p57Kip2 acts as a transcriptional corepressor to regulate intestinal stem cell fate and proliferation.

p57Kip2 is a cyclin/CDK inhibitor and a negative regulator of cell proliferation. Here, we report that p57 regulates intestinal stem cell (ISC) fate and proliferation in a CDK-independent manner during intestinal development. In the absence of p57, intestinal crypts exhibit an increased proliferation and an amplification of transit-amplifying cells and of Hopx+ ISCs, which are no longer quiescent, while Lgr5+ ISCs are unaffected. RNA sequencing (RNA-seq) analyses of Hopx+ ISCs show major gene expression changes in the absence of p57. We found that p57 binds to and inhibits the activity of Ascl2, a transcription factor critical for ISC specification and maintenance, by participating in the recruitment of a corepressor complex to Ascl2 target gene promoters. Thus, our data suggest that, during intestinal development, p57 plays a key role in maintaining Hopx+ ISC quiescence and repressing the ISC phenotype outside of the crypt bottom by inhibiting the transcription factor Ascl2 in a CDK-independent manner.

Small ORFs as New Regulators of Pri-miRNAs and miRNAs Expression in Human and Drosophila.

MicroRNAs (miRNAs) are small regulatory non-coding RNAs, resulting from the cleavage of long primary transcripts (pri-miRNAs) in the nucleus by the Microprocessor complex generating precursors (pre-miRNAs) that are then exported to the cytoplasm and processed into mature miRNAs. Some miRNAs are hosted in pri-miRNAs annotated as long non-coding RNAs (lncRNAs) and defined as MIRHGs (for miRNA Host Genes). However, several lnc pri-miRNAs contain translatable small open reading frames (smORFs). If smORFs present within lncRNAs can encode functional small peptides, they can also constitute cis-regulatory elements involved in lncRNA decay. Here, we investigated the possible involvement of smORFs in the regulation of lnc pri-miRNAs in Human and Drosophila, focusing on pri-miRNAs previously shown to contain translatable smORFs. We show that smORFs regulate the expression levels of human pri-miR-155 and pri-miR-497, and Drosophila pri-miR-8 and pri-miR-14, and also affect the expression and activity of their associated miRNAs. This smORF-dependent regulation is independent of the nucleotidic and amino acidic sequences of the smORFs and is sensitive to the ribosome-stalling drug cycloheximide, suggesting the involvement of translational events. This study identifies smORFs as new cis-acting elements involved in the regulation of pri-miRNAs and miRNAs expression, in both Human and Drosophila melanogaster.

Sphingosine 1-Phosphate Receptor 5 (S1P5) Deficiency Promotes Proliferation and Immortalization of Mouse Embryonic Fibroblasts.

Sphingosine 1-phosphate (S1P), a bioactive lipid, interacts with five widely expressed G protein-coupled receptors (S1P1-5), regulating a variety of downstream signaling pathways with overlapping but also opposing functions. To date, data regarding the role of S1P5 in cell proliferation are ambiguous, and its role in controlling the growth of untransformed cells remains to be fully elucidated. In this study, we examined the effects of S1P5 deficiency on mouse embryonic fibroblasts (MEFs). Our results indicate that lack of S1P5 expression profoundly affects cell morphology and proliferation. First, S1P5 deficiency reduces cellular senescence and promotes MEF immortalization. Second, it decreases cell size and leads to cell elongation, which is accompanied by decreased cell spreading and migration. Third, it increases proliferation rate, a phenotype rescued by the reintroduction of exogenous S1P5. Mechanistically, S1P5 promotes the activation of FAK, controlling cell spreading and adhesion while the anti-proliferative function of the S1P/S1P5 signaling is associated with reduced nuclear accumulation of activated ERK. Our results suggest that S1P5 opposes the growth-promoting function of S1P1-3 through spatial control of ERK activation and provides new insights into the anti-proliferative function of S1P5.

Functions of animal microRNA-encoded peptides: the race is on!

