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Chemical genetics approach to restoring p27Kip1 reveals novel compounds with antiproliferative activity in prostate cancer cells.

Rico-Bautista E, Yang CC, Lu L, Roth GP, Wolf DA - BMC Biol. (2010)

Bottom Line: SMIPs (small molecule inhibitors of p27 depletion) also upregulate p21(Cip)¹, inhibit cellular CDK2 activity, induce G1 delay, inhibit colony formation in soft agar and exhibit preferential cytotoxicity in LNCaP cells relative to normal human fibroblasts.Unlike SMIP001, SMIP004 was found to downregulate SKP2 and to stabilize p27, although neither SMIP is a proteasome inhibitor.Whereas the screening endpoint - nuclear p27 - was robustly modulated by the compounds, SMIP-mediated cell cycle arrest and apoptosis were not strictly dependent on p27 and p21 - a finding that is explained by parallel inhibitory effects of SMIPs on positive cell cycle regulators, including cyclins E and A, and CDK4.

View Article: PubMed Central - HTML - PubMed

Affiliation: Sanford-Burnham Medical Research Institute, La Jolla, CA 92037, USA. erico@sanfordburnham.org

ABSTRACT

Background: The cyclin-dependent kinase (CDK) inhibitor p27(Kip)¹ is downregulated in a majority of human cancers due to ectopic proteolysis by the ubiquitin-proteasome pathway. The expression of p27 is subject to multiple mechanisms of control involving several transcription factors, kinase pathways and at least three different ubiquitin ligases (SCF(SKP)², KPC, Pirh2), which regulate p27 transcription, translation, protein stability and subcellular localization. Using a chemical genetics approach, we have asked whether this control network can be modulated by small molecules such that p27 protein expression is restored in cancer cells.

Results: We developed a cell-based assay for measuring the levels of endogenous nuclear p27 in a high throughput screening format employing LNCaP prostate cancer cells engineered to overexpress SKP2. The assay platform was optimized to Z' factors of 0.48 - 0.6 and piloted by screening a total of 7368 chemical compounds. During the course of this work, we discovered two small molecules of previously unknown biological activity, SMIP001 and SMIP004, which increase the nuclear level of p27 at low micromolar concentrations. SMIPs (small molecule inhibitors of p27 depletion) also upregulate p21(Cip)¹, inhibit cellular CDK2 activity, induce G1 delay, inhibit colony formation in soft agar and exhibit preferential cytotoxicity in LNCaP cells relative to normal human fibroblasts. Unlike SMIP001, SMIP004 was found to downregulate SKP2 and to stabilize p27, although neither SMIP is a proteasome inhibitor. Whereas the screening endpoint - nuclear p27 - was robustly modulated by the compounds, SMIP-mediated cell cycle arrest and apoptosis were not strictly dependent on p27 and p21 - a finding that is explained by parallel inhibitory effects of SMIPs on positive cell cycle regulators, including cyclins E and A, and CDK4.

Conclusions: Our data provide proof-of-principle that the screening platform we developed, using endogenous nuclear p27 as an endpoint, presents an effective means of identifying bioactive molecules with cancer selective antiproliferative activity. This approach, when applied to larger and more diverse sets of compounds with refined drug-like properties, bears the potential of revealing both unknown cellular pathways globally impinging on p27 and novel leads for chemotherapeutics targeting a prominent molecular defect of human cancers.

