1028

CDKN1C

BWCR|BWS|KIP2|WBS|p57;cyclin-dependent kinase inhibitor 1C (p57, Kip2);CDKN1C;cyclin-dependent kinase inhibitor 1C (p57, Kip2)

cyclin-dependent kinase inhibitor 1C|cyclin-dependent kinase inhibitor p57|p57Kip2

11p15.5

protein-coding

Count: 4; Pubmed_search,TAG,Generif,UniProt

Cyclin-dependent kinases (Cdks) are positive regulators of cell proliferation, whereas Cdk inhibitors (CKIs) inhibit proliferation. We describe a new CKI, p57KIP2, which is related to p21CIP1 and p27KIP1. p57KIP2 is a potent, tight-binding inhibitor of several G1 cyclin/Cdk complexes, and its binding is cyclin dependent. Unlike CIP1, KIP2 is not regulated by p53. Overexpression of p57KIP2 arrests cells in G1. p57KIP2 proteins have a complex structure. Mouse p57KIP2 consists of four structurally distinct domains: an amino-terminal Cdk inhibitory domain, a proline-rich domain, an acidic-repeat region, and a carboxy-terminal domain conserved with p27KIP1. Human p57KIP2 appears to have conserved the amino- and carboxy-terminal domains but has replaced the internal regions with sequences containing proline-alanine repeats. In situ hybridization during mouse embryogenesis revealed that KIP2 mRNA displays a striking pattern of expression during development, showing high level expression in skeletal muscle, brain, heart, lungs, and eye. Most of the KIP2-expressing cells are terminally differentiated, suggesting that p57KIP2 is involved in decisions to exit the cell cycle during development and differentiation. Human KIP2 is located at 11p15.5, a region implicated in both sporadic cancers and Beckwith-Wiedemann syndrome, a familial cancer syndrome, marking it as a candidate tumor suppressor. The discovery of a new member of the p21CIP1 inhibitor family with novel structural features and expression patterns suggests a complex role for these proteins in cell cycle control and development.

SMARCB1 is deleted in rhabdoid tumor, an aggressive paediatric malignancy affecting the kidney and CNS. We hypothesized that the oncogenic pathway in rhabdoid tumors involved epigenetic silencing of key cell cycle regulators as a consequence of altered chromatin-remodelling, attributable to loss of SMARCB1, and that this hypothesis if proven could provide a biological rationale for testing epigenetic therapies in this disease. We used an inducible expression system to show that the imprinted cell cycle inhibitor CDKN1C is a downstream target for SMARCB1 and is transcriptionally activated by increased histone H3 and H4 acetylation at the promoter. We also show that CDKN1C expression induces cell cycle arrest, CDKN1C knockdown with siRNA is associated with increased proliferation, and is able to compete against the anti-proliferative effect of restored SMARCB1 expression. The histone deacetylase inhibitor (HDACi), Romidepsin, specifically restored CDKN1C expression in rhabdoid tumor cells through promoter histone H3 and H4 acetylation, recapitulating the effect of SMARCB1 on CDKNIC allelic expression, and induced cell cycle arrest in G401 and STM91-01 rhabdoid tumor cell lines. CDKN1C expression was also shown to be generally absent in clinical specimens of rhabdoid tumor, however CDKN1A and CDKN1B expression persisted. Our observations suggest that maintenance of CDKN1C expression plays a critical role in preventing rhabdoid tumor growth. Significantly, we report for the first time, parallels between the molecular pathways of SMARCB1 restoration and Romidepsin treatment, and demonstrate a biological basis for the further exploration of histone deacetylase inhibitors as relevant therapeutic reagents in the treatment of rhabdoid tumor.

