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Rb and the APC/C coactivator FZR1 determine CDK4/6-Cyclin D requirement in C. elegans and human cancer cells

ABSTRACT: Cyclin dependent kinase 4 and 6 (CDK4/6) in complex with D-type cyclins promote cell cycle entry, at least in part through phosphorylation of the retinoblastoma tumor suppressor protein (Rb). The CDK4/6-cyclin D-Rb pathway is commonly deregulated in human cancer, often through CDK4/6 or D-type cyclin overexpression, or inactivation of the CDK4/6 antagonist p16/CDKN2A. Importantly, a substantial fraction of cancers depend on continuous CDK4/6-cyclin D kinase activity and are sensitive to CDK4/6-specific inhibitors. Here, we investigate critical CDK4/6-cyclin D functions that may determine the sensitivity to CDK4/6 inhibitors, making use of the essential roles of CDK4/6 (CDK-4) and cyclin D (CYD-1) in the nematode C. elegans. In an unbiased screen, we found that simultaneous loss of C. elegans Rb (lin-35) and down-regulation of the APC/C substrate specificity factor FZR1/Cdh1 completely overcomes CDK-4/CYD-1 requirement. Furthermore, CDK-4/CYD-1 phosphorylates specific residues in the LIN-35 Rb spacer domain and FZR-1 N-terminus that correspond to inactivating phosphorylations of the human homologs. Thus, CDK-4/CYD-1 appears to promote cell cycle entry by antagonizing not only transcriptional repression by LIN-35 Rb but also protein degradation by APC/CFZR-1. Simultaneous knockdown of Rb and FZR1 in human breast cancer cells synergistically overcomes arrest by the CDK4/6-specific inhibitor PD 00332991. These results reveal APC/CFZR1 as a putative CDK4/6-Cyclin D target and important contributing factor in the response to CDK4/6-inhibitor treatment.

INSTRUMENT(S): LTQ Orbitrap Velos, Q Exactive

ORGANISM(S): Homo Sapiens (human) Caenorhabditis Elegans

SUBMITTER: Alba Cristobal

LAB HEAD: Albert J.R. Heck

PROVIDER: PXD001501 | Pride | 2016-07-05

REPOSITORIES: Pride

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Rb and FZR1/Cdh1 determine CDK4/6-cyclin D requirement in C. elegans and human cancer cells.

The Inge I Ruijtenberg Suzan S Bouchet Benjamin P BP Cristobal Alba A Prinsen Martine B W MB van Mourik Tim T Koreth John J Xu Huihong H Heck Albert J R AJ Akhmanova Anna A Cuppen Edwin E Boxem Mike M Muñoz Javier J van den Heuvel Sander S

Nature communications 20150106

Cyclin-dependent kinases 4 and 6 (CDK4/6) in complex with D-type cyclins promote cell cycle entry. Most human cancers contain overactive CDK4/6-cyclin D, and CDK4/6-specific inhibitors are promising anti-cancer therapeutics. Here, we investigate the critical functions of CDK4/6-cyclin D kinases, starting from an unbiased screen in the nematode Caenorhabditis elegans. We found that simultaneous mutation of lin-35, a retinoblastoma (Rb)-related gene, and fzr-1, an orthologue to the APC/C co-activa ...[more]

PMID: 25562820

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Project description:The retinoblastoma tumor suppressor protein (Rb) regulates early G1 phase checkpoints, including the DNA damage response, as well as cell cycle exit and differentiation. The widely accepted model of G1 cell cycle progression proposes that cyclin D:Cdk4/6 partially inactivates the Rb tumor suppressor during early G1 phase by progressive multi-phosphorylation, termed hypo-phosphorylation, resulting in release of E2F transcription factors. However, this model remains largely unproven biochemically and the biologically active form(s) of Rb remains unknown. Here we find that Rb is un-phosphorylated in G0 cells and becomes exclusively mono-phosphorylated throughout all of early G1 phase by cyclin D:Cdk4/6. Early G1 phase mono-phosphorylated Rb is composed of 14 independent isoforms that are all targeted by the E1a oncoprotein, but each shows a preferential binding pattern to specific E2F1-4 transcription factors. At the late G1 Restriction Point, cyclin E:Cdk2 inactivates Rb by a quantum hyper-phosphorylation (>12 phosphates/Rb). Cells undergoing a DNA damage response activate cyclin D:Cdk4/6 to generate mono-phosphorylated Rb that regulates global transcription. In contrast, a non-phosphorylatable ?Cdk-Rb allele was non-functional for regulating a DNA damage response, but functional for driving cell cycle exit and differentiation during myogenesis. These observations fundamentally change our understanding of G1 cell cycle progression and show that there is no progressive multi-phosphorylation or hypo-phosphorylation inactivation of Rb during early G1 phase by cyclin D:Cdk4/6. Instead, cyclin D:Cdk4/6 generates functionally active, mono-phosphorylated Rb that is the only Rb isoform present in cells during early G1 phase. Global transcriptional analysis of murine embryonic fibroblasts (MEFs) with conditional deletion of the endogenous RB gene by treatment with cell permeable TAT-Cre. Comparison to unaltered MEFs and MEFs with physiological level of exogenous wildtype or phospho-mutant RB expressed at time of RB gene deletion.

Project description:PD-0332991 is a selective inhibitor of the CDK4/6 kinases with the ability to block retinoblastoma (Rb) phosphorylation in the low nanomolar range. Here we investigate the role of CDK4/6 inhibition in human ovarian cancer. We examined the effects of PD-0332991 on proliferation, cell-cycle, apoptosis, and Rb phosphorylation using a panel of 40 established human ovarian cancer cell lines. Molecular markers for response prediction, including p16 and Rb, were studied using gene expression profiling, Western blot, and arrayCGH. Multiple drug effect analysis was used to study interactions with chemotherapeutic drugs. Expression of p16 and Rb was studied using immunohistochemistry in a large clinical cohort ovarian cancer patients. Concentration-dependent anti-proliferative effects of PD-0332991were seen in all ovarian cancer cell lines, but varied significantly between individual lines. Rb proficient cell lines with low p16 expression were most responsive to CDK4/6 inhibition. Copy number variations of CDKN2A, Rb, CCNE1, and CCND1 were associated with response to PD-0332991. CDK4/6 inhibition induced G0/G1 cell cycle arrest, blocked Rb phosphorylation in a concentration and time dependent manner, and enhanced the effects of chemotherapy. Rb proficiency with low p16 expression was seen in 97/262 (37%) of ovarian cancer patients and associated with adverse clinical outcome (progression free survival, adjusted relative risk 1.49, 95%CI 0.99-2.22, p =0.054). PD-0332991 shows promising biologic activity in ovarian cancer cell lines. Assessment of Rb and p16 expression may help select patients most likely to benefit from CDK4/6 inhibition in ovarian cancer. Gene expression of 40 individual ovarian cell lines relative to an ovarian cell line reference mix containing equal amounts of 41 ovarian cell lines (including OCC-1 which was later identified as originating from mouse). The expression data was correllated with cell line growth response to CDK 4/6 inhibitor PD-0332991 to identify genes associated with drug sensitivity and resistance.

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