Cisplatin-induced cell death increases the degradation of the MRE11-RAD50-NBS1 complex through the autophagy/lysosomal pathway.

Cisplatin and other platinum-based anticancer agents are among the most widely used chemotherapy drugs in the treatment of different types of cancer. However, it is common to find patients who respond well to treatment at first but later relapse due to the appearance of resistance to cisplatin. Among the mechanisms responsible for this phenomenon is the increase in DNA damage repair. Here, we elucidate the effect of cisplatin on the MRN (MRE11-RAD50-NBS1) DNA damage sensor complex. We found that the tumor suppressor FBXW7 is a key factor in controlling the turnover of the MRN complex by inducing its degradation through lysosomes. Inhibition of lysosomal enzymes allowed the detection of the association of FBXW7-dependent ubiquitylated MRN with LC3 and the autophagy adaptor p62/SQSTM1 and the localization of MRN in lysosomes. Furthermore, cisplatin-induced cell death increased MRN degradation, suggesting that this complex is one of the targets that favor cell death. These findings open the possibility of using the induction of the degradation of the MRN complex after genotoxic damage as a potential therapeutic strategy to eliminate tumor cells.

Bub1 is not essential for the checkpoint response to unattached kinetochores in diploid human cells.

Error-free chromosome segregation during mitosis depends on a functional spindle assembly checkpoint (SAC). The SAC is a multi-component signalling system that is recruited to unattached or incorrectly attached kinetochores to catalyse the formation of a soluble inhibitor, known as the Mitotic Checkpoint Complex (MCC), which binds and inhibits the anaphase promoting complex (APC/C) [1]. We have previously proposed that two separable pathways, composed of KNL1-Bub3-Bub1 (KBB) and Rod-Zwilch-Zw10 (RZZ), recruit Mad1-Mad2 complexes to human kinetochores to activate the SAC [2]. Although Bub1 is absolutely required for checkpoint signalling in yeast (which lack RZZ), there is conflicting evidence as to whether this is the case in human cells based on siRNA studies [2-5]. Here we show that, while Bub1 is required for recruitment of BubR1, it is not strictly required for the checkpoint response to unattached kinetochores in diploid human cells.

G1/S phase progression is regulated by PLK1 degradation through the CDK1/ßTrCP axis.

Polo-like kinase 1 (PLK1) is a serine/threonine kinase involved in several stages of the cell cycle, including the entry and exit from mitosis, and cytokinesis. Furthermore, it has an essential role in the regulation of DNA replication. Together with cyclin A, PLK1 also promotes CDH1 phosphorylation to trigger its ubiquitination and degradation, allowing cell cycle progression. The PLK1 levels in different type of tumors are very high compared to normal tissues, which is consistent with its role in promoting proliferation. Therefore, several PLK1 inhibitors have been developed and tested for the treatment of cancer. Here, we further analyzed PLK1 degradation and found that cytoplasmic PLK1 is ubiquitinated and subsequently degraded by the SCFßTrCP/proteasome. This procedure is triggered when heat shock protein (HSP) 90 is inhibited with geldanamycin, which results in misfolding of PLK1. We also identified CDK1 as the major kinase involved in this degradation. Our work shows for the first time that HSP90 inhibition arrests cell cycle progression at the G1/S transition. This novel mechanism inhibits CDH1 degradation through CDK1-dependent PLK1 destruction by the SCFßTrCP/proteasome. In these conditions, CDH1 substrates do not accumulate and cell cycle arrests, providing a novel pathway for regulation of the cell cycle at the G1-to-S boundary.-Giráldez, S., Galindo-Moreno, M., Limón-Mortés, M. C., Rivas, A. C., Herrero-Ruiz, J., Mora-Santos, M., Sáez, C., Japón, M. Á., Tortolero, M., Romero, F. G1/S phase progression is regulated by PLK1 degradation through the CDK1/ßTrCP axis.

ßTrCP controls the lysosome-mediated degradation of CDK1, whose accumulation correlates with tumor malignancy.

In mammals, cell cycle progression is controlled by cyclin-dependent kinases, among which CDK1 plays important roles in the regulation of the G2/M transition, G1 progression and G1/S transition. CDK1 is highly regulated by its association to cyclins, phosphorylation and dephosphorylation, changes in subcellular localization, and by direct binding of CDK inhibitor proteins. CDK1 steady-state protein levels are held constant throughout the cell cycle by a coordinated regulation of protein synthesis and degradation. We show that CDK1 is ubiquitinated by the E3 ubiquitin ligase SCFßTrCP and degraded by the lysosome. Furthermore, we found that DNA damage not only triggers the stabilization of inhibitory phosphorylation sites on CDK1 and repression of CDK1 gene expression, but also regulates ßTrCP-induced CDK1 degradation in a cell type-dependent manner. Specifically, treatment with the chemotherapeutic agent doxorubicin in certain cell lines provokes CDK1 degradation and induces apoptosis, whereas in others it inhibits destruction of the protein. These observations raise the possibility that different tumor types, depending on their pathogenic spectrum mutations, may display different sensitivity to ßTrCP-induced CDK1 degradation after DNA damage. Finally, we found that CDK1 accumulation in patients' tumors shows a negative correlation with ßTrCP and a positive correlation with the degree of tumor malignancy.