Short open reading frame (sORF)-encoded peptides (SEPs) recently emerged as new key players in biology. Pioneering work first established that sORFs encoded by long non-coding RNAs (lncRNAs) are efficiently translated and produce functional peptides. In plants, primary transcripts of microRNAs (pri-miRNAs) also produce sORF-encoded peptides called miPEPs, which are involved in specific transcriptional autoregulatory feedback loops (Lauressergues et al, 2015). To what extend are such mechanisms conserved in other species, especially in animals? In this issue of EMBO reports, Zhou et al show that pri-miR-31 encodes a miPEP promoting Treg differentiation and downregulating pri-miR-31 expression (Zhou et al, 2022).

Evidence That Regulation of Pri-miRNA/miRNA Expression Is Not a General Rule of miPEPs Function in Humans.

Some miRNAs are located in RNA precursors (pri-miRNAs) annotated as long non-coding (lncRNAs) due to absence of long open reading frames (ORFs). However, recent studies have shown that some lnc pri-miRNAs encode peptides called miPEPs (miRNA-encoded peptides). Initially discovered in plants, three miPEPs have also been identified in humans. Herein, we found that a dozen human pri-miRNAs potentially encode miPEPs, as revealed by ribosome profiling and proteomic databases survey. So far, the only known function of plant miPEPs is to enhance the transcription of their own pri-miRNAs, thereby increasing the level and activity of their associated miRNAs and downregulating the expression of their target genes. To date, in humans, only miPEP133 was shown to promote a positive autoregulatory loop. We investigated whether other human miPEPs are also involved in regulating the expression of their miRNAs by studying miPEP155, encoded by the lnc MIR155HG, miPEP497, a sORF-encoded peptide within lnc MIR497HG, and miPEP200a, encoded by the pri-miRNA of miR-200a/miR-200b. We show that overexpression of these miPEPs is unable to impact the expression/activity of their own pri-miRNA/miRNAs in humans, indicating that the positive feedback regulation observed with plant miPEPs and human miPEP133 is not a general rule of human miPEP function.

miR-497 suppresses cycle progression through an axis involving CDK6 in ALK-positive cells.

Anaplastic large-cell lymphoma, a T-cell neoplasm, is primarily a pediatric disease. Seventy-five percent of pediatric anaplastic large-cell lymphoma cases harbor the chromosomal translocation t(2;5)(p23;q35) leading to the ectopic expression of NPM-ALK, a chimeric tyrosine kinase. NPM-ALK consists of an N-terminal nucleophosmin (NPM) domain fused to an anaplastic lymphoma kinase (ALK) cytoplasmic domain. Pediatric NPM-ALK+ anaplastic large-cell lymphoma is often a disseminated disease and young patients are prone to chemoresistance or relapse shortly after chemotherapeutic treatment. Furthermore, there is no gold standard protocol for the treatment of relapses. To the best of our knowledge, this is the first study on the potential role of the microRNA, miR-497, in NPM-ALK+ anaplastic large-cell lymphoma tumorigenesis. Our results show that miR-497 expression is repressed in NPM-ALK+ cell lines and patient samples through the hypermethylation of its promoter and the activity of NPM-ALK is responsible for this epigenetic repression. We demonstrate that overexpression of miR-497 in human NPM-ALK+ anaplastic large-cell lymphoma cells inhibits cellular growth and causes cell cycle arrest by targeting CDK6, E2F3 and CCNE1, the three regulators of the G1 phase of the cell cycle. Interestingly, we show that a scoring system based on CDK6, E2F3 and CCNE1 expression could help to identify relapsing pediatric patients. In addition, we demonstrate the sensitivity of NPM-ALK+ cells to CDK4/6 inhibition using for the first time a selective inhibitor, palbociclib. Together, our findings suggest that CDK6 could be a therapeutic target for the development of future treatments for NPM-ALK+ anaplastic large-cell lymphoma.

Phosphorylation of CDC25A on SER283 in late S/G2 by CDK/cyclin complexes accelerates mitotic entry.

