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p27Kip1 and Tumors: Characterization of CDKN1B Variants Identified in MEN4 and Breast Cancer
Debora Bencivenga1, Emanuela Stampone1, Jahanzaib Azhar1
1Department of Precision Medicine, University of Campania "L. Vanvitelli", Via Luigi De Crecchio, 7, 80138 Naples, Italy.
Abstract:
p27Kip1 is a key cell cycle gatekeeper governing the timing of Cyclin-dependent kinase (CDK) activation/inactivation and, consequently, cell proliferation. Structurally, the protein is largely unfolded, a feature that strongly increases its plasticity and interactors and enhances the number of regulated cellular processes. p27Kip1, like other intrinsically unstructured proteins, is post-translationally modified on several residues. These modifications affect its cellular localization and address p27Kip1 for specific interactions/functions. Several germline or somatic CDKN1B (the p27Kip1 encoding gene) mutations have been demonstrated to be associated with multiple endocrine neoplasia type 4 (MEN4), hairy cell leukemia, small-intestine neuroendocrine tumors, and breast and prostate cancers. Here, we analyzed the effect of four CDKN1B missense and nonsense mutations found in patients affected by MEN4 or cancers, namely, c.349C>T, p.P117S; c.397C>A, p.P133T; c.487C>T, p.Q163*; and c.511G>T, p.E171*. By transfecting breast cancer cell lines, we observed increased growth and cell motility for all the investigated mutants compared to wild-type p27Kip1 transfected cells. Furthermore, we discovered that the mutant forms exhibited altered phosphorylation on key residues and different localization or degradation mechanisms in comparison to the wild-type protein and suggested a possible region as crucial for the lysosome-dependent degradation of the protein. Finally, the loss of p27Kip1 ability in blocking cell proliferation was in part explained through the different binding efficiency that mutant p27Kip1 forms exhibited with Cyclin/Cyclin-dependent Kinase complexes (or proteins involved indirectly in that binding) with respect to the WT.
Insights
Mutations in the p27Kip1 gene (CDKN1B) increase cancer cell growth and motility. These CDKN1B mutations alter protein function, localization, and interaction with cell cycle regulators, contributing to uncontrolled proliferation in MEN4 and various cancers.
Area of Science:
- Molecular Biology
- Cell Biology
- Genetics
Background:
- p27Kip1 is a crucial cell cycle regulator controlling CDK activity and cell proliferation.
- Intrinsically unstructured proteins like p27Kip1 undergo post-translational modifications affecting their function and localization.
- Germline and somatic CDKN1B mutations are linked to MEN4, leukemia, neuroendocrine tumors, and cancers.
Purpose of the Study:
- To investigate the functional impact of four specific CDKN1B mutations (p.P117S, p.P133T, p.Q163*, p.E171*) found in MEN4 and cancer patients.
- To analyze how these mutations affect p27Kip1 protein stability, localization, phosphorylation, and interaction with cell cycle machinery.
- To elucidate the mechanisms by which CDKN1B mutations contribute to increased cell proliferation and motility.
Main Methods:
- Transfection of breast cancer cell lines with wild-type and mutant CDKN1B constructs.
- Analysis of cell growth and motility.
- Assessment of protein phosphorylation, cellular localization, and degradation pathways.
- Evaluation of p27Kip1 binding efficiency with Cyclin/CDK complexes.
Main Results:
- All investigated CDKN1B mutants led to increased cell growth and motility compared to wild-type p27Kip1.
- Mutant p27Kip1 proteins showed altered phosphorylation patterns, different localization, and modified degradation mechanisms.
- A specific region was identified as potentially crucial for lysosome-dependent degradation of p27Kip1.
- The reduced ability of mutant p27Kip1 to inhibit cell proliferation correlated with decreased binding to Cyclin/CDK complexes.
Conclusions:
- The studied CDKN1B mutations disrupt p27Kip1's cell cycle-inhibitory functions, promoting cancer progression.
- Altered protein stability, localization, and interactions contribute to the oncogenic potential of these CDKN1B mutations.
- Understanding these mechanisms provides insights into therapeutic strategies for cancers associated with CDKN1B alterations.
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