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Inhibition of casein kinase 1-epsilon induces cancer-cell-selective, PERIOD2-dependent growth arrest.

Yang WS, Stockwell BR - Genome Biol. (2008)

Bottom Line: We employed a two-step screening strategy using human sarcoma cell lines and human fibroblast-derived isogenic cell lines, and found that short hairpin RNAs targeting CSNK1E, a clock gene that regulates circadian rhythms, can induce selective growth inhibition in engineered tumor cells.Treatment with IC261, a kinase domain inhibitor of casein kinase 1-epsilon (CK1epsilon), a protein product of CSNK1E, showed a similar degree of cancer-cell-selective growth inhibition.These data support the hypothesis that circadian clock genes can control the cell cycle and cell survival signaling, and emphasize a central role of CK1epsilon and PERIOD2 in linking these systems.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biological Sciences, Columbia University, Fairchild Center, Amsterdam Avenue, New York, NY 10027, USA.

ABSTRACT

Background: Kinases are under extensive investigation as targets for drug development. Discovering novel kinases whose inhibition induces cancer-cell-selective lethality would be of value. Recent advances in RNA interference have enabled the realization of this goal.

Results: We screened 5,760 short hairpin RNA clones targeting the human kinome to detect human kinases on which cancer cells are more dependent than normal cells. We employed a two-step screening strategy using human sarcoma cell lines and human fibroblast-derived isogenic cell lines, and found that short hairpin RNAs targeting CSNK1E, a clock gene that regulates circadian rhythms, can induce selective growth inhibition in engineered tumor cells. Analysis of gene-expression data revealed that CSNK1E is overexpressed in several cancer tissue samples examined compared to non-tumorigenic normal tissue, suggesting a positive role of CSNK1E in neogenesis or maintenance. Treatment with IC261, a kinase domain inhibitor of casein kinase 1-epsilon (CK1epsilon), a protein product of CSNK1E, showed a similar degree of cancer-cell-selective growth inhibition. In a search for substrates of CK1epsilon that mediate IC261-induced growth inhibition, we discovered that knocking down PER2, another clock gene involved in circadian rhythm control, rescues IC261-induced growth inhibition.

Conclusion: We identified CK1epsilon as a potential target for developing anticancer reagents with a high therapeutic index. These data support the hypothesis that circadian clock genes can control the cell cycle and cell survival signaling, and emphasize a central role of CK1epsilon and PERIOD2 in linking these systems.

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PERIOD2 is a key substrate of CK1ε that mediates IC261-induced growth inhibition. (a) IC261, a kinase inhibitor of CK1ε, induces growth inhibition in HT1080 cells. (b) IC261 treatment in BJ-derived cell lines showed a similar degree of cancer cell selective growth inhibition as shCSNK1E treatment. (c) Knocking down PER2 in HT1080 cells rescues growth inhibition induced by IC261. HT1080 cells were infected with indicated lentiviruses containing different shRNA clones targeting PER2 (per2_538, per2_539, per2_541, or per2_542). After two days of infection, cells were treated with the indicated concentration of IC261 and percent growth inhibition was determined using alamar blue. Values in (a-c) represent the mean ± standard deviation of triplicate data. (d) Cellular RNAs were prepared from the same set of virus infected cells in (c), and real-time PCR was performed with a PER2-specific primer set to monitor the efficiency of knock down by shRNA clones. (e) Proliferation rate of HT1080 cells infected with the same set of viruses as in (c) was determined using alamar blue assay. Error bars in (d,e) indicate one standard deviation of triplicate data. N.T., non-targeting shRNA clone.
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Figure 5: PERIOD2 is a key substrate of CK1ε that mediates IC261-induced growth inhibition. (a) IC261, a kinase inhibitor of CK1ε, induces growth inhibition in HT1080 cells. (b) IC261 treatment in BJ-derived cell lines showed a similar degree of cancer cell selective growth inhibition as shCSNK1E treatment. (c) Knocking down PER2 in HT1080 cells rescues growth inhibition induced by IC261. HT1080 cells were infected with indicated lentiviruses containing different shRNA clones targeting PER2 (per2_538, per2_539, per2_541, or per2_542). After two days of infection, cells were treated with the indicated concentration of IC261 and percent growth inhibition was determined using alamar blue. Values in (a-c) represent the mean ± standard deviation of triplicate data. (d) Cellular RNAs were prepared from the same set of virus infected cells in (c), and real-time PCR was performed with a PER2-specific primer set to monitor the efficiency of knock down by shRNA clones. (e) Proliferation rate of HT1080 cells infected with the same set of viruses as in (c) was determined using alamar blue assay. Error bars in (d,e) indicate one standard deviation of triplicate data. N.T., non-targeting shRNA clone.