The Cdc25A phosphatase is an essential activator of CDK-cyclin complexes at all steps of the eukaryotic cell cycle. The activity of Cdc25A is itself regulated in part by positive and negative feedback regulatory loops performed by its CDK-cyclin substrates that occur in G1 as well as during the G1/S and G2/M transitions. However, the regulation of Cdc25A during G2 phase progression before mitotic entry has not been intensively characterized. Here, we identify by mass spectrometry analysis a new phosphorylation event of Cdc25A on Serine283. Phospho-specific antibodies revealed that the phosphorylation of this residue appears in late S/G2 phase of an unperturbed cell cycle and is performed by CDK-cyclin complexes. Overexpression studies of wild-type and non-phosphorylatable mutant forms of Cdc25A indicated that Ser283 phosphorylation increases the G2/M-promoting activity of the phosphatase without impacting its stability or subcellular localization. Our results therefore identify a new positive regulatory loop between Cdc25A and its CDK-cyclin substrates which contributes to accelerate entry into mitosis through the regulation of Cdc25A activity in G2.

CDC25A governs proliferation and differentiation of FLT3-ITD acute myeloid leukemia.

We investigated cell cycle regulation in acute myeloid leukemia cells expressing the FLT3-ITD mutated tyrosine kinase receptor, an underexplored field in this disease. Upon FLT3 inhibition, CDC25A mRNA and protein were rapidly down-regulated, while levels of other cell cycle proteins remained unchanged. This regulation was dependent on STAT5, arguing for FLT3-ITD-dependent transcriptional regulation of CDC25A. CDC25 inhibitors triggered proliferation arrest and cell death of FLT3-ITD as well as FLT3-ITD/TKD AC-220 resistant cells, but not of FLT3-wt cells. Consistently, RNA interference-mediated knock-down of CDC25A reduced the proliferation of FLT3-ITD cell lines. Finally, the clonogenic capacity of primary FLT3-ITD AML cells was reduced by the CDC25 inhibitor IRC-083864, while FLT3-wt AML and normal CD34+ myeloid cells were unaffected. In good agreement, in a cohort of 100 samples from AML patients with intermediate-risk cytogenetics, high levels of CDC25A mRNA were predictive of higher clonogenic potential in FLT3-ITD+ samples, not in FLT3-wt ones.Importantly, pharmacological inhibition as well as RNA interference-mediated knock-down of CDC25A also induced monocytic differentiation of FLT3-ITD positive cells, as judged by cell surface markers expression, morphological modifications, and C/EBPα phosphorylation. CDC25 inhibition also re-induced monocytic differentiation in primary AML blasts carrying the FLT3-ITD mutation, but not in blasts expressing wild type FLT3. Altogether, these data identify CDC25A as an early cell cycle transducer of FLT3-ITD oncogenic signaling, and as a promising target to inhibit proliferation and re-induce differentiation of FLT3-ITD AML cells.

Targeting CHK1 inhibits cell proliferation in FLT3-ITD positive acute myeloid leukemia.

CHK1 Ser/Thr kinase, a well characterized regulator of DNA damage response, is also involved in normal cell cycle progression. In this study, we investigate how CHK1 participates to proliferation of acute myeloid leukemia cells expressing the mutated FLT3-ITD tyrosine kinase receptor. Pharmacological inhibition of CHK1 as well as its shRNA mediated down regulation reduced the proliferation rate of FLT-ITD expressing leukemic cell lines in a cytostatic manner. Flow cytometry analysis revealed no accumulation in a specific phase of the cell cycle upon CHK1 inhibition. Accordingly, lentiviral-mediated CHK1 overexpression increased the proliferation rate of FLT3-ITD expressing cells, as judged by cell viability and [3H] thymidine incorporation experiments. By contrast, expression of a ser280 mutant did not, suggesting that phosphorylation of this residue is an important determinant of CHK1 proliferative function. Clonogenic growth of primary leukemic cells from patients in semi-solid medium was reduced upon CHK1 inhibition, confirming the data obtained with leukemic established cell lines. Surprisingly, 3 out of 4 CHK1 inhibitory compounds tested in this study were also potent inhibitors of the FLT3-ITD tyrosine kinase receptor. Altogether, these data identify CHK1 as a regulator of FLT3-ITD-positive leukemic cells proliferation, and they open interesting perspectives in terms of new therapeutic strategies for these pathologies.