BACKGROUND: CDKN1C (also known as p57KIP2) is a cyclin-dependent kinase inhibitor previously implicated in several types of human cancer. Its family members (CDKN1A/p21CIP1 and B/p27KIP1) have been implicated in breast cancer, but information about CDKN1C's role is limited. We hypothesized that decreased CDKN1C may be involved in human breast carcinogenesis in vivo. METHODS: We determined rates of allele imbalance or loss of heterozygosity (AI/LOH) in CDKN1C, using an intronic polymorphism, and in the surrounding 11p15.5 region in 82 breast cancers. We examined the CDKN1C mRNA level in 10 cancers using quantitative real-time PCR (qPCR), and the CDKN1C protein level in 20 cancers using immunohistochemistry (IHC). All samples were obtained using laser microdissection. Data were analyzed using standard statistical tests. RESULTS: AI/LOH at 11p15.5 occurred in 28/73 (38%) informative cancers, but CDKN1C itself underwent AI/LOH in only 3/16 (19%) cancers (p = ns). In contrast, CDKN1C mRNA levels were reduced in 9/10 (90%) cancers (p < 0.0001), ranging from 2-60% of paired normal epithelium. Similarly, CDKN1C protein staining was seen in 19/20 (95%) cases' normal epithelium but in only 7/14 (50%) cases' CIS (p < 0.004) and 5/18 (28%) cases' IC (p < 0.00003). The reduction appears primarily due to loss of CDKN1C expression from myoepithelial layer cells, which stained intensely in 17/20 (85%) normal lobules, but in 0/14 (0%) CIS (p < 0.00001). In contrast, luminal cells displayed less intense, focal staining fairly consistently across histologies. Decreased CDKN1C was not clearly associated with tumor grade, histology, ER, PR or HER2 status. CONCLUSION: CDKN1C is expressed in normal epithelium of most breast cancer cases, mainly in the myothepithelial layer. This expression decreases, at both the mRNA and protein level, in the large majority of breast cancers, and does not appear to be mediated by AI/LOH at the gene. Thus, CDKN1C may be a breast cancer tumor suppressor.

To investigate if the tumor suppressor properties of p57KIP2 are dependent on its DNA methylation status, we studied the impact of several stress stimuli in leukemic cell lines with different p57KIP2 promoter DNA methylation levels. p57KIP2 reactivation was observed after stimulation with transforming growth factor-beta, other cytokines, high-density culture or serum withdrawal in p57KIP2 promoter unmethylated cells but not in methylated cells. In these cells, p57KIP2 reactivation required the use of a hypomethylating agent or a histone deacetylase inhibitor. Overexpression of p57KIP2 in p57KIP2 promoter methylated leukemic cell lines resulted in cell growth arrest and the induction of apoptosis. In contrast, overexpression of p57KIP2 in partially methylated cells only resulted in a moderate inhibition of cell growth and had no impact on apoptosis. Transduction of unmethylated cells expressing high levels of p57KIP2 with p57KIP2 short hairpin RNA resulted in increased cell proliferation. These results suggest that the tumor suppressive properties of p57KIP2 in leukemia may depend on the intrinsic promoter DNA methylation status of the gene.

BACKGROUND: The RB1 gene was the first tumor suppressor gene cloned from humans by studying genetic lesions in families with retinoblastoma. Children who inherit one defective copy of the RB1 gene have an increased susceptibility to retinoblastoma. Several years after the identification of the human RB1 gene, a targeted deletion of Rb was generated in mice. Mice with one defective copy of the Rb gene do not develop retinoblastoma. In this manuscript, we explore the different roles of the Rb family in human and mouse retinal development in order to better understand the species-specific difference in retinoblastoma susceptibility. RESULTS: We found that the Rb family of proteins (Rb, p107 and p130) are expressed in a dynamic manner during mouse retinal development. The primary Rb family member expressed in proliferating embryonic retinal progenitor cells in mice is p107, which is required for appropriate cell cycle exit during retinogenesis. The primary Rb family member expressed in proliferating postnatal retinal progenitor cells is Rb. p130 protein is expressed redundantly with Rb in postmitotic cells of the inner nuclear layer and the ganglion cell layer of the mouse retina. When Rb is inactivated in an acute or chronic manner during mouse retinal development, p107 is upregulated in a compensatory manner. Similarly, when p107 is inactivated in the mouse retina, Rb is upregulated. No changes in p130 expression were seen when p107, Rb or both were inactivated in the developing mouse retina. In the human retina, RB1 was the primary family member expressed throughout development. There was very little if any p107 expressed in the developing human retina. In contrast to the developing mouse retina, when RB1 was acutely inactivated in the developing human fetal retina, p107 was not upregulated in a compensatory manner. CONCLUSION: We propose that intrinsic genetic compensation between Rb and p107 prevents retinoblastoma in Rb- or p107-deficient mice, but this compensation does not occur in humans. Together, these data suggest a model that explains why humans are susceptible to retinoblastoma following RB1 loss, but mice require both Rb and p107 gene inactivation.