Connie Wun Chun Hui

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.

FGF8b oncogene mediates proliferation and invasion of Epstein–Barr virus-associated nasopharyngeal carcinoma cells: implication for viral-mediated FGF8b upregulation

The fibroblast growth factor 8b (FGF8b) oncogene is known to be primarily involved in the tumorigenesis and progression of hormone-related cancers. Its role in other epithelial cancers has not been investigated, except for esophageal cancer, in which FGF8b overexpression was mainly found in tumor biopsies of male patients. These observations were consistent with previous findings in these cancer types that the male sex-hormone androgen is responsible for FGF8b expression. Nasopharyngeal carcinoma (NPC) is a highly metastatic cancer of head and neck commonly found in Asia. It is etiologically associated with Epstein–Barr Virus (EBV) infection, inflammatory tumor microenvironment and relatively higher male predominance. Here, we reported for the first time that FGF8b is overexpressed in this EBV-associated non-hormone-related cancer of the head and neck, NPC. More importantly, overexpression of FGF8b mRNA and protein was detected in a large majority of NPC tumors from both male and female genders, in addition to multiple NPC cell lines. We hypothesized that FGF8b overexpression may contribute to NPC tumorigenesis. Using EBV-associated NPC cell lines, we demonstrated that specific knockdown of FGF8b by small interfering RNA inhibited cell proliferation, migration and invasion, whereas exogenous FGF8b stimulated these multiple phenotypes. Further mechanistic investigation revealed that in addition to NF-κB signaling (a major inflammatory signaling pathway known to be activated in NPC), an important EBV oncoprotein, the latent membrane protein 1 (LMP1), was found to be a direct inducer of FGF8b overexpression in NPC cells, whereas androgen (testosterone) has minimal effect on FGF8b expression in EBV-associated NPC cells. In summary, our study has identified LMP1 as the first viral oncogene capable of directly inducing FGF8b (an important cellular oncogene) expression in human cancer cells. This novel mechanism of viral-mediated FGF8 upregulation may implicate a new role of oncoviruses in human carcinogenesis.

Preclinical evaluation of the mTOR–PI3K inhibitor BEZ235 in nasopharyngeal cancer models

The dual PI3K-mTOR inhibitor BEZ235 was evaluated in preclinical models of nasopharyngeal carcinoma (NPC). The IC50 value of BEZ235 for growth was in the nanomolar range in vitro, induce G1 cycle arrest and apoptosis, and inhibited AKT and mTOR signaling in most NPC cell lines. No synergistic effect was observed when BEZ235 was combined with chemotherapy. BEZ235 increased MAPK activation in vitro but not in vivo. A daily schedule was more effective than a weekly schedule on tumor growth and inhibition of downstream mTOR signaling in vivo. The activity of BEZ235 maybe independent of the PIK3CA amplification and mutation status.

Lung ischaemia-reperfusion induced gene expression.

OBJECTIVES Pulmonary dysfunction following lung ischaemia-reperfusion is a well-known phenomenon, which may contribute to post-cardiac surgical morbidity. The process is associated with pulmonary inflammatory response and cellular apoptosis. Early molecular mechanisms leading to such lung injury remain largely unknown. We examined whether lung ischaemia and reperfusion cause significant expression changes in numerous genes in the lungs involved in pulmonary apoptosis and other cellular processes by using oligonucleotide microarrays in an experimental model of rodent lung ischaemia-reperfusion injury. METHODS Sprague-Dawley rodents (n=5 in each group) were anaesthetised and underwent controlled ventilation, with varying durations of warm lung ischaemia (60 and 90 min) followed by a short reperfusion period. The right middle lobe of the lung was harvested. Gene expression changes in the lungs were analysed by rodent DNA microarray chips, and reverse transcription polymerase chain reaction (RT-PCR) performed to validate changes in gene expression. RESULTS Significant expression changes, with reference to false discovery rate (FDR) controls, were detected in over 80 genes following controlled lung ventilation, and more than 50 were up-regulated more than 2-fold. Lung ischaemia-reperfusion caused expression changes in over 50 additional genes, including many novel genes not previously associated with lung ischaemia-reperfusion. Up-regulated genes identified include those associated with apoptosis, inflammation and cell-cycle control. CONCLUSIONS Large numbers of genes relating to cell metabolism, transcription control, inflammation and apoptosis were significantly up- and down-regulated following controlled ventilation and early lung ischaemia-reperfusion, consistent with previous studies. In addition, novel genes related to lung injury were identified. These genetic signatures provide new insights into early molecular mechanisms of ischaemia-reperfusion lung injury and help refine therapeutic strategies to lessen pulmonary dysfunction following cardiac surgery.

Ventilation during cardiopulmonary bypass: impact on neutrophil activation and pulmonary sequestration.

Background: Cardiopulmonary bypass (CPB) is associated with neutrophil activation, pulmonary sequestration, and release of inflammatory mediators leading to pulmonary dysfunction. We investigate the effect of continuous ventilation during cardiopulmonary bypass on neutrophil activation and pulmonary sequestration. Methods: Forty-six patients undergoing coronary artery bypass grafting with cardiopulmonary bypass were prospectively randomized to continuous ventilation and nonventilation groups. Blood samples were collected, and bronchoalveolar lavage (BAL) was performed following induction of anesthesia and at 4 hr after aortic declamping. Differential white cell count was measured, and flow cytometry to determine cell count numbers and quantify CD45 and CD11b leukocyte cell surface adhesion molecule expression was performed on the blood and BAL samples. Results: Twenty-three patients were randomized to standard nonventilated CPB and 23 patients to ventilation throughout CPB. Significant increases in blood and BAL neutrophil numbers were detected at 4 hr following aortic declamping in both groups (Blood: NV p <. 0001, V p <. 0001; BAL: NV p =. 017, V p =. 0007). No significant inter-group differences in BAL and blood neutrophil numbers were found. Significantly higher blood neutrophil CD11b mean fluorescent intensity levels were present 4 hr following declamping compared with baseline in both groups (NV Blood, p =. 021; V Blood p <. 0001). No significant inter- or intragroup differences in BAL neutrophil CD11b mean fluorescent intensity levels were found. There was no death or major complication. Conclusions: Cardiopulmonary bypass during coronary artery bypass grafting is associated with increased neutrophil pulmonary sequestration, and blood neutrophil CD11b activation. Continuous ventilation during cardiopulmonary bypass does not significantly reduce neutrophil pulmonary sequestration or activation.

