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Profiling global kinome signatures of the radioresistant MCF-7/C6 breast cancer cells using MRM-based targeted proteomics.

Guo L, Xiao Y, Fan M, Li JJ, Wang Y - J. Proteome Res. (2014)

Bottom Line: We rigorously quantified 120 kinases, of which (1)/3 exhibited significant differences in expression levels or ATP binding affinities.Several kinases involved in cell cycle progression and DNA damage response were found to be overexpressed or hyperactivated, including checkpoint kinase 1 (CHK1), cyclin-dependent kinases 1 and 2 (CDK1 and CDK2), and the catalytic subunit of DNA-dependent protein kinase.The unique kinome profiling results also afforded potential effective targets for resensitizing radioresistant cancer cells and counteracting deleterious effects of ionizing radiation exposure.

View Article: PubMed Central - PubMed

Affiliation: Environmental Toxicology Graduate Program and ‡Department of Chemistry, University of California , Riverside, California 92521-0403, United States.

ABSTRACT
Ionizing radiation is widely used in cancer therapy; however, cancer cells often develop radioresistance, which compromises the efficacy of cancer radiation therapy. Quantitative assessment of the alteration of the entire kinome in radioresistant cancer cells relative to their radiosensitive counterparts may provide important knowledge to define the mechanism(s) underlying tumor adaptive radioresistance and uncover novel target(s) for effective prevention and treatment of tumor radioresistance. By employing a scheduled multiple-reaction monitoring analysis in conjunction with isotope-coded ATP affinity probes, we assessed the global kinome of radioresistant MCF-7/C6 cells and their parental MCF-7 human breast cancer cells. We rigorously quantified 120 kinases, of which (1)/3 exhibited significant differences in expression levels or ATP binding affinities. Several kinases involved in cell cycle progression and DNA damage response were found to be overexpressed or hyperactivated, including checkpoint kinase 1 (CHK1), cyclin-dependent kinases 1 and 2 (CDK1 and CDK2), and the catalytic subunit of DNA-dependent protein kinase. The elevated expression of CHK1, CDK1, and CDK2 in MCF-7/C6 cells was further validated by Western blot analysis. Thus, the altered kinome profile of radioresistant MCF-7/C6 cells suggests the involvement of kinases on cell cycle progression and DNA repair in tumor adaptive radioresistance. The unique kinome profiling results also afforded potential effective targets for resensitizing radioresistant cancer cells and counteracting deleterious effects of ionizing radiation exposure.

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GO and KEGG pathway analysis of kinases detected in theLC–MRManalysis. The number of kinases in the (A) KEGG pathways and (B) GOcellular components with p-values <0.05 are listed.Displayed are also the significantly (C) down- and (D) up-regulatedkinases involved KEGG pathways in MCF-7/C6 relative to MCF-7/WT cells.
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fig4: GO and KEGG pathway analysis of kinases detected in theLC–MRManalysis. The number of kinases in the (A) KEGG pathways and (B) GOcellular components with p-values <0.05 are listed.Displayed are also the significantly (C) down- and (D) up-regulatedkinases involved KEGG pathways in MCF-7/C6 relative to MCF-7/WT cells.

Mentions: In the current study, we wereable to rigorously quantify 120 kinases from one forward and two reverselabeling experiments (Table S2, Supporting Information). The average relative standard deviation for all the quantifiedkinases is 14%. Thus, we employed cutoff ratios of >1.5 and <0.67to define the significantly up- and down-regulated kinases, respectively.With the use of this criterion, 24 and 13 of the quantified kinaseswere significantly up- and down-regulated in MCF-7/C6 relative toMCF-7/WT cells, respectively (Table S3, SupportingInformation and Figure 3). To the bestof our knowledge, this represents the first in-depth analysis of thealteration of the entire kinome after breast cancer cells developradioresistance. Kinases from all kinase families except that theRGC and PKL families were successfully detected. These kinases covera major part of cellular components and are involved in a large collectionof cellular pathways, as revealed by GO and KEGG pathway analysis(Figure 4). Figure 4, panels A and B list the GO cellular components and KEGG pathwaysas well as the number of kinases in each component or pathway witha p-value <0.05. For example, 25, 10, and 8 keykinase modulators involved in MAPK, Toll-like receptor (TLR), andErbB signaling pathways were successfully quantified with our method.Among them, TLR signaling pathway is well-known for its critical rolein innate immune response.32 Some membersof TLRs such as TLR5 and TLR9 were found to trigger cancer recurrenceafter radiotherapy.33,34


Profiling global kinome signatures of the radioresistant MCF-7/C6 breast cancer cells using MRM-based targeted proteomics.