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Validation of prioritized small molecule inhibitors of p27 depletions (SMIPs). (a) Accumulation of p27 upon treatment with SMIPs. LNCaP-S14 cells were treated for 18 h with the respective SMIPs (40 μM), DMSO (-) or the positive control MG132 (M). The data is representative of three independent experiments. (b) Quantification of three independent immunoblotting experiments by film densitometry and normalization to the loading control tubulin. (c) Target pathway selectivity was evaluated in LNCaP cells stably transfected with NKX3.1-YFP. Cells were treated with 40 μM SMIPs for 18 h. YFP fluorescence was normalized to cell numbers and plotted relative to DMSO treated cells. The graph represents the mean +/- standard deviation (SD) from 16 replicates. (d) Cell type specificity was evaluated by measuring the accumulation of p27Luc in Hela_p27Luc cells. Luciferase activity was measured in total cell lysate from cells treated with 40 μM of the respective SMIPs. The graph is representative of three independent experiments and shows the mean +/- SD from eight replicate measurements. Proteasome inhibitors MG132 and epoxomycin were used as positive controls.
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Figure 4: Validation of prioritized small molecule inhibitors of p27 depletions (SMIPs). (a) Accumulation of p27 upon treatment with SMIPs. LNCaP-S14 cells were treated for 18 h with the respective SMIPs (40 μM), DMSO (-) or the positive control MG132 (M). The data is representative of three independent experiments. (b) Quantification of three independent immunoblotting experiments by film densitometry and normalization to the loading control tubulin. (c) Target pathway selectivity was evaluated in LNCaP cells stably transfected with NKX3.1-YFP. Cells were treated with 40 μM SMIPs for 18 h. YFP fluorescence was normalized to cell numbers and plotted relative to DMSO treated cells. The graph represents the mean +/- standard deviation (SD) from 16 replicates. (d) Cell type specificity was evaluated by measuring the accumulation of p27Luc in Hela_p27Luc cells. Luciferase activity was measured in total cell lysate from cells treated with 40 μM of the respective SMIPs. The graph is representative of three independent experiments and shows the mean +/- SD from eight replicate measurements. Proteasome inhibitors MG132 and epoxomycin were used as positive controls.

Mentions: Several SMIPs also induced p27 accumulation as determined by immunoblotting (Figure 4a and 4b), although the effects were generally weaker (~twofold at 40 μM). While seemingly small, such an increase is biologically significant because p27 levels do not vary more than two- to threefold during a normal cell cycle ([40] and our own unpublished observation). This apparent discrepancy between the immunofluorescence and immunoblotting data, which we had encountered previously during assay development (with positive control compounds roscovitine and MG132; Figure 1c and data not shown), is explained by the different metrics of the assays: While the immunofluorescence assay determines the percentage of cells with nuclear p27 staining above a threshold determined by staining with secondary antibody alone, the immunoblotting assay measures the total amount of p27 that can be extracted from a cell population (no threshold).


Chemical genetics approach to restoring p27Kip1 reveals novel compounds with antiproliferative activity in prostate cancer cells.

Rico-Bautista E, Yang CC, Lu L, Roth GP, Wolf DA - BMC Biol. (2010)

Validation of prioritized small molecule inhibitors of p27 depletions (SMIPs). (a) Accumulation of p27 upon treatment with SMIPs. LNCaP-S14 cells were treated for 18 h with the respective SMIPs (40 μM), DMSO (-) or the positive control MG132 (M). The data is representative of three independent experiments. (b) Quantification of three independent immunoblotting experiments by film densitometry and normalization to the loading control tubulin. (c) Target pathway selectivity was evaluated in LNCaP cells stably transfected with NKX3.1-YFP. Cells were treated with 40 μM SMIPs for 18 h. YFP fluorescence was normalized to cell numbers and plotted relative to DMSO treated cells. The graph represents the mean +/- standard deviation (SD) from 16 replicates. (d) Cell type specificity was evaluated by measuring the accumulation of p27Luc in Hela_p27Luc cells. Luciferase activity was measured in total cell lysate from cells treated with 40 μM of the respective SMIPs. The graph is representative of three independent experiments and shows the mean +/- SD from eight replicate measurements. Proteasome inhibitors MG132 and epoxomycin were used as positive controls.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3025922&req=5

