Abstract 2750: Conditional RNAi-mediated knockdown of TPX2 synergizes with vincristine and docetaxel cytotoxicity against HeLa cell sub-clones in vitro and in vivo
Authors: Allmendinger Annette J. , Schönig Kai , Gruss Oliver J. , Berger Stefan , Berger Martin R. 
Published: May 2015

Microtubuli-associated proteins could be potent novel targets for anti-cancer therapy. To test this hypothesis, a HeLa cell line was generated, in which the reverse tetracycline-dependent transactivator rtTA2S-M2 drives the expression of eGFP and a miRNA, inducing RNAi-dependent inhibition of TPX2 expression, from the bidirectional tet-regulated promoter (HeLa EM2-11-TPX2). As a negative control for respective experiments a similar cell line was generated (HeLa EM2-11), in which the presence of the inducer doxycycline (dox) activates expression of the genes mCherry and firefly luciferase. First, the effect of TPX2 knockdown on in vitro proliferation was determined by MTT assay. Here, the respective cells were treated with either a single agent therapy consisting of vincristine (VC) (0.5-2.6 ng/ml) or docetaxel (DCX) (0.125-4 nmol/l) or dox (0.2 μg/ml), or a combination of dox and VC or DCX, and cell viability was assessed after 72h. Treatment with VC or DCX caused a concentration-dependent decrease in proliferation in vitro, whereas knockdown of TPX2 reduced cell growth by 35-45%. The proliferation of HeLa EM2-11-TPX2 was synergistically reduced by the combination treatment dox and VC and additively reduced by dox and DCX when compared to the single agent exposures. Next, the efficiency of tumor growth inhibition following knockdown of TPX2 alone or in combination with VC or DCX was tested in a nude mouse xenograft model. Here, the size of tumors growing in nude mice following s.c. implantation was monitored over a period of 4-5 weeks. Dox (0.2 mg/ml in drinking water) was given continuously from day 14 after cell injection until the end of the observation period. The antimitotic agents VC (1 mg/kg) or DCX (22.3 mg/kg) were applied twice on days 14 and 21. In nude mice, TPX2-knockdown alone caused a 71% reduction in tumor growth. VC or DCX reduced tumor growth by 59% or 76%, respectively. The combination of dox and VC or DCX inhibited tumor growth by 93% or 97%, respectively. Both combination treatments were significantly superior to the respective single treatment arms (p&lt;0.05). We could demonstrate that persistent inactivation of TPX2 expression was highly effective in inhibiting tumor growth, both in vitro and in vivo. The combination of the conditional TPX2-knockdown and VC showed a synergistic therapeutic effect in vitro, whereas the combination of TPX2-knockdown and DCX showed an additive therapeutic effect in vitro. Both combination treatments were significantly superior to the respective single agent treatments in vivo.

It is concluded that TPX2 is a valuable treatment target, causing effective tumor growth inhibition, which can be successfully combined with other anti-mitotic agents.

Citation Format: Annette J. Allmendinger, Kai Schönig, Oliver J. Gruss, Stefan Berger, Martin R. Berger. Conditional RNAi-mediated knockdown of TPX2 synergizes with vincristine and docetaxel cytotoxicity against HeLa cell sub-clones in vitro and in vivo. [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 2750. doi:10.1158/1538-7445.AM2014-2750