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. 2014 Jun 16:13:152.
doi: 10.1186/1476-4598-13-152.

HDAC-inhibition counteracts everolimus resistance in renal cell carcinoma in vitro by diminishing cdk2 and cyclin A

Eva Juengel, Snigdha Nowaz, Jasmina Makarevi, Iyad Natsheh, Isabella Werner, Karen Nelson, Michael Reiter, Igor Tsaur, Jens Mani, Sebastian Harder, Georg Bartsch, Axel Haferkamp, Roman A Blaheta  1
Affiliations

HDAC-inhibition counteracts everolimus resistance in renal cell carcinoma in vitro by diminishing cdk2 and cyclin A

Eva Juengel et al. Mol Cancer. .

Abstract

Background: Targeted therapies have improved therapeutic options of treating renal cell carcinoma (RCC). However, drug response is temporary due to resistance development.

Methods: Functional and molecular changes in RCC Caki-1 cells, after acquired resistance to the mammalian target of rapamycin (mTOR)-inhibitor everolimus (Cakires), were investigated with and without additional application of the histone deacetylase (HDAC)-inhibitor valproic acid (VPA). Cell growth was evaluated by MTT assay, cell cycle progression and apoptosis by flow cytometry. Target molecules of everolimus and VPA, apoptotic and cell cycle regulating proteins were investigated by western blotting. siRNA blockade was performed to evaluate the functional relevance of the proteins.

Results: Everolimus resistance was accompanied by significant increases in the percentage of G2/M-phase cells and in the IC50. Akt and p70S6K, targets of everolimus, were activated in Cakires compared to drug sensitive cells. The most prominent change in Cakires cells was an increase in the cell cycle activating proteins cdk2 and cyclin A. Knock-down of cdk2 and cyclin A caused significant growth inhibition in the Cakires cells. The HDAC-inhibitor, VPA, counteracted everolimus resistance in Cakires, evidenced by a significant decrease in tumor growth and cdk2/cyclin A.

Conclusion: It is concluded that non-response to everolimus is characterized by increased cdk2/cyclin A, driving RCC cells into the G2/M-phase. VPA hinders everolimus non-response by diminishing cdk2/cyclin A. Therefore, treatment with HDAC-inhibitors might be an option for patients with advanced renal cell carcinoma and acquired everolimus resistance.

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Figures

Figure 1
Figure 1
Dose response analysis of Cakipar and Cakires. (A) Growth efficacy of increasing concentrations of everolimus on Cakipar and Cakires cell growth from 24 h to 72 h. *Indicates significant difference to controls, set to 100% (SD ≤ 16%, n = 6). IC50-values of Cakipar and Cakires cells were calculated from dose response analysis. (B) Cell cycle analysis of unsynchronized and synchronized Cakipar and Cakires cells in subconfluent cell cultures. The cell phase is expressed as percentage of total cells. The figure shows one of five separate experiments.
Figure 2
Figure 2
Cell cycle analysis of Cakipar (A) and Cakires (B) after 24 h exposure to 1, 5 and 50 nM everolimus in subconfluent cell cultures. The cell population is expressed as percentage of the total cells analyzed. Untreated cells served as controls. One representative experiment of six is shown.
Figure 3
Figure 3
Western blot analysis of cell cycle regulating proteins and everolimus target molecules. Cakipar and Cakires treated with 1 nM everolimus for 24 h. Controls remained untreated. β-actin served as the internal control. The figure shows one representative from three separate experiments.
Figure 4
Figure 4
Cell growth analysis. Growth inhibitory efficacy of 1 nM or 1 mM VPA in Cakipar and Cakires cells treated for one (A) or two weeks (B). *Indicates significant difference to untreated controls, set at 100%. #Indicates significant difference between Cakipar + VPA and Cakires + VPA (SD ≤ 22%, n = 6).
Figure 5
Figure 5
Western blot analysis of Cakires exposed to 1 mM VPA for one (1 wk) or two weeks (2 wks) showing cell cycle regulating proteins and target molecules of mTOR- (A) and HDAC-inhibitors (B). VPA untreated cells served as controls. β-actin served as the internal control. The figure shows one representative from three separate experiments.
Figure 6
Figure 6
siRNA knock-down controls. siRNA blockade of cdk2 or cyclin A (A) and HDAC1 or HDAC2 (B) were confirmed by western blotting. Cakipar and Cakires cells were transfected with cdk2, cyclin A, HDAC1 or HDAC2 siRNA. Untreated cells and with control siRNA transfected cells served as controls. One representative from three experiments is shown.
Figure 7
Figure 7
Influence of cdk2, cyclin A, HDAC1 or HDAC2 knock-down on tumor cell growth. Cakipar(A) and Cakires(B) cells were transfected with cdk2, cyclin A HDAC1 or HDAC2 siRNA and then subjected to the MTT cell growth assay. Untreated cells and with control siRNA transfected cells served as controls. Cell number of control Cakipar or Cakires cells and the corresponding knock-down cells were measured. *Indicates significant difference to untreated and siRNA controls (SD ≤ 18%, n = 6).
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