Activation of autophagy triggers mitochondrial loss and changes acetylation profile relevant for mechanotransduction in bladder cancer cells

Abstract

Bladder cells are constantly exposed to multiple xenobiotics and bioactive metabolites. In addition to this challenging chemical environment, they are also exposed to shear stress originating from urine and interstitial fluids. Hence, physiological function of bladder cells relies on a high biochemical and biomechanical adaptive competence, which, in turn, is largely supported via autophagy-related mechanisms. As a negative side of this plasticity, bladder cancer cells are known to adapt readily to chemotherapeutic programs. At the molecular level, autophagy was described to support resistance against pharmacological treatments and to contribute to the maintenance of cell structure and metabolic competence. In this study, we enhanced autophagy with rapamycin (1-100 nM) and assessed its effects on the motility of bladder cells, as well as the capability to respond to shear stress. We observed that rapamycin reduced cell migration and the mechanical-induced translocation potential of Krüppel-like transcription factor 2 (KLF2). These effects were accompanied by a rearrangement of cytoskeletal elements and mitochondrial loss. In parallel, intracellular acetylation levels were decreased. Mechanistically, inhibition of the NAD + -dependent deacetylase sirtuin-1 (SIRT1) with nicotinamide (NAM; 0.1-5 mM) restored acetylation levels hampered by rapamycin and cell motility. Taken together, we described the effects of rapamycin on cytoskeletal elements crucial for mechanotransduction and the dependency of these changes on the mitochondrial turnover caused by autophagy activation. Additionally, we could show that targeted metabolic intervention could revert the outcome of autophagy activation, reinforcing the idea that bladder cells can easily adapt to multiple xenobiotics and circumvent in this way the effects of single chemicals.

Keywords: Acetylation; Migration; Mitophagy; Rapamycin; Shear stress (fluid); T24 bladder cancer cells.

Fig. 1

Changes in migration of T24 cells when exposed to rapamycin (RAPA) and/or shear stress a quantification of the gap-closure assay, results are given as area healed [µm²], calculated as the difference between the initial area of the scratch minus the area at the same coordinates after 24 h incubation (n = 12 optical fields); *indicates a significant difference at Mann–Whitney Test (*p < 0.05; **p < 0.01; ***p < 0.001); b Representative phase contrast images at both t = 0 and t = 24 h, shear stress incubation is indicated by curved arrows (scale bar: 1000 µm); c, d Quantification of the mean fluorescence intensity of KLF2 in cytoplasmic c and nuclear d area taken from the evaluation of n > 50 cells; n.s. indicates no statistical significant difference to control according to the Student's t-test (p > 0.05) * indicates a significant difference in comparison to controls and § among treatments (*§ p < 0.05; **§§ p < 0.01; ***§§§ p < 0.001); e Representative images of actin and KLF2 following 24 h incubation with RAPA (1–10-100 nM) and control cells. f Images depicting shear stress incubation (SHEAR; 2.7 dyn/cm²) or YODA1 (5 µM) treatment respectively; DAPI stained nuclei are depicted in blue, actin in white, KLF2 in red (scale bar: 20 µm)

Fig. 2

Cytoskeletal response to rapamycin. a Representative images of actin and caveolin1 and corresponding quantification, following 24 h incubation with rapamycin (RAPA) and control cells. Data is given as mean fluorescence per ROIs, taken from n > 50 cells; n.s. indicates no significant difference (p > 0.05) for Student’s t-test; Actin is depicted in red, caveolin in teal and nuclei in white; (scale bar: 10 µm). b Comparisons of actin and integrin levels in T24 cells following 24 h RAPA treatment, representative images (scale bar: 10 µm), and quantification of n > 60 ROIs, results are given as mean fluorescence intensity, n.s. indicates no significant difference (p > 0.05) for Student's t-test statistical significance is shown via ** (p < 0.01), *** (p < 0.001) by listing the corresponding p value; Actin is depicted in white, integrins in teal and nuclei in blue. c Representative image of T24 cells after 100 nM RAPA treatment for 24 h, with increased magnification (scale bar: 5 µm); actin is shown as white integrins in teal. d Quantification of the percentage of cell area covered by actin signal n > 60 cells statistical significance is shown via Student’s t-test: * (p < 0.05), ** (p < 0.01), *** (p < 0.001)

