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.
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 BYL719's 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
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
Background: Arginine deprivation is a novel approach to limit arginine-dependent tumour growth. The presence of enzymes involved in the de novo synthesis of arginine from citrulline, argininosuccinate synthetase (ASS), argininosuccinate lyase (ASL) and ornithine transcarbamylase (OTC), can influence the sensitivity of tumour to arginine depletion. We studied the preclinical efficacy of PEG-BCT-100 (also known as rhArg1peg5000), a PEGylated recombinant human arginase 1 on sarcoma cell lines. In this study, ASS, ASL and OTC expression were established in sarcoma cell lines and the sensitivity of each cell line to PEG-BCT-100 treatment was determined in order to establish their suitability as predictive markers to determine responsiveness to PEG-BCT-100 treatment. Methods: Cells from 5 representative soft-tissue sarcoma (STS) cell lines (RD, GCT, HT-1080, SW872, SW982) and 1 osteosarcoma cell line (SJSA-1), either incubated in full culture medium (control), arginine free medium or PEG-BCT-100 (1U/ml) treated medium. They were monitored by live cell imaging for 72 hours. Their corresponding IC50 were determined by cell viability assay. The expression of ASS, ASL and OTC were determined by Western blotting. Results: All sarcoma cell lines were sensitive to PEG-BCT-100 and demonstrated significant cell proliferation inhibition. Their IC50, as determined by cell viability assay were 0.023 U/ml, 0.028 U/ml, 0.023 U/ml, 0.026 U/ml, 0.20 U/ml, 0.053 U/ml, and 0.03 U/ml for GCT, HT-1080, RD, SJSA-1, SW872 and SW982 respectively. Western blot analysis confirmed strong basal protein expression of ASL and low OTC expression in all sarcoma cell lines, suggesting sensitivity to PEG-BCT-100 is not determined by ASL but rather, OTC. On the contrary, low ASS basal protein expression was revealed in all sarcoma cell lines except for RD. A strongly expressed ASS was observed in RD despite of its sensitivity to PEG-BCT-100, implying that ASS might be less important in the response towards PEG-BCT-100. Conclusion: Arginine deprivation by PEG-BCT-100 is effective in suppressing STS cell growth in vitro, suggesting arginine auxotrophism in STSs. Moreover, arginase treatment can be an effective strategy against STS. Low expression of OTC, instead of ASS and ASL, may be a more important predictive biomarker for response to treatment. Further in-vivo and clinical studies are warranted. Citation Format: Chi Tung Choy, Hio Teng Cheong, Kin Pong U, Chi Hang Wong, Herbert Ho Fung Loong. Preclinical evaluation of the recombinant human arginase PEG-BCT-100 leading to arginine deprivation in sarcomas. [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 5512. doi:10.1158/1538-7445.AM2015-5512
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