Identification of N-terminally truncated stable nuclear isoforms of CDC25B that are specifically involved in G2/M checkpoint recovery.

CDC25B phosphatases must activate cyclin B-CDK1 complexes to restart the cell cycle after an arrest in G2 phase caused by DNA damage. However, little is known about the precise mechanisms involved in this process, which may exert considerable impact on cancer susceptibility and therapeutic responses. Here we report the discovery of novel N-terminally truncated CDC25B isoforms, referred to as ΔN-CDC25B, with an exclusively nuclear and nonredundant function in cell cycle re-initiation after DNA damage. ΔN-CDC25B isoforms are expressed from a distinct promoter not involved in expression of canonical full-length isoforms. Remarkably, in contrast to the high lability and spatial dynamism of the full-length isoforms, ΔN-CDC25B isoforms are highly stable and exclusively nuclear, strongly suggesting the existence of two pools of CDC25B phosphatases in the cell that have functionally distinct properties. Using isoform-specific siRNA, we found that depleting full-length isoforms, but not ΔN-CDC25B isoforms, delays entry into mitosis. Thus, in an unperturbed cell cycle, the full-length isoforms are exclusively responsible for activating cyclin B-CDK1. Strikingly, in the late response to DNA damage, we found a CHK1-dependent shift in accumulation of CDC25B isoforms toward the ΔN-CDC25B species. Under this physiological stress condition, the ΔN-CDC25B isoform was found to play a crucial, nonredundant function in restarting the cell cycle after DNA damage-induced G2 phase arrest. Our findings reveal the existence of a previously unrecognized CDC25B isoform that operates specifically in the nucleus to reinitiate G2/M transition after DNA damage.

CDC25B associates with a centrin 2-containing complex and is involved in maintaining centrosome integrity.

BACKGROUND INFORMATION: CDC25 (cell division cycle 25) phosphatases function as activators of CDK (cyclin-dependent kinase)-cyclin complexes to regulate progression through the CDC. We have recently identified a pool of CDC25B at the centrosome of interphase cells that plays a role in regulating centrosome numbers. RESULTS: In the present study, we demonstrate that CDC25B forms a close association with Ctn (centrin) proteins at the centrosome. This interaction involves both N- and C-terminal regions of CDC25B and requires CDC25B binding to its CDK-cyclin substrates. However, the interaction is not dependent on the enzyme activity of CDC25B. Although CDC25B appears to bind indirectly to Ctn2, this association is pertinent to CDC25B localization at the centrosome. We further demonstrate that CDC25B plays a role in maintaining the overall integrity of the centrosome, by regulating the centrosome levels of multiple centrosome proteins, including that of Ctn2. CONCLUSIONS: Our results therefore suggest that CDC25B associates with a Ctn2-containing multiprotein complex in the cytoplasm, which targets it to the centrosome, where it plays a role in maintaining the centrosome levels of Ctn2 and a number of other centrosome components.

BRCA1 is regulated by Chk2 in response to spindle damage.