Fig. 3

Cholesterol and PMP70 response after 24 h treatment with increasing rapamycin (RAPA) concentrations a representative images of cholesterol staining in control cells and after incubation with rapamycin; cholesterol in blue (x/y axis in 20 µm segmentation) b quantification of the mean fluorescence intensity of cholesterol (n > 60 ROIs) and membrane fluidity (n = 3), taken from the evaluation of 3 independent experiments, n.s. indicates no statistical significant difference to control according to the Student’s and ANOVA tests (p > 0.05), c PMP70 response in control cells and after incubation with RAPA; PMP70 in red, nuclei in blue (scale bar: 10 µm) d quantification of the mean fluorescence intensity of PMP70, from n > 60 cells. Statistically significant differences were calculated via Student’s t-test (p < 1.84E-4)

Fig. 4

TOM20 and CLUH response after 24 h treatment with increasing rapamycin (RAPA) concentrations a representative images of control cells, as well as b cells after incubation with RAPA; TOM20 is depicted in red and CLUH in green, and nuclei in blue (scale bar: 20 µm) c quantification of the mean fluorescence intensity of TOM20 and the ratio of the mitochondrial area to the overall cell area, taken from the evaluation of n > 60 cells., * indicates statistical significant difference to control according to the Student’s t-test * (p < 0.05), or *** (p < 0.001) by listing the corresponding p value.  d CLUH response in control cells and after incubation with RAPA, quantification of the mean fluorescence intensity of CLUH, from n > 60 ROIs, from 3 independent experiments, statistical significant differences were calculated via Student’s t-test as (*p < 0.05), e quantification of the CLUH/TOM20 ratio in control cells and RAPA treated cells, from at least 9 random optical fields, from 3 independent experiments (n > 60 ROIs) statistical significant differences were calculated via Student’s t-test (p < 5.49E-5)

Fig. 5

Changes in acetylation following 24 h rapamycin (RAPA) incubation in T24 cells. a, b Quantification of acetylated lysine in the nuclear (a) and cytoplasmic region of the cell (b). Quantification of cell viability (c) measured as cell biomass (crystal violet assay; n = 3). Changes in acetylation following 24 h incubation with bafilomycin (BAFI) and combination of BAFI and RAPA in T24 cells in the nuclear (d) and cytoplasmic region of the cell (e), results are given as mean fluorescent intensity per cell, n = 27 randomly selected optical field were quantified statistical significance is shown via Student's t-test: *** (p < 0.001). Quantification of the gap-closure assay (f), results are given area healed [µm²], calculated from the initial area of the wound minus the area at the same coordinates after 24 h incubation with BAFI or BAFI and RAPA in combination; * indicates a significant difference at Mann–Whitney test (***: p < 0.001); g Representative images at both t = 0 and t = 24 hours, taken with phase contrast (scale bar: 1000 µm)

Fig. 6

Changes in acetylation and migration following co-incubation with nicotinamide (NAM) and rapamycin (RAPA). a, b Quantification of acetylation in T24 cells; cells were incubated with increasing NAM concentrations with and without RAPA; results are given as the ratio of mean fluorescence intensity of acetylated lysine of the treatment and the average of the control (n = 27 optical fields) statistical significance is shown via Student’s t-test: *** (p < 0.001); cell count is given as average number of DAPI stained nuclei per quantified image c representative images of the experiment; DAPI is shown in blue (nuclei), acetylated lysine in red, (scale bar: 200 µm). e Quantification of the gap-closure assay, results are given area healed [µm²], calculated from the initial area of the wound minus the area at the same coordinates after 24 h incubation; * indicates a significant difference to controls and § among treatments at the Mann–Whitney Test (*p < 0.05; §§§p < 0.001); e Representative images at both t = 0 and t = 24 hours, taken with phase contrast (scale bar: 1000 µm)