Faculty Bibliography

D-type cyclins are central regulators of the cell division cycle and are among the most frequently deregulated therapeutic targets in human cancer¹, but the mechanisms that regulate their turnover are still being debated^2,3. Here, by combining biochemical and genetics studies in somatic cells, we identify CRL4^AMBRA1 (also known as CRL4^DCAF3) as the ubiquitin ligase that targets all three D-type cyclins for degradation. During development, loss of Ambra1 induces the accumulation of D-type cyclins and retinoblastoma (RB) hyperphosphorylation and hyperproliferation, and results in defects of the nervous system that are reduced by treating pregnant mice with the FDA-approved CDK4 and CDK6 (CDK4/6) inhibitor abemaciclib. Moreover, AMBRA1 acts as a tumour suppressor in mouse models and low AMBRA1 mRNA levels are predictive of poor survival in cancer patients. Cancer hotspot mutations in D-type cyclins abrogate their binding to AMBRA1 and induce their stabilization. Finally, a whole-genome, CRISPR-Cas9 screen identified AMBRA1 as a regulator of the response to CDK4/6 inhibition. Loss of AMBRA1 reduces sensitivity to CDK4/6 inhibitors by promoting the formation of complexes of D-type cyclins with CDK2. Collectively, our results reveal the molecular mechanism that controls the stability of D-type cyclins during cell-cycle progression, in development and in human cancer, and implicate AMBRA1 as a critical regulator of the RB pathway.

Mammalian development, adult tissue homeostasis and the avoidance of severe diseases including cancer require a properly orchestrated cell cycle, as well as error-free genome maintenance. The key cell-fate decision to replicate the genome is controlled by two major signalling pathways that act in parallel-the MYC pathway and the cyclin D-cyclin-dependent kinase (CDK)-retinoblastoma protein (RB) pathway^1,2. Both MYC and the cyclin D-CDK-RB axis are commonly deregulated in cancer, and this is associated with increased genomic instability. The autophagic tumour-suppressor protein AMBRA1 has been linked to the control of cell proliferation, but the underlying molecular mechanisms remain poorly understood. Here we show that AMBRA1 is an upstream master regulator of the transition from G1 to S phase and thereby prevents replication stress. Using a combination of cell and molecular approaches and in vivo models, we reveal that AMBRA1 regulates the abundance of D-type cyclins by mediating their degradation. Furthermore, by controlling the transition from G1 to S phase, AMBRA1 helps to maintain genomic integrity during DNA replication, which counteracts developmental abnormalities and tumour growth. Finally, we identify the CHK1 kinase as a potential therapeutic target in AMBRA1-deficient tumours. These results advance our understanding of the control of replication-phase entry and genomic integrity, and identify the AMBRA1-cyclin D pathway as a crucial cell-cycle-regulatory mechanism that is deeply interconnected with genomic stability in embryonic development and tumorigenesis.

Cell-cell fusion is essential to the development of multicellular organisms and is driven by remodeling of the actin cytoskeleton. In this issue of Developmental Cell, Rodríguez-Pérez et al. reveal how CRL3-dependent mono-ubiquitylation modulates cell fusion by controlling the dynamics of cytoskeletal rearrangements.

The SCF^SKP2 ubiquitin ligase relieves G1 checkpoint control of CDK-cyclin complexes by promoting p27KIP1 degradation. We describe reconstitution of stable complexes containing SKP1-SKP2 and CDK1-cyclin B or CDK2-cyclin A/E, mediated by the CDK regulatory subunit CKS1. We further show that a direct interaction between a SKP2 N-terminal motif and cyclin A can stabilize SKP1-SKP2-CDK2-cyclin A complexes in the absence of CKS1. We identify the SKP2 binding site on cyclin A and demonstrate the site is not present in cyclin B or cyclin E. This site is distinct from but overlapping with features that mediate binding of p27KIP1 and other G1 cyclin regulators to cyclin A. We propose that the capacity of SKP2 to engage with CDK2-cyclin A by more than one structural mechanism provides a way to fine tune the degradation of p27KIP1 and distinguishes cyclin A from other G1 cyclins to ensure orderly cell cycle progression.

Epigenetic reprogramming is emerging as a key mechanism for metastasis development. Our study identified a novel regulatory mechanism whereby promoter methylation-mediated epigenetic silencing of the gene encoding the ubiquitin ligase subunit F-box/LRR-repeat protein 7 (FBXL7) induces accumulation of active c-SRC, which, in turn, activates epithelial-to-mesenchymal transition and supports cancer cell invasion and metastasis.