Mentions: These results suggest that chemotherapeutic reagents targeting CK1ε may induce growth arrest and apoptosis with some degree of cancer cell selectivity. To test this hypothesis, we examined the effect of IC261, a kinase inhibitor of CK1ε, in cell culture. IC261 was reported to selectively inhibit casein kinase 1 compared to other protein kinases, by an ATP-competitive mechanism. Moreover, it showed an order of magnitude greater selectivity for CK1δ and CK1ε over other casein kinase 1 isoforms [23]. Treatment with IC261 started to inhibit the growth of HT1080 cells at submicromolar concentrations (Figure 5a). When we tested IC261 in BJ-TERT and BJ-TERT/LT/ST/RASV12 cells, the sensitivity of BJ-TERT/LT/ST/RASV12 cells was greater than that of BJ-TERT cells, which was consistent with the results obtained with shCSNK1E (Figures 5b and 2a). These data suggest that inhibition of the kinase activity of CK1ε is crucial for the observed growth arrest and apoptosis, as opposed to other functions of this protein, such as those mediated by protein-protein interactions. Moreover, as shRNAs targeting CK1δ were not effective in suppressing cell growth during primary screening, the cancer-cell-selective activity of IC261 can likely be attributed to its inhibition of CK1ε (Additional data file 1).


Inhibition of casein kinase 1-epsilon induces cancer-cell-selective, PERIOD2-dependent growth arrest.

Yang WS, Stockwell BR - Genome Biol. (2008)

PERIOD2 is a key substrate of CK1ε that mediates IC261-induced growth inhibition. (a) IC261, a kinase inhibitor of CK1ε, induces growth inhibition in HT1080 cells. (b) IC261 treatment in BJ-derived cell lines showed a similar degree of cancer cell selective growth inhibition as shCSNK1E treatment. (c) Knocking down PER2 in HT1080 cells rescues growth inhibition induced by IC261. HT1080 cells were infected with indicated lentiviruses containing different shRNA clones targeting PER2 (per2_538, per2_539, per2_541, or per2_542). After two days of infection, cells were treated with the indicated concentration of IC261 and percent growth inhibition was determined using alamar blue. Values in (a-c) represent the mean ± standard deviation of triplicate data. (d) Cellular RNAs were prepared from the same set of virus infected cells in (c), and real-time PCR was performed with a PER2-specific primer set to monitor the efficiency of knock down by shRNA clones. (e) Proliferation rate of HT1080 cells infected with the same set of viruses as in (c) was determined using alamar blue assay. Error bars in (d,e) indicate one standard deviation of triplicate data. N.T., non-targeting shRNA clone.
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Figure 5: PERIOD2 is a key substrate of CK1ε that mediates IC261-induced growth inhibition. (a) IC261, a kinase inhibitor of CK1ε, induces growth inhibition in HT1080 cells. (b) IC261 treatment in BJ-derived cell lines showed a similar degree of cancer cell selective growth inhibition as shCSNK1E treatment. (c) Knocking down PER2 in HT1080 cells rescues growth inhibition induced by IC261. HT1080 cells were infected with indicated lentiviruses containing different shRNA clones targeting PER2 (per2_538, per2_539, per2_541, or per2_542). After two days of infection, cells were treated with the indicated concentration of IC261 and percent growth inhibition was determined using alamar blue. Values in (a-c) represent the mean ± standard deviation of triplicate data. (d) Cellular RNAs were prepared from the same set of virus infected cells in (c), and real-time PCR was performed with a PER2-specific primer set to monitor the efficiency of knock down by shRNA clones. (e) Proliferation rate of HT1080 cells infected with the same set of viruses as in (c) was determined using alamar blue assay. Error bars in (d,e) indicate one standard deviation of triplicate data. N.T., non-targeting shRNA clone.
Mentions: These results suggest that chemotherapeutic reagents targeting CK1ε may induce growth arrest and apoptosis with some degree of cancer cell selectivity. To test this hypothesis, we examined the effect of IC261, a kinase inhibitor of CK1ε, in cell culture. IC261 was reported to selectively inhibit casein kinase 1 compared to other protein kinases, by an ATP-competitive mechanism. Moreover, it showed an order of magnitude greater selectivity for CK1δ and CK1ε over other casein kinase 1 isoforms [23]. Treatment with IC261 started to inhibit the growth of HT1080 cells at submicromolar concentrations (Figure 5a). When we tested IC261 in BJ-TERT and BJ-TERT/LT/ST/RASV12 cells, the sensitivity of BJ-TERT/LT/ST/RASV12 cells was greater than that of BJ-TERT cells, which was consistent with the results obtained with shCSNK1E (Figures 5b and 2a). These data suggest that inhibition of the kinase activity of CK1ε is crucial for the observed growth arrest and apoptosis, as opposed to other functions of this protein, such as those mediated by protein-protein interactions. Moreover, as shRNAs targeting CK1δ were not effective in suppressing cell growth during primary screening, the cancer-cell-selective activity of IC261 can likely be attributed to its inhibition of CK1ε (Additional data file 1).

