LIMK2 affects microtubule acetylation and TPPP1 levels.

<p>(A) LIMK2-depleted cells have reduced amounts of polymerized tubulin. SHEP cells were transfected with LIMK2 or non-targeting control (NT) siRNA and 72 hours later the soluble (S) and polymerized (P) tubulin fractions were separated by centrifugation and analyzed by immunoblotting. A representative immunoblot of three experiments is shown. The efficiency of the LIMK2 knockdown and the loading control is shown on the left panel. (B) LIMK2 knockdown cells show reduced levels of acetylated tubulin. The numbers below the second panel represent the relative level of acetylated tubulin. Cells transfected with the indicated siRNAs were analyzed by immunoblotting and immunofluorescent staining. Bar = 20 µm. (C) SHEP cells overexpressing LIMK2 have increased levels of acetylated tubulin. SHEP cells overexpressing LIMK2a and LIMK2b as well as BE(2)-C and BE/VCR10 cells were analyzed by immunoblotting. The numbers below the second panel represent the relative level of acetylated tubulin. (D) LIMK2 knockdown cells are more sensitive to microtubule depolymerization induced by microtubule-targeted drugs. SHEP cells transfected with the indicated siRNAs were treated with 0.5 µg/ml nocodazole (Noc) or 0.1 µM vincristine (VCR) for 24 hours and analyzed by immunoblotting. The graph indicates the relative acetylated tubulin levels compared with the NT siRNA control (vehicle) represented as mean ± S.E.M of three independent experiments (*, p < 0.05). (E) LIMK2 and TPPP1 interact in SHEP cells. LIMK2 or TPPP1 were immunoprecipitated from SHEP cell lysates and the respective co-immunoprecipitated TPPP1 (left panel) or LIMK2 (right panel) were detected by immunoblotting. (F) LIMK2 modulates TPPP1 protein levels. Lysates from SHEP cells transfected with LIMK2 or control (NT) siRNA (left panels) and LIMK2a or LIMK2b overexpressing SHEP cells (right panels) were analyzed by immunoblotting. Two different exposures of TPPP1 immunoblot are shown (low and high). The numbers below the top panels in B, C, and F represent the folds change in the indicated protein levels.</p

LIMK2 overexpression results in increased number of multinucleated cells.

<p>(A) A high percentage of BE/VCR10 cells are multinucleated. Fixed BE(2)-C and BE/VCR10 cells were stained with FITC-conjugated anti-α-tubulin antibody and Hoechst. White arrowheads indicate multinucleated cells. Scale bar = 20 µm. The percentage of multinucleated cells shown on the right panel is represented as mean ± S.E.M of three independent experiments (***, p < 0.0001, unpaired t-test). The blots on the left show whole cell lysates of the BE(2)-C and BE/VCR10 cells analyzed by immunoblotting with the indicated antibodies. The numbers below the top panel represent the fold changes in the indicated protein levels. (B) Overexpression of LIMK2a or LIMK2b proteins in SHEP cells increases ploidy. Stable SHEP cell lines expressing HA-tagged LIMK2a or LIMK2b or vector control (pMSCV) were generated by transduction with retroviruses. The left panel shows the relative expression of the ectopic LIMK2 proteins. Cells expressing LIMK2a or LIMK2b were fixed and stained with an anti-HA antibody and Hoechst (middle panel). White arrowheads indicate multinucleated cells. Scale bar = 20 µm. The percentage of multinucleated cells is shown on the right panel represented as mean ± S.E.M of three independent experiments (**, p < 0.001, unpaired t-test). (C) LIMK2 localizes to the mitotic spindle microtubules. An unsynchronized population of NIH-3T3 cells was fixed and immuno-stained with an anti-LIMK2 antibody (red), FITC-conjugated anti-α-tubulin antibody (green) and Hoechst (blue). Scale bar = 20 µm. (D) LIMK2 levels do not change during cell division. SHEP cells were treated with 0.5 µM taxol, 0.1 µM vincristine (VCR) or 1 µM dimethylenastron (DIMEN) for 20 hours and mitotic cells were collected by mitotic shake-off. Half of the cells were used to prepare whole cell lysates that were immunoblotted with anti-LIMK2 as well as anti-GAPDH antibodies. The rest of the cells were fixed, stained with propidium iodide and analyzed by flow cytometry (bottom panels). The cell cycle profiles show the G2/M arrest with the different treatments.</p

LIMK2 regulates microtubule drug-dependent cell cycle arrest.