Numerous anti-cancer drugs perturb thymidylate biosynthesis and lead to genomic uracil incorporation contributing to their antiproliferative effect. Still, it is not yet characterized if uracil incorporations have any positional preference. Here, we aimed to uncover genome-wide alterations in uracil pattern upon drug treatments in human cancer cell line models derived from HCT116. We developed a straightforward U-DNA sequencing method (U-DNA-Seq) that was combined with in situ super-resolution imaging. Using a novel robust analysis pipeline, we found broad regions with elevated probability of uracil occurrence both in treated and non-treated cells. Correlation with chromatin markers and other genomic features shows that non-treated cells possess uracil in the late replicating constitutive heterochromatic regions, while drug treatment induced a shift of incorporated uracil towards segments that are normally more active/functional. Data were corroborated by colocalization studies via dSTORM microscopy. This approach can be applied to study the dynamic spatio-temporal nature of genomic uracil.

During mitosis, the chromosomal passenger complex (CPC) ensures the faithful transmission of the genome. The CPC is composed of the enzymatic component Aurora B (AURKB) and the three regulatory and targeting components borealin, INCENP, and survivin (also known as BIRC5). Although the CPC is known to be involved in diverse mitotic events, it is still unclear how CPC function terminates after mitosis. Here we show that borealin is ubiquitylated by the anaphase promoting complex/cyclosome (APC/C) and its cofactor Cdh1 (also known as FZR1) and is subsequently degraded in G1 phase. Cdh1 binds to regions within the N terminus of borealin that act as a non-canonical degron. Aurora B has also been shown previously to be degraded by the APC/C^Cdh1 from late mitosis to G1. Indeed, Cdh1 depletion sustains an Aurora B activity with stable levels of borealin and Aurora B throughout the cell cycle, and causes reduced efficiency of DNA replication after release from serum starvation. Notably, inhibition of Aurora B kinase activity improves the efficiency of DNA replication in Cdh1-depleted cells. We thus propose that APC/C^Cdh1 terminates CPC activity upon mitotic exit and thereby contributes to proper control of DNA replication.

Epigenetic plasticity is a pivotal factor that drives metastasis. Here, we show that the promoter of the gene that encodes the ubiquitin ligase subunit FBXL7 is hypermethylated in advanced prostate and pancreatic cancers, correlating with decreased FBXL7 mRNA and protein levels. Low FBXL7 mRNA levels are predictive of poor survival in patients with pancreatic and prostatic cancers. FBXL7 mediates the ubiquitylation and proteasomal degradation of active c-SRC after its phosphorylation at Ser 104. The DNA-demethylating agent decitabine recovers FBXL7 expression and limits epithelial-to-mesenchymal transition and cell invasion in a c-SRC-dependent manner. In vivo, FBXL7-depleted cancer cells form tumours with a high metastatic burden. Silencing of c-SRC or treatment with the c-SRC inhibitor dasatinib together with FBXL7 depletion prevents metastases. Furthermore, decitabine reduces metastases derived from prostate and pancreatic cancer cells in a FBXL7-dependent manner. Collectively, this research implicates FBXL7 as a metastasis-suppressor gene and suggests therapeutic strategies to counteract metastatic dissemination of pancreatic and prostatic cancer cells.

BACKGROUND:The SMC5/6 complex, cohesin and condensin are the three mammalian members of the structural maintenance of chromosomes (SMC) family, large ring-like protein complexes that are essential for genome maintenance. The SMC5/6 complex is the least characterized complex in mammals; however, it is known to be involved in homologous recombination repair (HRR) and chromosome segregation. RESULTS:In this study, a yeast two-hybrid screen was used to help elucidate novel interactions of the kleisin subunit of the SMC5/6 complex, NSMCE4A. This approach discovered an interaction between NSMCE4A and GPS1, a COP9 signalosome (CSN) component, and this interaction was further confirmed by co-immunoprecipitation. Additionally, GPS1 and components of SMC5/6 complex colocalize during interphase and mitosis. CSN is a cullin deNEDDylase and is an important factor for HRR. Depletion of GPS1, which has been shown to negatively impact DNA end resection during HRR, caused an increase in SMC5/6 levels at sites of laser-induced DNA damage. Furthermore, inhibition of the dennedylation function of CSN increased SMC5/6 levels at sites of laser-induced DNA damage. CONCLUSION/CONCLUSIONS:Taken together, these data demonstrate for the first time that the SMC5/6 and CSN complexes interact and provides evidence that the CSN complex influences SMC5/6 functions during cell cycle progression and response to DNA damage.