Bottom Line: We employed a two-step screening strategy using human sarcoma cell lines and human fibroblast-derived isogenic cell lines, and found that short hairpin RNAs targeting CSNK1E, a clock gene that regulates circadian rhythms, can induce selective growth inhibition in engineered tumor cells.Treatment with IC261, a kinase domain inhibitor of casein kinase 1-epsilon (CK1epsilon), a protein product of CSNK1E, showed a similar degree of cancer-cell-selective growth inhibition.These data support the hypothesis that circadian clock genes can control the cell cycle and cell survival signaling, and emphasize a central role of CK1epsilon and PERIOD2 in linking these systems.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biological Sciences, Columbia University, Fairchild Center, Amsterdam Avenue, New York, NY 10027, USA.

ABSTRACT

Background: Kinases are under extensive investigation as targets for drug development. Discovering novel kinases whose inhibition induces cancer-cell-selective lethality would be of value. Recent advances in RNA interference have enabled the realization of this goal.

Results: We screened 5,760 short hairpin RNA clones targeting the human kinome to detect human kinases on which cancer cells are more dependent than normal cells. We employed a two-step screening strategy using human sarcoma cell lines and human fibroblast-derived isogenic cell lines, and found that short hairpin RNAs targeting CSNK1E, a clock gene that regulates circadian rhythms, can induce selective growth inhibition in engineered tumor cells. Analysis of gene-expression data revealed that CSNK1E is overexpressed in several cancer tissue samples examined compared to non-tumorigenic normal tissue, suggesting a positive role of CSNK1E in neogenesis or maintenance. Treatment with IC261, a kinase domain inhibitor of casein kinase 1-epsilon (CK1epsilon), a protein product of CSNK1E, showed a similar degree of cancer-cell-selective growth inhibition. In a search for substrates of CK1epsilon that mediate IC261-induced growth inhibition, we discovered that knocking down PER2, another clock gene involved in circadian rhythm control, rescues IC261-induced growth inhibition.

Conclusion: We identified CK1epsilon as a potential target for developing anticancer reagents with a high therapeutic index. These data support the hypothesis that circadian clock genes can control the cell cycle and cell survival signaling, and emphasize a central role of CK1epsilon and PERIOD2 in linking these systems.

Show MeSH
Related in: MedlinePlus