Guo L, Xiao Y, Fan M, Li JJ, Wang Y - J. Proteome Res. (2014)

GO and KEGG pathway analysis of kinases detected in theLC–MRManalysis. The number of kinases in the (A) KEGG pathways and (B) GOcellular components with p-values <0.05 are listed.Displayed are also the significantly (C) down- and (D) up-regulatedkinases involved KEGG pathways in MCF-7/C6 relative to MCF-7/WT cells.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4286165&req=5

fig4: GO and KEGG pathway analysis of kinases detected in theLC–MRManalysis. The number of kinases in the (A) KEGG pathways and (B) GOcellular components with p-values <0.05 are listed.Displayed are also the significantly (C) down- and (D) up-regulatedkinases involved KEGG pathways in MCF-7/C6 relative to MCF-7/WT cells.
Mentions: In the current study, we wereable to rigorously quantify 120 kinases from one forward and two reverselabeling experiments (Table S2, Supporting Information). The average relative standard deviation for all the quantifiedkinases is 14%. Thus, we employed cutoff ratios of >1.5 and <0.67to define the significantly up- and down-regulated kinases, respectively.With the use of this criterion, 24 and 13 of the quantified kinaseswere significantly up- and down-regulated in MCF-7/C6 relative toMCF-7/WT cells, respectively (Table S3, SupportingInformation and Figure 3). To the bestof our knowledge, this represents the first in-depth analysis of thealteration of the entire kinome after breast cancer cells developradioresistance. Kinases from all kinase families except that theRGC and PKL families were successfully detected. These kinases covera major part of cellular components and are involved in a large collectionof cellular pathways, as revealed by GO and KEGG pathway analysis(Figure 4). Figure 4, panels A and B list the GO cellular components and KEGG pathwaysas well as the number of kinases in each component or pathway witha p-value <0.05. For example, 25, 10, and 8 keykinase modulators involved in MAPK, Toll-like receptor (TLR), andErbB signaling pathways were successfully quantified with our method.Among them, TLR signaling pathway is well-known for its critical rolein innate immune response.32 Some membersof TLRs such as TLR5 and TLR9 were found to trigger cancer recurrenceafter radiotherapy.33,34

Bottom Line: We rigorously quantified 120 kinases, of which (1)/3 exhibited significant differences in expression levels or ATP binding affinities.Several kinases involved in cell cycle progression and DNA damage response were found to be overexpressed or hyperactivated, including checkpoint kinase 1 (CHK1), cyclin-dependent kinases 1 and 2 (CDK1 and CDK2), and the catalytic subunit of DNA-dependent protein kinase.The unique kinome profiling results also afforded potential effective targets for resensitizing radioresistant cancer cells and counteracting deleterious effects of ionizing radiation exposure.

View Article: PubMed Central - PubMed

Affiliation: Environmental Toxicology Graduate Program and ‡Department of Chemistry, University of California , Riverside, California 92521-0403, United States.

ABSTRACT
Ionizing radiation is widely used in cancer therapy; however, cancer cells often develop radioresistance, which compromises the efficacy of cancer radiation therapy. Quantitative assessment of the alteration of the entire kinome in radioresistant cancer cells relative to their radiosensitive counterparts may provide important knowledge to define the mechanism(s) underlying tumor adaptive radioresistance and uncover novel target(s) for effective prevention and treatment of tumor radioresistance. By employing a scheduled multiple-reaction monitoring analysis in conjunction with isotope-coded ATP affinity probes, we assessed the global kinome of radioresistant MCF-7/C6 cells and their parental MCF-7 human breast cancer cells. We rigorously quantified 120 kinases, of which (1)/3 exhibited significant differences in expression levels or ATP binding affinities. Several kinases involved in cell cycle progression and DNA damage response were found to be overexpressed or hyperactivated, including checkpoint kinase 1 (CHK1), cyclin-dependent kinases 1 and 2 (CDK1 and CDK2), and the catalytic subunit of DNA-dependent protein kinase. The elevated expression of CHK1, CDK1, and CDK2 in MCF-7/C6 cells was further validated by Western blot analysis. Thus, the altered kinome profile of radioresistant MCF-7/C6 cells suggests the involvement of kinases on cell cycle progression and DNA repair in tumor adaptive radioresistance. The unique kinome profiling results also afforded potential effective targets for resensitizing radioresistant cancer cells and counteracting deleterious effects of ionizing radiation exposure.

Show MeSH
Related in: MedlinePlus