Inherited mutations of the breast cancer susceptibility gene 1 (BRCA1) confer an increased risk for breast, ovarian and prostate cancer. BRCA1 has been involved in regulation of cell cycle progression, DNA damage signaling and repair, maintenance of genome integrity, ubiquitination and regulation of transcription. Aside from its essential functions in the DNA damage response BRCA1 has been also involved in the cellular response to microtubule damage. Emerging evidence indicates that BRCA1 regulates the duplication and the function of centrosomes, participates in mitotic spindle assembly and is required in the spindle checkpoint. Given BRCA1 distinct functions in microtubule-dependent pathways, we hypothesized that BRCA1 might be regulated following microtubule damage. In the present study, we report the novel finding that BRCA1 is phosphorylated by the checkpoint kinase Chk2 on the previously identified site Ser988 following anti-mitotic treatment in human cancer cells. Ser988-phosphorylated BRCA1 accumulates at centrosomes in response to microtubule damage but Ser988 is not essential for BRCA1 localization at the microtubule-organizing centers. We further demonstrate that the Ser988 phosphorylation is important for the inhibiting microtubule nucleation activity of BRCA1 and for BRCA1 function in cell survival following microtubule damage. These findings reveal a striking outcome of BRCA1 phosphorylation by Chk2 on its role in microtubule-dependent pathways and suggest a fine cross-talk between DNA damage and spindle damage responses.

NanoLC-MS/MS analysis provides new insights into the phosphorylation pattern of Cdc25B in vivo: full overlap with sites of phosphorylation by Chk1 and Cdk1/cycB kinases in vitro.

NanoLC-MS/MS analysis was used to characterize the phosphorylation pattern in vivo of CDC25B3 (phosphatase splice variant 1) expressed in a human cell line and to compare it to the phosphorylation of CDC25B3 by Cdk1/cyclin B and Chk1 in vitro. Cellular CDC25B3 was purified from U2OS cells conditionally overexpressing the phosphatase. Eighteen sites were detectably phosphorylated in vivo. Nearly all existing (S/T)P sites were phosphorylated in vivo and in vitro. Eight non(S/T)P sites were phosphorylated in vivo. All these sites could be phosphorylated by kinase Chk1, which phosphorylated a total of 11 sites in vitro, with consensus sequence (R/K) X(2-3) (S/P)-non P. Nearly half of the sites identified in this study were not previously described and were not homologous to sites reported to be phosphorylated in other CDC25 species. We also show that in vivo a significant part of CDC25B molecules can be hyperphosphorylated, with up to 13 phosphates per phosphatase molecule.

CHK1 phosphorylates CDC25B during the cell cycle in the absence of DNA damage.

CDC25B is one of the three human phosphatases that activate the CDK-cyclin complexes, thereby triggering cell-cycle progression and division. Commitment to early mitotic events depends on the activation of a centrosomal pool of CDK1-cyclin-B1, and CDC25B is thought to be involved in initiating this centrosomal CDK1-cyclin-B1 activity. Centrosome-associated checkpoint kinase 1 (CHK1) has been proposed to contribute to the proper timing of a normal cell division cycle by inhibiting the activation of the centrosomal pool of CDK1. Here, we show that CDC25B is phosphorylated by CHK1 in vitro on multiple residues, including S230 and S563. We demonstrate these phosphorylations occur in vivo and that they are dependent on CHK1 activity. S230 CHK1-mediated phosphorylation is detected in cell extracts during S phase and G2 phase in the absence of DNA damage. We show that the S230-phosphorylated form of CDC25B is located at the centrosome from early S phase until mitosis. Furthermore, mutation of S230 to alanine increases the mitotic-inducing activity of CDC25B. Our results support a model in which, under normal cell cycle conditions and in the absence of DNA damage, CHK1 constitutively phosphorylates CDC25B during interphase and thus prevents the premature initiation of mitosis by negatively regulating the activity of CDC25B at the centrosome.

The when and wheres of CDC25 phosphatases.

The CDC25 phosphatases are key regulators of normal cell division and the cell's response to DNA damage. Earlier studies suggested non-overlapping roles for each isoform during a specific cell cycle phase. However, recent data suggest that multiple CDC25 isoforms cooperate to regulate each cell cycle transition. For instance, although CDC25A was initially thought to exclusively regulate the G(1)-S transition, recent data demonstrate a significant role for CDC25A in the G(2)-M transition. Further evidence demonstrates that in addition to the ATM/ATR-CHK pathway, a p38-MAPKAP pathway is also involved in controlling CDC25 activity during G(2)/M checkpoint activation. Together with the fact that CDC25 overexpression is reported in many cancers, these data highlight the significance of developing specific CDC25 inhibitors for cancer therapy.