<p>(A) LIMK2 knockdown enhances the G2/M block induced by microtubule-targeted drugs. Cell cycle analysis of SHEP cells transfected with LIMK2 or non-targeting control (NT) siRNA for 72 hours followed by treatment with 0.5 µM taxol or 0.1 µM vincristine (VCR) for 24 hours. The extent of the G2/M arrest, represented by G2/G1 ratio, is shown on the right as mean ± S.E.M of three independent experiments (*, p < 0.05, unpaired t-test). (B) LIMK2 overexpressing cells reduce the G2/M arrest induced by microtubule-targeted drugs. Cell cycle analysis of LIMK2a or LIMK2b expressing cells treated with 0.5 µM taxol or 0.1 µM vincristine (VCR) for 24 hours. The extent of the G2/M arrest, represented by G2/G1 ratio, is shown on the right as mean ± S.E.M of three independent experiments (*, p < 0.05; **, p < 0.001; ns, non significant, unpaired t-test). (C) LIMK2 overexpressing cells recover faster from the cell cycle block induced by microtubule-targeted drugs. Analysis of the cell cycle recovery after drug wash-out of SHEP cells expressing LIMK2a or LIMK2b treated with 0.5 µM taxol or 0.1 µM vincristine (VCR) for 24 hours. The right panels show the cell cycle profile of the cells 8 hours after drug removal.</p

LIMK2 levels are upregulated by DNA damage agents but not by microtubule-targeted drugs.

<p>(A) LIMK2 expression is not regulated at the transcriptional level by microtubule-targeted drugs. SHEP cells were treated with the indicated drugs for 16 hours. The levels of LIMK2a and LIMK2b mRNA were quantified by qRT-PCR, normalized to the housekeeping gene L32, and plotted as relative expression to the control ± S.E.M of three independent experiments (*, p < 0.05; **, p < 0.001, unpaired t-test). (B) LIMK2 protein levels are induced by genotoxic stress but not by treatment with microtubule-targeted drugs. SHEP cells were treated with 1 µM doxorubicin (Doxo), 10 µM etoposide (Eto) or with taxol or vincristine (VCR) at the indicated concentrations for 24 hours before immunoblotting analysis. Two different exposures of LIMK2 immunoblot are shown (low and high). The numbers below the top panel represent the fold-change in the indicated protein levels. (C) Microtubule-targeted drugs do not affect the stability of LIMK2a or LIMK2b proteins. SHEP cells were transiently transfected with GST-LIMK2a or GST-LIMK2b and 24 hours later, they were incubated with 25 nM taxol or 2 nM vincristine (VCR) for 10 hours. Cells were then pulse-labeled with [³⁵S]-methionine/cysteine and chased in the presence of the microtubule-targeted drugs for the indicated time periods. The GST-tagged proteins were purified with glutathione sepharose beads and analyzed by autoradiography (AR) and immunoblotting with anti-GST antibody. Quantification of the level of GST-LIMK2a and GST-LIMK2b proteins from three separate assays is expressed as mean ± S.E.M of percentage values of the samples at zero time.</p

LIMK2 participates in DNA damage response.

<p>(A) BE/VCR10 cells are more resistant to genotoxic stress compared with the BE(2)-C parental cell line. Confluent monolayers of the BE(2)-C and BE/VCR10 cells were irradiated with 8 mJ/cm² ultraviolet B (UV-B). After 24 hours recovery, adherent and non-adherent cells were collected, stained with propidium iodide and analyzed by flow cytometry. Cell death is represented as the mean percentage of propidium iodide positive cells ± S.E.M of three independent experiments (*, p < 0.05, unpaired t-test) (B) Knockdown of LIMK2 in SHEP cells increases their sensitivity to genotoxic stress. SHEP cells were transfected with LIMK2 or non-targeting control (NT) siRNA and 72 hours later they were treated with 10 µM doxorubicin (Doxo) or 10 µM etoposide (Eto) for 48 hours before cell viability was analyzed by MTT assay. Results are expressed as percentage of the control (vehicle). Mean ± S.E.M of three independent experiments (*, p < 0.05; **, p < 0.001, unpaired t-test). (C) LIMK2 knockdown sensitizes cells to apoptosis induced by DNA damage agents. SHEP cells transfected with the indicated siRNAs were treated with 10 µM doxorubicin (Doxo) or 100 µM etoposide (Eto) for 24 hours and analyzed by immunoblotting. Two different exposures of the cleaved PARP immunoblot are shown (low and high). The graph indicates the relative cleaved PARP levels compared to control (vehicle) and is represented as the mean ± S.E.M of three independent experiments (*, p < 0.05, unpaired t-test). (D) SHEP cells transfected with the indicated siRNAs were treated as in A for 72 hours and apoptosis was analyzed by flow cytometry. The percentage of apoptotic cells is represented by the mean percentage of AnnexinV+/PI- cells ± S.E.M of three independent experiments (*, p < 0.05; **, p < 0.001, unpaired t-test). (E) LIMK2 knockdown promotes G2/M arrest induced by DNA damage. Cell cycle analysis of SHEP cells transfected with the indicated siRNAs and treated with 10 µM doxorubicin (Doxo) for 24 hours. The G2/G1 ratio of three independent experiments is shown on the right as the mean ± S.E.M (*, p < 0.05, unpaired t-test). (F) Cell cycle analysis of LIMK2a or LIMK2b overexpressing cells treated with 10 µM doxorubicin (Doxo) for 24 hours. The extent of the G2/M arrest, represented by G2/G1 ratio, is shown on the right as mean ± S.E.M of three independent experiments (*, p < 0.05; ***, p < 0.0001, unpaired t-test). (G) LIMK2 overexpressing cells are more resistant to DNA damage-induced cell cycle arrest. SHEP cells expressing LIMK2a or LIMK2b were treated with 10 µM doxorubicin (Doxo) for 24 hours before washing out the drugs and the cell cycle recovery was analyzed by flow cytometry. The panel on the right depicts the cells cycle profile 8 hours after drug removal.</p

