Author of

• 11515

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  P2.05 - Poster Session 2 - Preclinical Models of Therapeutics/Imaging (ID 158)

  • Event: WCLC 2013
  • Type: Poster Session
  • Track: Biology
  • Presentations: 1
  • Moderators:
  • Coordinates: 10/29/2013, 09:30 - 16:30, Exhibit Hall, Ground Level

  • 11528

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    P2.05-013 - Silencing the <em>TUBB3</em> gene i<em>n vivo</em> sensitises non-small cell lung cancer to cisplatin (ID 2178)

    09:30 - 09:30  |  Author(s): A. Gobel
    • Abstract

    Background
    Lung cancer is the most common cause of cancer death. Despite advances in treatment of other cancers, overall 5-year survival rates for advanced non-small cell lung cancer (NSCLC) are dismal. Expression of βIII-tubulin (encoded by TUBB3 gene) is associated with resistance to tubulin-binding agents in a range of tumor types including NSCLC[1]. We identified a multifactorial role for βIII-tubulin in chemosensitivity by showing that it can mediate response not only to tubulin-binding agents, but also DNA damaging agents in NSCLC[2, 3], validating βIII-tubulin as a therapeutic target in NSCLC. We hypothesized that in vivo targeting of the TUBB3 gene would sensitise NSCLC tumours to chemotherapy.

    Methods
    DNA-directed RNA interference (ddRNAi) is a potent gene silencing approach that can achieve prolonged suppression of target genes. We developed a triple cassette shRNA vector containing three unique targets against the TUBB3 gene (βIII~TRP~) and a control vector containing three non-targeting sequences (Cont~TRP~). Gene and protein analysis was achieved using RT-qPCR and western blotting respectively. An orthotopic mouse xenograft model of NSCLC was used to evaluate gene ddRNAi delivery, efficacy and chemo sensitisation. Mouse tumour growth was monitored using non-invasive xenogen imaging. Statistical analysis included Mann–Whitney test and Log rank (Mantel-Cox).

    Results
    Transient transfection of NSCLC H460 cells with the βIII~TRP~ vector resulted in >40% decrease of TUBB3 gene expression and a concomitant decrease in βIII-tubulin protein level compared to the Cont~TRP~ vector. This led to increased in vitro sensitivity to both paclitaxel and cisplatin. Using a clinically relevant orthotopic model of NSCLC we treated H460 tumour-bearing mice with the βIII~TRP~ vector encapsulated in a lipophilic delivery agent and this resulted in a potent decrease (71.6%) in TUBB3 expression in tumours compared to Cont~TRP~ vector (P=0.03). Median survival increased by 6 days in the Cont~TRP~~+ cisplatin~ versus Cont~TRP~ groups although this increase was not significant. In contrast, median survival increased by 21 days in the βIII~TRP + cisplatin~ versus βIII~TRP+ vehicle~ groups (P=0.003). Importantly, in vivo suppression of TUBB3 combined with cisplatin treatment led to a significant increase in overall survival compared to βIII~TRP + vehicle~ control mice (P=0.0174).

    Conclusion
    This study provides the first evidence that a vector-based gene suppression approach can potently silence TUBB3 expression and increase chemosensitivity in vivo, highlighting this as a promising treatment strategy for drug refractory NSCLC. References 1. Kavallaris M. Microtubules and resistance to tubulin-binding agents. Nat Rev Cancer. 2010;10:194-204. 2. McCarroll JA, Gan PP, Liu M, Kavallaris M. betaIII-tubulin is a multifunctional protein involved in drug sensitivity and tumorigenesis in non-small cell lung cancer. Cancer Res. 2010;70:4995-5003. 3. Gan PP, Pasquier E, Kavallaris M. Class III beta-tubulin mediates sensitivity to chemotherapeutic drugs in non small cell lung cancer. Cancer Res. 2007;67:9356-63.