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J.C.M. Ho



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    P1.01 - Poster Session 1 - Cancer Biology (ID 143)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P1.01-009 - The Role of Autophagy Induced by Tyrosine Kinase Inhibitors in Non-small Cell Lung Carcinoma (ID 1618)

      09:30 - 16:30  |  Author(s): J.C.M. Ho

      • Abstract

      Background
      Background and Aim of Study: Non-small cell lung cancer (NSCLC) is one of the leading causes of cancer deaths. Erlotinib (tyrosine kinase inhibitor, TKI) is widely used as a specific treatment targeting epidermal growth factor receptor (EGFR), while crizotinib serves as a c-MET and anaplastic lymphoma kinase (ALK) inhibitor. Autophagy is a regulated cellular catabolic process in response to stress. This study aimed to investigate whether autophagy could confer acquired resistance to TKI in NSCLC.

      Methods
      Methods: Two NSCLC cell lines (HCC827 and HCC4006) with EGFR mutations (exon 19 deletions) and two NSCLC cell lines (H1993 and H2228) with c-MET amplification or ALK rearrangement were selected. Cell proliferation (MTT) and annexin-V binding assays were performed to determine cell proliferation and apoptotic cell death upon TKI treatment respectively. Autophagy was determined by conversion of LC3I to LC3II and p62 degradation using Western blot. Acidic vesicular organelle (AVO) formation was shown by acridine orange staining. Autophagy inhibitor (chloroquine) was used to study the functional significance of TKI-induced autophagy.

      Results
      Results: MTT assay confirmed that all cell lines were sensitive to corresponding TKI after 72 hours incubation (IC~50~ <0.5 μM). Erlotinib or crizotinib increased LC3II expression, p62 degradation and formation of AVO, compatible with induction of autophagy. Combination of 10 μM chloroquine (autophagy inhibitor) with 0.2 μM erlotinib or crizotinib for 48 hours increased apoptotic events compared to single treatment (Table 1)

      Table 1. Apoptotic events in NSCLC cell lines after different treatments.
      Cell line Chloroquine + Erlotinib Erlotinib Chloroquine p-value
      HCC827 52.3 ± 2.8% 21.2 ± 4.2% 13.0 ± 2.5% <0.01
      HCC4006 49.3 ± 4.8% 9.4 ± 2.4% 9.0 ± 1.8% <0.01
      Chloroquine + Crizotinib Crizotinib Chloroquine
      H1993 42.6 ± 12.0% 17.3 ± 5.1% 10.5 ± 5.4% <0.01
      H2228 34.9 ± 10.1% 12.2 ± 2.6% 7.6 ± 1.2% <0.01

      Conclusion
      Conclusions: TKI induced both apoptosis and autophagy in EGFR-mutated, c-MET-amplified and ALK-rearranged NSCLC cell lines. Inhibition of autophagy with chloroquine enhanced TKI-induced cell death. Autophagy may serve as a protective mechanism in NSCLC upon treatment with TKI.

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    P3.01 - Poster Session 3 - Cancer Biology (ID 147)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 2
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      P3.01-001 - Downregulation of Thymidylate Synthase with Arsenic Trioxide in Non-Small Cell Lung Cancer: in vitro and in vivo (ID 1341)

      09:30 - 16:30  |  Author(s): J.C.M. Ho

      • Abstract

      Background
      Background: Thymidylate synthase (TYMS) is an important chemotherapeutic target in non-small cell lung cancer (NSCLC). Recently, arsenic trioxide (ATO) has been shown to downregulate TYMS in a colonic cancer model. Therefore, we examined the effect of TYMS suppression by ATO in NSCLC.