CDC25B phosphorylation by Aurora-A occurs at the G2/M transition and is inhibited by DNA damage.

CDC25B is one of the three human dual-specificity phosphatases involved in the activation of cyclin-dependent kinases at key stages of the cell division cycle. CDC25B that is responsible for the activation of CDK1-cyclin B1 is regulated by phosphorylation. The STK15/Aurora-A kinase locally phosphorylates CDC25B on serine 353 at the centrosome during the G2/M transition. Here we have investigated this phosphorylation event during the cell cycle, and in response to activation of the G2 DNA damage checkpoint. We show that accumulation of the S353-phosphorylated form of CDC25B at the centrosome correlates with the relocalization of cyclin B1 to the nucleus and the activation of CDK1 at entry into mitosis. Upon activation of the G2/M checkpoint by DNA damage, we demonstrate that Aurora-A is not activated and consequently CDC25B is not phosphorylated. We show that ectopic expression of Aurora-A results in a bypass of the checkpoint that was partially overcome by a S353A mutant of CDC25B. Finally, we show that bypass of the G2/M checkpoint by the CHK1 kinase inhibitor UCN-01 results in the activation of Aurora-A and phosphorylation of CDC25B on S353. These results strongly suggest that Aurora-A-mediated phosphorylation of CDC25B at the centrosome is an important step contributing to the earliest events inducing mitosis, upstream of CDK1-cyclin B1 activation.

Regulation of Chk2 phosphorylation by interaction with protein phosphatase 2A via its B' regulatory subunit.

Chk2 is a key player of the DNA damage signalling pathway. To identify new regulators of this kinase, we performed a yeast two-hybrid screen and found that Chk2 associated with the B' regulatory subunit of protein phosphatase PP2A. In vitro GST-Chk2 pulldowns demonstrated that B'gamma isoforms bound to Chk2 with the strongest apparent affinity. This was confirmed in cellulo by co-immunoprecipitation after overexpression of the respective partners in HEK293 cells. The A and C subunits of PP2A were present in the complexes, suggesting that Chk2 was associated with a functionnal PP2A. In vitro kinase assays showed that B'gamma3 was a potent Chk2 substrate. This phosphorylation increased the catalytic phosphatase activity of PP2A measured on MAP kinase-phosphorylated myelin basic protein as well as on autophosphorylated Chk2. Finally, we demonstrated that overexpressing B'gamma3 in HEK293 suppressed the phosphorylation of Chk2 induced by a genotoxic treatment, suggesting that PP2A may counteract the action of the checkpoint kinase in living cells.

Phosphorylation of CDC25B by Aurora-A at the centrosome contributes to the G2-M transition.

Aurora-A protein kinase, which is the product of an oncogene, is required for the assembly of a functional mitotic apparatus and the regulation of cell ploidy. Overexpression of Aurora-A in tumour cells has been correlated with cancer susceptibility and poor prognosis. Aurora-A activity is required for the recruitment of CDK1-cyclin B1 to the centrosome prior to its activation and the commitment of the cell to mitosis. In this report, we demonstrate that the CDC25B phosphatase, an activator of cyclin dependent kinases at mitosis, is phosphorylated both in vitro and in vivo by Aurora-A on serine 353 and that this phosphorylated form of CDC25B is located at the centrosome during mitosis. Knockdown experiments by RNAi confirm that the centrosome phosphorylation of CDC25B on S353 depends on Aurora-A kinase. Microinjection of antibodies against phosphorylated S353 results in a mitotic delay whilst overexpression of a S353 phosphomimetic mutant enhances the mitotic inducing effect of CDC25B. Our results demonstrate that Aurora-A phosphorylates CDC25B in vivo at the centrosome during mitosis. This phosphorylation might locally participate in the control of the onset of mitosis. These findings re-emphasise the role of the centrosome as a functional integrator of the pathways contributing to the triggering of mitosis.