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. 2017 Nov 6;8(66):110016-110028.
doi: 10.18632/oncotarget.22454. eCollection 2017 Dec 15.

HDAC inhibition as a treatment concept to combat temsirolimus-resistant bladder cancer cells

Eva Juengel  1   2 , Ramin Najafi  1 , Jochen Rutz  1 , Sebastian Maxeiner  1 , Jasmina Makarevic  1 , Frederik Roos  1   2 , Igor Tsaur  1   2 , Axel Haferkamp  1   2 , Roman A Blaheta  1
Affiliations

HDAC inhibition as a treatment concept to combat temsirolimus-resistant bladder cancer cells

Eva Juengel et al. Oncotarget. .

Abstract

Introduction: Although the mechanistic target of rapamycin (mTOR) might be a promising molecular target to treat advanced bladder cancer, resistance develops under chronic exposure to an mTOR inhibitor (everolimus, temsirolimus). Based on earlier studies, we proposed that histone deacetylase (HDAC) blockade might circumvent resistance and investigated whether HDAC inhibition has an impact on growth of bladder cancer cells with acquired resistance towards temsirolimus.

Results: The HDAC inhibitor valproic acid (VPA) significantly inhibited growth, proliferation and caused G0/G1 phase arrest in RT112res and UMUC-3res. cdk1, cyclin B, cdk2, cyclin A and Skp1 p19 were down-regulated, p27 was elevated. Akt-mTOR signaling was deactivated, whereas acetylation of histone H3 and H4 in RT112res and UMUC-3res increased in the presence of VPA. Knocking down cdk2 or cyclin A resulted in a significant growth blockade of RT112res and UMUC-3res.

Materials and methods: Parental (par) and resistant (res) RT112 and UMUC-3 cells were exposed to the HDAC inhibitor VPA. Tumor cell growth, proliferation, cell cycling and expression of cell cycle regulating proteins were then evaluated. siRNA blockade was used to investigate the functional impact of the proteins.

Conclusions: HDAC inhibition induced a strong response of temsirolimus-resistant bladder cancer cells. Therefore, the temsirolimus-VPA-combination might be an innovative strategy for bladder cancer treatment.

Keywords: HDAC inhibition; bladder cancer; temsirolimus-resistance; tumor growth; valproic acid (VPA).

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Conflict of interest statement

CONFLICTS OF INTEREST None.

Figures

Figure 1
Figure 1
Growth of parental (par) and temsirolimus-resistant (res) bladder cancer cells, RT112 (A) and UMUC-3 (B). Temsirolimus-resistant cells were exposed to 1 μmol/ml temsirolimus three times a week. Cells were treated with VPA [1 mmol/ml] in the 96-well-plates for 24 h, 48 h and 72 h. Controls remained untreated. Cell number was set to 100% after 24h incubation. Bars indicate standard deviation (SD). *indicates significant difference to untreated control cells, p ≤ 0.05. n = 5.
Figure 2
Figure 2. Proliferation of RT112par and RT112res
Temsirolimus-resistant cells were exposed to temsirolimus [1 μmol/ml] three times a week. Tumor cells were further treated with VPA [1 mmol/ml] in the BrdU assay for 24 h or 48 h. Controls remained untreated. (A) BrdU incorporation [RFU] for each sample. (B) % difference of VPA treated cells to controls without VPA. Bars indicate standard deviation (SD). *indicates significant difference to control, #indicates significant difference to parental cells, p ≤ 0.05. n = 5.
Figure 3
Figure 3. Proliferation of UMUC-3par and UMUC-3res
Temsirolimus-resistant cells were exposed to 1 μmol/ml temsirolimus three times a week. Tumor cells were further treated with VPA [1 mmol/ml] in the BrdU assay for 24 h or 48 h. Controls remained untreated. (A) BrdU incorporation [RFU] for each sample. (B) % difference of VPA treated cells to controls without VPA. Bars indicate standard deviation (SD). *indicates significant difference to control, #indicates significant difference to parental cells, p ≤ 0.05. n = 5.
Figure 4
Figure 4. Cell distribution in the different cell cycle phases
(A) Percentage of parental and resistant RT112 in G01/1, S and G2/M phase is indicated. Bladder cancer cells were pre-treated with VPA [1 mmol/ml] for 3 days. Controls remained untreated. One representative of three separate experiments is shown. (B) % difference of RT112par and RT112res exposed to VPA [1 mmol/ml] compared with the corresponding untreated controls. Control phases were set to 100%. Bars indicate standard deviation (SD). *indicates significant difference to control, p ≤ 0.05. n = 5.
Figure 5
Figure 5. Cell distribution in the different cell cycle phases
(A) Percentage of parental and resistant UMUC-3 in G01/1, S and G2/M phase is indicated. Bladder cancer cells were pre-treated with VPA [1 mmol/ml] for 3 days. Controls remained untreated. One representative of three separate experiments is shown. (B) % difference of UMUC-3par and UMUC-3res exposed to VPA [1 mmol/ml] compared with the corresponding untreated controls. Control phases were set to 100%. Bars indicate standard deviation (SD). *indicates significant difference to control, p ≤ 0.05. n = 5.
Figure 6
Figure 6. Protein expression profile of cell cycle regulating and targeted proteins in parental and temsirolimus-resistant RT112 (left) and UMUC-3 (right) cells after 3 days exposure to VPA [1 mmol/ml] and untreated controls
ß-actin served as internal control. One representative of three separate experiments is shown.
Figure 7
Figure 7. Functional blocking with siRNA targeting cdk2 and cyclin A of RT112 (upper panel) and UMUC-3 (lower panel)
All Stars Negative Control siRNA served as transfection control (mock). Controls remained untreated. (A) and (B) Protein expression profile of cell cycle regulating proteins of RT112 and UMUC-3 cells after functional blocking with siRNA targeting cdk2 and cyclin A. ß-actin served as internal control. One representative of three separate experiments is shown. (C–F) Tumor cell growth of blocked bladder cancer cells, (C) RT112par, (D) UMUC-3par, (E) RT112res and (F) UMUC-3res. Bars indicate standard deviation (SD). *indicates significant difference to control, p ≤ 0.05. n = 5.
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