      Methods
      Methods: A panel of seven NSCLC cell lines (NCI-H23, NCI-H358, HCC827, NCI-H1650, NCI-H1975, HCC2935 and HCC4006, from ATCC) was used to determine the effects of ATO treatment on cell viability, TYMS protein and mRNA expression, E2F1 protein expression and TYMS activity. TYMS knockdown and overexpression were performed to confirm the importance of TYMS in cell survival and resistance to ATO respectively. Tumor growth inhibition in vivo was studied using a nude mice xenograft model.

      Results
      Results: ATO showed antiproliferative effects with clinically achievable concentrations (around 1.1-6.9 μM after 72 hours incubation) in NSCLC cell lines. Baseline TYMS protein expression was detected in H23, H358, H1650 and H1975 cells. Among them, downregulation of TYMS protein and mRNA expression, reduced total TYMS activity, and suppressed E2F1 expression were demonstrated in H23, H358, and H1650 cells with ATO. Cell viability was reduced by 15-50% with TYMS knockdown (to less than 30% protein expression) (Fig. 1). Overexpression of TYMS led to a 2.7-fold increase in IC~50~ value with ATO treatment for 72 hours in H358 cells, but not H23 cells. Using a xenograft model with H358 cell line, relative tumor volume was reduced to 44% that of control after 8 days of treatment with 7.5 mg/kg intraperitoneal ATO, and associated with significant downregulation of TYMS protein expression in tumor xenografts (Fig. 2). Figure 1 Figure 2

      Conclusion
      Conclusion: ATO has potent in vitro and in vivo activity in NSCLC, and is partially mediated by transcriptional downregulation of TYMS.

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      P3.01-016 - Genetic Alterations and Cellular Properties of Acquired Arsenic Resistance in Small Cell Lung Cancer (ID 1624)

      09:30 - 16:30  |  Author(s): J.C.M. Ho

      • Abstract

      Background
      Background: Small cell lung cancer (SCLC) is notoriously a highly fatal disease, with rapid emergence of resistance to first-line chemotherapy. Arsenic trioxide (ATO) has been used as a standard treatment for acute promyelocytic leukemia for the past decade. Our recent data have demonstrated in vitro activity of ATO, with clinically relevant concentrations, in SCLC cell line model. Although induced oxidative stress has been implicated as a possible mechanism of cytotoxicity, the exact mechanism of action of ATO in SCLC is not fully elucidated. In this study, we further explore the potential genetic changes and biological properties of acquired resistance to ATO in SCLC.

      Methods
      Methods: Using H841 SCLC cell line as a model, a daughter cell line resistant to ATO (H841AR) was established by culturing H841 cells in medium with progressively increasing concentrations of ATO for 12 months. Resistant clones were then selected and cultured in HITIS medium containing 20 μM ATO. Total RNA was extracted from both H841 and H841AR cells, followed by reverse transcription and hybridization with Affymetrix EXON 1.0 ST array. Gene chip signals were analyzed by Gene Spring 12 software. Cell proliferation (MTT) assay, wound healing (migration) experiment and invasion assay were performed to compare between H841 and H841AR cells.

      Results
      Results: Exponential growth of H841AR cells was shown in HITIS medium with 20 μM ATO. Comparing with H841 cells, 17 genes were upregulated by at least 5-fold in H841AR cells, consisting of stress response factors, immune system regulators and proliferation-associated or transcriptional factors. Similarly, 45 genes were downregulated by more than 5-fold in H841AR compared with H841 cells, mainly involving angiogenesis, signal transduction and neurodifferentiation. Apart from resistance to ATO (resistant index (RI) = 12.5 comparing H841AR cells with H841 cells), H841AR cells were also slightly resistant to cisplatin (RI = 2) compared with H841 cells. H841AR cells demonstrated faster proliferation, with greater migration and invasion properties.

      Conclusion
      Conclusion: There are distinct genetic alterations and biological properties in SCLC cell line (H841AR) with acquired resistance to ATO. Further analysis of the functional significance of various genetic alterations in ATO-resistant SCLC cell line is warranted. This study is partly funded by the CRCG Small Project Funding from the University of Hong Kong