In:
Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 109, No. 17 ( 2012-04-24)
Abstract:
Our chemical-genetic approach allows for inhibition of the activity of a kinase of interest with unparalleled specificity. Our study challenges the notion that CDK2 is dispensable. Instead, we identify CDK2 as a potentially useful therapeutic target to inhibit anchorage-independent proliferation of tumor cells that are driven by a variety of oncogenic signals. Additionally, we asked whether CDK2 inhibition would affect MEFs transformed by a variety of different oncogenes. We observed that CDK2 inhibition failed to disrupt cellular proliferation in adherent culture conditions. Interestingly, in nonadherent culture, treatment with 1NM-PP1 drastically reduced the number and size of colonies formed from the analog-sensitive cell lines, regardless of which oncogene was used. These results indicate that specific inhibition of CDK2 can significantly reduce anchorage-independent growth, a hallmark of cancer cells, in the context of multiple oncogenic signals. We next examined the effects of specific CDK2 kinase inhibition in the context of oncogenic transformation, or the progression to cancer. We placed CDK2 AS in the endogenous genomic locus within HCT116 cells, a human colon cancer cell line. We found that, similar to the nontransformed or noncancerous cells, small-molecule inhibition of CDK2 disrupted cellular proliferation in HCT116 cells. Additionally, we determined whether this inhibition of CDK2 affected the ability of HCT116 cells to form tumorspheres when grown on low-adherent plates, a hallmark of cancerous transformation. We indeed found a significant decrease in anchorage-independent tumorsphere formation ( Fig. P1 B ). To investigate this possibility, we examined protein levels of CDK1 and CDK2 following treatment with either siRNAs or 1NM-PP1. As expected, siRNA treatment decreased CDK2 protein expression. Interestingly, CDK2 knockdown also dramatically increased CDK1 protein levels. In contrast, treatment with 1NM-PP1 caused relatively little change in CDK2 protein levels and no appreciable increase in CDK1 expression. We therefore concluded that small-molecule inhibition of CDK2 disrupts MEF cell proliferation, whereas CDK2 knockdown does not, likely attributable to CDK1 compensation. These observations highlight the differences between genetic ablation and small-molecule inhibition of CDK2 and suggest that CDK2 kinase activity may be required for normal cellular proliferation. We generated MEF cells in which the mouse CDK2 is functionally replaced with either a normal WT human CDK2 WT or an analog-sensitive CDK2 (CDK2 AS ) form. Treatment with the inhibitor 1NM-PP1 dramatically decreased proliferation in CDK2 AS cells, whereas CDK2 WT cells were unaffected ( Fig. P1 A , Lower Left ), demonstrating the specificity of the approach. We next treated the MEFs with siRNAs against CDK2. We found that siRNA knockdown of CDK2 actually increased proliferation rates ( Fig. P1 A , Lower Right ), and we hypothesized that this effect might be attributable to compensation by CDK1. In this study, we used a chemical-genetic approach in which we modified kinases so they can be selectively inhibited by engineered ATP analogs, thus rendering these kinases “analog-sensitive.” This approach involves mutating a bulky amino acid residue within the ATP-binding site of the kinase into a smaller residue, thus creating a unique, expanded pocket ( Fig. P1 A , Upper ). Small molecules can be designed to fit in the new pocket and selectively inhibit the modified kinase without affecting other kinases ( 5 ). Here, we use this approach to directly compare the cellular consequences of small-molecule inhibition of CDK2 activity compared with knockdown of CDK2 levels by previous methods that have used siRNA molecules. Additionally, we investigate whether such inhibition can disrupt cellular proliferation in the context of various cancer genes. Prior studies have shown that deletion of CDK2 in mice failed to disrupt cellular proliferation ( 2 ), with similar results obtained using colon cancer cells ( 3 ). One possible explanation is that the mitotic kinase, CDK1, may compensate by binding to the cyclin partners of CDK2 ( 4 ). Small-molecule inhibitors disrupt kinase activity without acutely affecting protein expression. Therefore, these inhibitors can be used to study CDK2 inactivation without potential compensation by other CDKs. Available small molecules against CDK2, however, also inhibit other CDKs. An alternative strategy is therefore needed. Eukaryotic cell division is regulated by a family of enzymes known as cyclin-dependent kinases (CDKs). One member of this family, CDK2, has been implicated to function during the G1/S transition and S phase of the cell cycle, whereby DNA is replicated before mitosis ( 1 ). Because of a proposed role for CDK2 in cell cycle progression, small-molecule CDK2 inhibitors have been pursued as a potential therapy for various cancers. Recent studies, however, have suggested that CDK2 may have a redundant role in cell cycle progression, challenging its potential as a therapeutic target. In this study, we sought to investigate the significance of CDK2 in normal as well as tumor cell proliferation, by adopting a chemical-genetic approach to inhibit CDK2 function selectively in mammalian cells. In contrast to prior studies suggesting that CDK2 was dispensable for cellular proliferation, we found that acute and selective inhibition of CDK2 kinase activity can attenuate the growth of human colon cancer cells or mouse embryo fibroblasts (MEFs) transformed by a variety of different oncogenes grown in nonadherent conditions.
Type of Medium:
Online Resource
ISSN:
0027-8424
,
1091-6490
DOI:
10.1073/pnas.1111317109
Language:
English
Publisher:
Proceedings of the National Academy of Sciences
Publication Date:
2012
detail.hit.zdb_id:
209104-5
detail.hit.zdb_id:
1461794-8
SSG:
11
SSG:
12
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