cell lines including C666-1, CNE-2, HK1, HK1-EBV, HONE-1, HONE-1-LMP were selected for this preclinical study. All cell lines had a basal expression in both phosphorylated and total forms of Akt, mTOR and p70S6K which are the downstream pathways of PI3K. MTT assay was used to evaluate the cytotoxicity of BYL719 on NPC, cells were incubated in BYL719 with escalating concentration (10nM, 50nM, 0.1µM, 0.5µM, 1µM, 5µM and 10µM) for 48 or 72 hours in culture medium. The maximum growth inhibition was attained in 72 hours of BYL719 incubation, the IC50s of all cell lines with 72-hour BYL719 treatment were in micromolar range (C666-1=1.85µM, CNE-2=0.90µM, HONE-1=0.56µM, HONE-1-LMP=0.95µM, HK-1-EBV=1.50µM and HK-1=1.53µM). Two sensitive cell lines CNE-2 and HONE-1 were selected for further analysis on apoptosis, cell cycle and BYL7199s synergistic effect by 3D cell culture system. After 72 hours of treatment, BYL719 up-regulated mTOR, EGFR, p-EGFR (Y1086), p-STAT3 (Y705) and p-p44/22 MAPK (T202/Y204) in CNE-2 but not in HONE-1, however, it down-regulates mTOR in HONE-1. This suggested that BYL719 is more effective to HONE-1 in inhibiting mTOR downstream pathways and accordingly anti-proliferation. HONE-1 was then used to examine the synergistic effect of BYL719 combined with cisplatin or MEK inhibitor (AZD6244). Drug treatment commenced 72 hours after cell plating and growth inhibition was determined by MTT assay on day 9. As supported by cell viability assay and western blot, strong synergistic effect was expressed when BYL719 was combined with AZD6244. In comparison with using BYL719 alone, there was a 2-fold increase in growth inhibition observed in combination of BYL719 and AZD6244. The combination indices (CI) of 0.6µM BYL719 plus 0.325µM AZD6244 and 0.6µM BYL719 plus 0.65µM AZD6244 are 0.17 and 0.13 respectively, indicating strong synergism in these drug combinations. On the other hand, there was also mild synergistic effect observed when BYL719 was combined with cisplatin, with CI of 0.585 for 0.6µM BYL719 plus 1.2µg/ml cisplatin. The growth inhibition for BYL719 combined with cisplatin was comparable to the effect of high dose cisplatin (1.2µg/ml) administration. BYL719 is effective in NPC growth inhibition, its synergistic effect with MEK inhibitor does provide a great advantage in NPC treatment and is worthwhile for further studies. Acknowledgement: This work is supported by Novartis * Denotes co-authorship. Citation Format: Hio Teng Cheong, Chi Hang Wong, Connie Wun Chun Hui, Anthony Tak Cheung Chan, Brigette Buig Yue Ma. Preclinical evaluation of PI3K inhibitor BYL719 as a single agent and its synergism in combination with cisplatin or MEK inhibitor in nasopharyngeal carcinoma (NPC) using 3D cell culture system. [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 5499. doi:10.1158/1538-7445.AM2014-5499

Abstract 2811: Preclinical evaluation of combined TKI258 and RAD001 in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the most lethal cancers. Signaling pathways including AKT-mTOR axis is often dysregulated in HCC. RAD001 principally targets at the mTOR complex 1. mTOR inhibition is reportedly associated with activation of feedback loop and parallel pathways. TKI258 is a potent tyrosine kinase inhibitor targeting the FGFRs and VEGFRs, which blocks subsequent PI3K/AKT signaling pathways in cancer cells. In the current study, we aim to study the in-vitro and in-vivo effects of the drug combination with reference to the parallel and upstream pathway of AKT-mTOR axis. We have treated the HCC cell line Hep3B with TKI258 and RAD001. RAD001 could suppress the phosphorylation of down stream signal mediators of mTOR pathway including p70S6 kinase 1 (S6K) and eukaryotic initiation factor binding protein 1 (4E-BP1) and led to G1 phase arrest. The results demonstrated a significant increase in suppression of cell proliferation in vitro with the combination of TKI258 (300nM) and RAD001 (either 200nM or 200pM) compared with either drug alone. Although the addition of the TKI258 only slightly suppressed the AKT positive feedback loop induced by RAD001, the combination of drugs could significantly suppress the phosphorylation of mTOR (Ser2448), ERK1/2 (Ser217/221) and p38 (Thr180/Tyr182) as well as their endogenous protein expression levels. The expression levels of FGFR1 and FGFR2 were also suppressed. The overall protein reduction was due to the synergistic inhibition of p70S6 (Ser240/244) and 4E-BP1 (Ser65) phosphorylation, which indicated a strong suppression in translation and protein synthesis. Pro-caspases 3 and PARP cleavage were not observed at 24 hours and only slightly detected at 48 hours post-treatment, suggesting that the drug combination did not further increase cytotoxic effects but mainly increased in cytostatic arrest ability. In Hep3B derived xenograft model, TKI258 (15mg/ml) and RAD001 (2.5mg/kg) had shown a synergistic inhibition of tumor growth in volume and weight. In addition, there was a significant reduction in microvessel density (MVD) in the xenograft, which indicated an improved effect in angiogenesis inhibition. Our data showed that the combination of RAD001 and TKI258 were active in controlling HCC proliferation via double inhibition of both AKT-mTOR axis and its parallel pathway. Acknowledge: The works are supported by Novartis Oncology. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2811. doi:1538-7445.AM2012-2811