Figure 4: Validation of prioritized small molecule inhibitors of p27 depletions (SMIPs). (a) Accumulation of p27 upon treatment with SMIPs. LNCaP-S14 cells were treated for 18 h with the respective SMIPs (40 μM), DMSO (-) or the positive control MG132 (M). The data is representative of three independent experiments. (b) Quantification of three independent immunoblotting experiments by film densitometry and normalization to the loading control tubulin. (c) Target pathway selectivity was evaluated in LNCaP cells stably transfected with NKX3.1-YFP. Cells were treated with 40 μM SMIPs for 18 h. YFP fluorescence was normalized to cell numbers and plotted relative to DMSO treated cells. The graph represents the mean +/- standard deviation (SD) from 16 replicates. (d) Cell type specificity was evaluated by measuring the accumulation of p27Luc in Hela_p27Luc cells. Luciferase activity was measured in total cell lysate from cells treated with 40 μM of the respective SMIPs. The graph is representative of three independent experiments and shows the mean +/- SD from eight replicate measurements. Proteasome inhibitors MG132 and epoxomycin were used as positive controls.
Mentions: Several SMIPs also induced p27 accumulation as determined by immunoblotting (Figure 4a and 4b), although the effects were generally weaker (~twofold at 40 μM). While seemingly small, such an increase is biologically significant because p27 levels do not vary more than two- to threefold during a normal cell cycle ([40] and our own unpublished observation). This apparent discrepancy between the immunofluorescence and immunoblotting data, which we had encountered previously during assay development (with positive control compounds roscovitine and MG132; Figure 1c and data not shown), is explained by the different metrics of the assays: While the immunofluorescence assay determines the percentage of cells with nuclear p27 staining above a threshold determined by staining with secondary antibody alone, the immunoblotting assay measures the total amount of p27 that can be extracted from a cell population (no threshold).

Bottom Line: SMIPs (small molecule inhibitors of p27 depletion) also upregulate p21(Cip)¹, inhibit cellular CDK2 activity, induce G1 delay, inhibit colony formation in soft agar and exhibit preferential cytotoxicity in LNCaP cells relative to normal human fibroblasts.Unlike SMIP001, SMIP004 was found to downregulate SKP2 and to stabilize p27, although neither SMIP is a proteasome inhibitor.Whereas the screening endpoint - nuclear p27 - was robustly modulated by the compounds, SMIP-mediated cell cycle arrest and apoptosis were not strictly dependent on p27 and p21 - a finding that is explained by parallel inhibitory effects of SMIPs on positive cell cycle regulators, including cyclins E and A, and CDK4.

View Article: PubMed Central - HTML - PubMed

Affiliation: Sanford-Burnham Medical Research Institute, La Jolla, CA 92037, USA. erico@sanfordburnham.org

ABSTRACT

Background: The cyclin-dependent kinase (CDK) inhibitor p27(Kip)¹ is downregulated in a majority of human cancers due to ectopic proteolysis by the ubiquitin-proteasome pathway. The expression of p27 is subject to multiple mechanisms of control involving several transcription factors, kinase pathways and at least three different ubiquitin ligases (SCF(SKP)², KPC, Pirh2), which regulate p27 transcription, translation, protein stability and subcellular localization. Using a chemical genetics approach, we have asked whether this control network can be modulated by small molecules such that p27 protein expression is restored in cancer cells.

Results: We developed a cell-based assay for measuring the levels of endogenous nuclear p27 in a high throughput screening format employing LNCaP prostate cancer cells engineered to overexpress SKP2. The assay platform was optimized to Z' factors of 0.48 - 0.6 and piloted by screening a total of 7368 chemical compounds. During the course of this work, we discovered two small molecules of previously unknown biological activity, SMIP001 and SMIP004, which increase the nuclear level of p27 at low micromolar concentrations. SMIPs (small molecule inhibitors of p27 depletion) also upregulate p21(Cip)¹, inhibit cellular CDK2 activity, induce G1 delay, inhibit colony formation in soft agar and exhibit preferential cytotoxicity in LNCaP cells relative to normal human fibroblasts. Unlike SMIP001, SMIP004 was found to downregulate SKP2 and to stabilize p27, although neither SMIP is a proteasome inhibitor. Whereas the screening endpoint - nuclear p27 - was robustly modulated by the compounds, SMIP-mediated cell cycle arrest and apoptosis were not strictly dependent on p27 and p21 - a finding that is explained by parallel inhibitory effects of SMIPs on positive cell cycle regulators, including cyclins E and A, and CDK4.

Conclusions: Our data provide proof-of-principle that the screening platform we developed, using endogenous nuclear p27 as an endpoint, presents an effective means of identifying bioactive molecules with cancer selective antiproliferative activity. This approach, when applied to larger and more diverse sets of compounds with refined drug-like properties, bears the potential of revealing both unknown cellular pathways globally impinging on p27 and novel leads for chemotherapeutics targeting a prominent molecular defect of human cancers.

Show MeSH
Related in: MedlinePlus