Abstract 1846: Co-treatment with simvastatin and cetuximab in KRAS mutant LoVo cells decreases PTK activity effectively and increases STK activity to overcome the anti-EGFR resistance
Authors: Krens Lisanne , Pablo-Baak Renee , Mommersteeg Monique , Hilhorst Maria , Ruijtenbeek Rob , Guchelaar Henk-Jan , Straaten Tahar van der 
Published: May 2015

Abstract
Background:
40% of colorectal cancer patients have mutations in the KRAS gene, resulting in auto activation of KRAS and EGFR independent proliferation. Those patients are excluded from therapy with anti-EGFR antibodies. Activation of KRAS depends on its prenylation. The synthesis of prenyl groups is reduced by statins that are used as cholesterol lowering therapy by inhibiting HMG-CoA reductase. We hypothesize that KRAS mutated cell lines may become susceptible for cetuximab in the presence of simvastatin. To further elucidate the effect of these drugs on signal transduction we established kinase activity profiles using dynamic peptide microarrays.
Methods:
The cell lines LoVo, A431 and HCT116 were incubated for 24 hours with vehicle, simvastatin, cetuximab or combinations followed by lysis. Serine/threonine (STK) and tyrosine (PTK) kinase activity profiles were determined for all lysates in vitro using Pamgene's PTK and STK peptide microarrays comprising each 144 peptides derived from human protein phosphorylation sites.
Results:
In the KRAS mutant colorectal cancer cell line LoVo, the combination of simvastatin and cetuximab resulted in a synergistic growth reduction of 50%. No synergistic effects were observed in A431 (EGFR amplification) and only a small effect in HTC116 (KRAS mutant, PI3K mutant)
For LoVo, treatment with cetuximab reduced the tyrosine kinase activity, whereas simvastatin had no significant effect. The combination treatment reduced tyrosine kinase activity even further. The treatment with either cetuximab or simvastatin increased activity of serine/threonine kinases. The combination resulted in an increase slightly less than for the single agent treatments.
The kinomic results from the reference cell lines A431 and HTC116 were consistent with their proliferation responses as single agent or combination treatment.
Conclusions:
In LoVo cells, the synergistic decrease of growth by simvastatin and cetuximab could be explained by the block of EGFR and downstream PTK signalling by cetuximab. This inhibition, by the single agent is sufficient in A431, however, in case of LoVo cells, insufficient to reduce cell proliferation probably because of the constitutively active KRAS. By preventing KRAS prenylation by simvastatin, cell proliferation in LoVo is effectively reduced. The STK activity is increased in all treatment conditions and this could be due to stress mechanisms or in case of the combination therapy due to initiation of STK controlled apoptosis pathways leading to reduced cell proliferation. From these results, we conclude that in KRAS mutated cells, co-treatment with simvastatin and cetuximab decreases PTK activity effectively and increases STK activity which provides a molecular explanation for overcoming the anti-EGFR resistance.
Citation Format: Lisanne Krens, Renee Pablo-Baak, Monique Mommersteeg, Maria Hilhorst, Rob Ruijtenbeek, Henk-Jan Guchelaar, Tahar van der Straaten. Co-treatment with simvastatin and cetuximab in KRAS mutant LoVo cells decreases PTK activity effectively and increases STK activity to overcome the anti-EGFR resistance. [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 1846. doi:10.1158/1538-7445.AM2014-1846