Abstract 3773: Preclinical evaluation of the CDK4/6 inhibitor LEE011 in nasopharyngeal carcinoma (NPC) cell lines

LEE011 is a specific CDK4/6 inhibitor that induces G1 cycle arrest by blocking the formation of cyclin D1-CDK4/6 complex and inhibiting Rb phosphorylation. Cyclin D1 is overexpressed in > 90% of NPC and CCND1 gene activation is implicated in pathogenesis. The preclinical activity of LEE011 was evaluated in 4 NPC cell lines (C666-1, HK1, HK1-LMP1, HONE-1) and the immortalized nasopharyngeal epithelial cell line NP69. Under basal condition, phosphorylated (p) Rb was strongly expressed in HK1 and HK1-LMP1, moderately in HONE-1 and NP69, and weakly in C666-1. Cyclin D1, CDK4 and 6 were expressed in all cell lines. The IC50 concentrations for cell growth inhibition after 72 hours of exposure to LEE011 in the respective cell lines were: HK1-LMP1 = 4.10±1.12μM; HK1 = 5.07±1.37μM, C666-1 = 12.25±1.63μM, NP69 = 15.23±1.93μM, HONE-1 = 19.58±1.54μM. LEE011 could induce over 95% cell growth inhibition in all NPC cell lines. Three representative cell lines (HK1 as the most sensitive to LEE011, and C666-1 and HONE-1 as less sensitive) were chosen to evaluate the effect of LEE011 on kinase signaling, apoptosis and cell cycle. Treatment of these cell lines at or above their respective IC50 concentrations for up to 48 hrs showed a dose-dependent reduction in p- and total Rb expression. A slight increase in the expression of CDK4, CDK6 and cyclinD1 were observed in HK1 and HONE1 cells, but not in C666-1 cells. G0/G1 population was increased by more than 20% in C666-1 and HK-1 cells at up to 48 hrs of exposure to LEE011. The effect of combining LEE011 with the alpha-specific PI3K inhibitor BYL719 on cell growth was studied in C666-1, HK1 and HONE-1 cells. A strong synergistic effect on growth inhibition was seen in C666-1 and HK1, but not HONE-1 at 72hrs, with the respective combination index (CI) of ED50 less than 0.5. Additionally, the combination of LEE011 and cisplatin at different sequences was investigated on their effect on cell growth. The sequential administration of cisplatin followed by LEE011 was the most optimal sequence on cell growth inhibition in C666-1 and HONE-1, but not in HK1 cells. Preliminary result suggests that this schedule was associated with better pRB inhibition than other schedules in C666-1 cells. The IC50 of LEE011 for a cisplatin-resistant HK1-LMP1-cis cell line at 72 hr was similar to its parental HK1-LMP1 cell line. In summary, LEE011 displayed dose- and time-dependent growth inhibitory effect in over 95% of NPC cells examined. A synergistic inhibitory effect on cancer cell growth was observed when LEE011 was combined with BYL719 in vitro and sequential administration of cisplatin followed by LEE011 has shown an optimal inhibitory effect. These results should be confirmed in xenograft models of NPC. Citation Format: Brigette B. Ma, Chi-Hang Wong, Connie Hui, Edwin P. Hui, Anthony T.C. Chan. Preclinical evaluation of the CDK4/6 inhibitor LEE011 in nasopharyngeal carcinoma (NPC) cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3773.

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

Preclinical evaluation of ribociclib and its synergistic effect in combination with alpelisib in non-keratinizing nasopharyngeal carcinoma

Ribociclib is a specific cyclin dependent kinase (Cdk) 4/6 inhibitor that induces G1 arrest by blocking the formation of cyclin D1-Cdk4/6 complex and inhibiting retinoblastoma (RB) phosphorylation. Cyclin D1 is overexpressed in over 90% of nasopharyngeal carcinoma (NPC) and CCND1 gene activation plays a critical role in NPC pathogenesis. This study evaluated the preclinical activities of ribociclib in NPC cell lines and patient derived xenograft (PDX) models. Over 95% cell growth inhibition was observed at 96 hours after ribociclib treatment. (IC50 concentrations: HK1 = 1.42 ± 0.23 µM; HK1-LMP1 = 2.18 ± 0.70 µM and C666-1 = 8.26 ± 0.92 µM). HK1 and C666-1 cells were chosen for analysis of ribociclib on kinase signaling, apoptosis and cell cycle. Treatment with ribociclib for 48 hours consistently showed a dose-dependent reduction in phosphorylated and total RB expression and G1 cycle arrest was only observed. Combining ribociclib with the alpha-specific PI3K inhibitor alpelisib showed a synergistic effect in two NPC PDX models in nude mice. The co-treatment induced a significant reduction in tumor volume in both xeno-666 and xeno-2117 compared with ribociclib treatment alone and control (p < 0.01). In summary, ribociclib is active in NPC models and the effect on growth inhibition was augmented when combined with alpelisib. This study supports the clinical evaluation of ribociclib in NPC.

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 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