SCF(FBXW7α) modulates the intra-S-phase DNA-damage checkpoint by regulating Polo like kinase-1 stability.

The intra-S-checkpoint is essential to control cell progression through S phase under normal conditions and in response to replication stress. When DNA lesions are detected, replication fork progression is blocked allowing time for repair to avoid genomic instability and the risk of cancer. DNA replication initiates at many origins of replication in eukaryotic cells, where a series of proteins form pre-replicative complexes (pre-RCs) that are activated to become pre-initiation complexes and ensure a single round of replication in each cell cycle. PLK1 plays an important role in the regulation of DNA replication, contributing to the regulation of pre-RCs formation by phosphorylating several proteins, under both normal and stress conditions. Here we report that PLK1 is ubiquitinated and degraded by SCFFBXW7α/proteasome. Moreover, we identified a new Cdc4 phosphodegron in PLK1, conserved from yeast to humans, whose mutation prevents PLK1 destruction. We established that endogenous SCFFBXW7α degrades PLK1 in the G1 and S phases of an unperturbed cell cycle and in S phase following UV irradiation. Furthermore, we showed that FBXW7α overexpression or UV irradiation prevented the loading of proteins onto chromatin to form pre-RCs and, accordingly, reduced cell proliferation. We conclude that PLK1 degradation mediated by SCFFBXW7α modulates the intra-S-phase checkpoint.

A single mutation in Securin induces chromosomal instability and enhances cell invasion.

Pituitary tumour transforming gene (pttg1) encodes Securin, a protein involved in the inhibition of sister chromatid separation binding to Separase until the onset of anaphase. Separase is a cysteine-protease that degrades cohesin to segregate the sister chromatids to opposite poles of the cell. The amount of Securin is strongly regulated because it should allow Separase activation when it is degraded by the anaphase promoting complex/cyclosome, should arrest the cell cycle after DNA damage, when it is degraded through SKP1-CUL1-ßTrCP ubiquitin ligase, and its overexpression induces tumour formation and correlates with metastasis in multiple tumours. Securin is a phosphoprotein that contains 32 potentially phosphorylatable residues. We mutated and analysed most of them, and found a single mutant, hSecT60A, that showed enhanced oncogenic properties. Our fluorescence activated cell sorting analysis, fluorescence in situ hybridisation assays, tumour cell migration and invasion experiments and gene expression by microarrays analysis clearly involved hSecT60A in chromosomal instability and cell invasion. These results show, for the first time, that a single mutation in pttg1 is sufficient to trigger the oncogenic properties of Securin. The finding of this point mutation in patients might be used as an effective strategy for early detection of cancer.

Glycogen synthase kinase-3beta (GSK3beta) negatively regulates PTTG1/human securin protein stability, and GSK3beta inactivation correlates with securin accumulation in breast tumors.

PTTG1, also known as securin, is an inactivating partner of separase, the major effector for chromosome segregation during mitosis. At the metaphase-to-anaphase transition, securin is targeted for proteasomal destruction by the anaphase-promoting complex or cyclosome, allowing activation of separase. In addition, securin is overexpressed in metastatic or genomically instable tumors, suggesting a relevant role for securin in tumor progression. Stability of securin is regulated by phosphorylation; some phosphorylated forms are degraded out of mitosis, by the action of the SKP1-CUL1-F-box protein (SCF) complex. The kinases targeting securin for proteolysis have not been identified, and mechanistic insight into the cause of securin accumulation in human cancers is lacking. Here, we demonstrate that glycogen synthase kinase-3ß (GSK3ß) phosphorylates securin to promote its proteolysis via SCF(ßTrCP) E3 ubiquitin ligase. Importantly, a strong correlation between securin accumulation and GSK3ß inactivation was observed in breast cancer tissues, indicating that GSK3ß inactivation may account for securin accumulation in breast cancers.

UV-induced degradation of securin is mediated by SKP1-CUL1-beta TrCP E3 ubiquitin ligase.

Securin is a chaperone protein with bifunctional properties. It binds to separase to inhibit premature sister chromatid separation until the onset of anaphase, and it also takes part in cell-cycle arrest after UV irradiation. At metaphase-to-anaphase transition, securin is targeted for proteasomal destruction by the anaphase-promoting complex or cyclosome (APC/C), allowing activation of separase. However, although securin is reported to undergo proteasome-dependent degradation after UV irradiation, the ubiquitin ligase responsible for securin ubiquitylation has not been well characterized. In this study, we show that UV radiation induced a marked reduction of securin in both the nucleus and cytoplasm. Moreover, we show that GSK-3beta inhibitors prevent securin degradation, and that CUL1 and betaTrCP are involved in this depletion. We also confirmed that SKP1-CUL1-betaTrCP (SCF(betaTrCP)) ubiquitylates securin in vivo, and identified a conserved and unconventional betaTrCP recognition motif (DDAYPE) in the securin primary amino acid sequence of humans, nonhuman primates and rodents. Furthermore, downregulation of betaTrCP caused an accumulation of securin in non-irradiated cells. We conclude that SCF(betaTrCP) is the E3 ubiquitin ligase responsible for securin degradation after UV irradiation, and that it is involved in securin turnover in nonstressed cells.