Discovery of a small molecule TUBB3/βIII-tubulin modulator in lung cancer — ASN Events
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Discovery of a small molecule TUBB3/βIII-tubulin modulator in lung cancer (#126)

Felicity Kao 1 2 , Greg Arndt 2 3 , Tim Failes 2 3 , Murray Norris 2 , Maria Kavallaris 1 2
  1. Australian Centre for Nanomedicine, School of Chemical Sciences and Engineering, UNSW, Sydney, NSW, Australia
  2. Children's Cancer Institute Australia, Randwick, NSW, Australia
  3. ACRF Drug Discovery Centre for Childhood Cancer, CCIA, UNSW , Randwick, NSW, Australia

Background: Non-small Cell Lung Cancer (NSCLC) survival rates are dismal and chemotherapy resistance is a significant clinical problem. bIII-tubulin (encoded by TUBB3 gene) is aberrantly expressed and is associated with chemoresistance and tumor aggressiveness in NSCLC1,where it has been identified as a bona fide target for chemosensitisation2,3. In order to understand how TUBB3/bIII-tubulin is regulated in NSCLC cells we sought to identify chemical small molecules that can modulate its expression.

Methods: H460 cells expressing a TUBB3 or GAPDH promoter-luciferase reporter construct were generated and used in drug screening and promoter activity testing. To isolate modulators of TUBB3 promoter activity, a cell-based screen of diverse chemical small molecules and FDA-approved bioactives was performed. Cell viability and proliferation were measured using standard methods. Cell cycle was assessed using flow cytometry, gene and protein expression by RT-PCR and Western blotting, respectively. Microtubule morphology was assessed using immunostaining and confocal microscopy. Drug-treated clonogenic assays were used to quantitate changes in drug sensitivity.

Results: Based upon their ability to modulate TUBB3 promoter activity, we identified two hit compounds, CCI01 and CCI02, and a bioactive compound, RITA. For all three leads we observed: 1) repression in TUBB3 promoter activity which was not a result of cell cytotoxicity; 2) significantly enhanced TUBB3 expression in a time and dose-dependent manner. TUBB3 gene enhancement was translated at the protein level in CCI01 treated H460, but not in CCI02 or RITA treated cells. Additionally, CCI01 did not alter microtubule morphology but enhanced βIII-tubulin immunostaining in two independent NSCLC cell lines, H460 and H1299, compared to control. Importantly, CCI01 enhanced βIII-tubulin expression was functional and led to a significant decrease in in vitro sensitivity to DNA-damaging and tubulin-binding agents in H460 cells.

Conclusion: A novel small molecule TUBB3/βIII-tubulin enhancer has been identified that is able to increase expression of βIII-tubulin in NSCLC and significantly reduce chemosensitivity. Identification of a modulator of TUBB3/βIII-tubulin expression will provide a valuable research tool to probe βIII-tubulin regulation.

  1. 1 Kavallaris. Nature Rev Cancer, 10:194-204, 2010
  2. 2 McCarroll et al., Cancer Res 70:4995-5003, 2010
  3. 3 Gan et al., Cancer Res. 67:9356-9363, 2007