LIMK2 knockdown sensitizes cells to apoptosis induced by microtubule-targeted drugs.

<p>(A) LIMK2 knockdown increases the sensitivity of SHEP cells to microtubule-targeted drugs. SHEP cells were transfected with LIMK2 or non-targeting control (NT) siRNA. 72 hours after transfection, cells were treated with 0.5 µM taxol or 0.1 µM vincristine (VCR) for 48 hours and cell viability was analyzed by MTT assay. Results are expressed as percentage of the control (vehicle) and represented as mean ± S.E.M of three independent experiments (*, p < 0.05, unpaired t-test). The efficiency of the LIMK2 knockdown in a representative experiment is shown on the right panel. (B) LIMK2-depleted cells show enhanced apoptosis induced by microtubule-targeted drugs. SHEP cells transfected with the indicated siRNAs were treated with 0.5 µg/ml nocodazole (Noc), 0.5 µM taxol or 0.1 µM vincristine (VCR) for 24 hours and analyzed by immunoblots probed with the indicated antibodies. The graph on the right that indicates the relative cleaved PARP levels compared to control (vehicle) as mean ± S.E.M of three independent experiments (*, p < 0.05; **, p < 0.001, unpaired t-test). (C) SHEP cells were transfected with the indicated siRNAs and treated with 0.5 µM taxol or 0.1 µM vincristine (VCR) for 72 hours. Apoptosis was determined by flow cytometry and is shown as the mean percentage of AnnexinV+/PI- cells ± S.E.M of three independent experiments (*, p < 0.05; **, p < 0.001, unpaired t-test).</p

Targeting MDM4 as a Novel Therapeutic Approach in Prostate Cancer Independent of p53 Status

Metastatic prostate cancer is a lethal disease in patients incapable of responding to therapeutic interventions. Invasive prostate cancer spread is caused by failure of the normal anti-cancer defense systems that are controlled by the tumour suppressor protein, p53. Upon mutation, p53 malfunctions. Therapeutic strategies to directly re-empower the growth-restrictive capacities of p53 in cancers have largely been unsuccessful, frequently because of a failure to discriminate responses in diseased and healthy tissues. Our studies sought alternative prostate cancer drivers, intending to uncover new treatment targets. We discovered the oncogenic potency of MDM4 in prostate cancer cells, both in the presence and absence of p53 and also its mutation. We uncovered that sustained depletion of MDM4 is growth inhibitory in prostate cancer cells, involving either apoptosis or senescence, depending on the cell and genetic context. We identified that the potency of MDM4 targeting could be potentiated in prostate cancers with mutant p53 through the addition of a first-in-class small molecule drug that was selected as a p53 reactivator and has the capacity to elevate oxidative stress in cancer cells to drive their death

Targeting MDM4 as a Novel Therapeutic Approach in Prostate Cancer Independent of p53 Status

Metastatic prostate cancer is a lethal disease in patients incapable of responding to therapeutic interventions. Invasive prostate cancer spread is caused by failure of the normal anti-cancer defense systems that are controlled by the tumour suppressor protein, p53. Upon mutation, p53 malfunctions. Therapeutic strategies to directly re-empower the growth-restrictive capacities of p53 in cancers have largely been unsuccessful, frequently because of a failure to discriminate responses in diseased and healthy tissues. Our studies sought alternative prostate cancer drivers, intending to uncover new treatment targets. We discovered the oncogenic potency of MDM4 in prostate cancer cells, both in the presence and absence of p53 and also its mutation. We uncovered that sustained depletion of MDM4 is growth inhibitory in prostate cancer cells, involving either apoptosis or senescence, depending on the cell and genetic context. We identified that the potency of MDM4 targeting could be potentiated in prostate cancers with mutant p53 through the addition of a first-in-class small molecule drug that was selected as a p53 reactivator and has the capacity to elevate oxidative stress in cancer cells to drive their death