Characterization of POLE c.1373A > T p.(Tyr458Phe), causing high cancer risk.

The cancer syndrome polymerase proofreading-associated polyposis results from germline mutations in the POLE and POLD1 genes. Mutations in the exonuclease domain of these genes are associated with hyper- and ultra-mutated tumors with a predominance of base substitutions resulting from faulty proofreading during DNA replication. When a new variant is identified by gene testing of POLE and POLD1, it is important to verify whether the variant is associated with PPAP or not, to guide genetic counseling of mutation carriers. In 2015, we reported the likely pathogenic (class 4) germline POLE c.1373A > T p.(Tyr458Phe) variant and we have now characterized this variant to verify that it is a class 5 pathogenic variant. For this purpose, we investigated (1) mutator phenotype in tumors from two carriers, (2) mutation frequency in cell-based mutagenesis assays, and (3) structural consequences based on protein modeling. Whole-exome sequencing of two tumors identified an ultra-mutator phenotype with a predominance of base substitutions, the majority of which are C > T. A SupF mutagenesis assay revealed increased mutation frequency in cells overexpressing the variant of interest as well as in isogenic cells encoding the variant. Moreover, exonuclease repair yeast-based assay supported defect in proofreading activity. Lastly, we present a homology model of human POLE to demonstrate structural consequences leading to pathogenic impact of the p.(Tyr458Phe) mutation. The three lines of evidence, taken together with updated co-segregation and previously published data, allow the germline variant POLE c.1373A > T p.(Tyr458Phe) to be reclassified as a class 5 variant. That means the variant is associated with PPAP.

Role of POLE and POLD1 in familial cancer.

PURPOSE: Germline pathogenic variants in the exonuclease domain (ED) of polymerases POLE and POLD1 predispose to adenomatous polyps, colorectal cancer (CRC), endometrial tumors, and other malignancies, and exhibit increased mutation rate and highly specific associated mutational signatures. The tumor spectrum and prevalence of POLE and POLD1 variants in hereditary cancer are evaluated in this study. METHODS: POLE and POLD1 were sequenced in 2813 unrelated probands referred for genetic counseling (2309 hereditary cancer patients subjected to a multigene panel, and 504 patients selected based on phenotypic characteristics). Cosegregation and case-control studies, yeast-based functional assays, and tumor mutational analyses were performed for variant interpretation. RESULTS: Twelve ED missense variants, 6 loss-of-function, and 23 outside-ED predicted-deleterious missense variants, all with population allele frequencies <1%, were identified. One ED variant (POLE p.Met294Arg) was classified as likely pathogenic, four as likely benign, and seven as variants of unknown significance. The most commonly associated tumor types were colorectal, endometrial and ovarian cancers. Loss-of-function and outside-ED variants are likely not pathogenic for this syndrome. CONCLUSIONS: Polymerase proofreading-associated syndrome constitutes 0.1-0.4% of familial cancer cases, reaching 0.3-0.7% when only CRC and polyposis are considered. ED variant interpretation is challenging and should include multiple pieces of evidence.

Novel POLE pathogenic germline variant in a family with multiple primary tumors results in distinct mutational signatures.

We describe a family in which four siblings exhibited multiple or classic colonic polyposis with or without colorectal carcinoma (CRC). One female developed three primary tumors, including CRC and carcinomas of the ovary and breast. Whole-exome sequencing of germline DNA from affected and unaffected individuals revealed a novel missense mutation in the exonuclease domain of POLE (c.833C>A; p.Thr278Lys) associated with a highly penetrant, autosomal-dominant inheritance pattern. Functional studies in yeast and demonstration of a high mutational burden in the available tumors confirmed the pathogenicity of the novel variant. Prominent POLE-deficient somatic mutational signatures were seen in the CRCs, but in contrast, a mutational signature typical of concomitant tumoral loss of POLE and mismatch-repair function (POLE-exo* /MSI) was noted in the breast cancer. The breast cancer also showed distinctive pathological characteristics that reflect the presence of both the germline POLE variant and the secondary somatic MMR alterations.

Role of p27Kip1 as a transcriptional regulator.

The protein p27Kip1 is a member of the Cip/Kip family of cyclin-dependent kinase (Cdk) inhibitors. It interacts with both the catalytic and the regulatory subunit (cyclin) and introduces a region into the catalytic cleave of the Cdk inducing its inactivation. Its inhibitory capacity can be modulated by specific tyrosine phosphorylations. p27Kip1 also behaves as a transcriptional regulator. It associates with specific chromatin domains through different transcription factors. ChIP on chip, ChIP-seq and expression microarray analysis allowed the identification of the transcriptional programs regulated by p27Kip1. Thus, important cellular functions as cell division cycle, respiration, RNA processing, translation and cell adhesion, are under p27Kip1 regulation. Moreover, genes involved in pathologies as cancer and neurodegeneration are also regulated by p27Kip1, suggesting its implication in these pathologies. The carboxyl moiety of p27Kip1 can associate with different proteins, including transcriptional regulators. In contrast, its NH2-terminal region specifically interacts with cyclin-Cdk complexes. The general mechanistic model of how p27Kip1 regulates transcription is that it associates by its COOH region to the transcriptional regulators on the chromatin and by the NH2-domain to cyclin-Cdk complexes. After Cdk activation it would phosphorylate the specific targets on the chromatin leading to gene expression. This model has been demonstrated to apply in the transcriptional regulation of p130/E2F4 repressed genes involved in cell cycle progression. We summarize in this review our current knowledge on the role of p27Kip1 in the regulation of transcription, on the transcriptional programs under its regulation and on its relevance in pathologies as cancer and neurodegeneration.

p27Kip1 regulates alpha-synuclein expression.

Alpha-synuclein (α-SYN) is the main component of anomalous protein aggregates (Lewy bodies) that play a crucial role in several neurodegenerative diseases (synucleinopathies) like Parkinson's disease and multiple system atrophy. However, the mechanisms involved in its transcriptional regulation are poorly understood. We investigated here the role of the cyclin-dependent kinase (Cdk) inhibitor and transcriptional regulator p27Kip1 (p27) in the regulation of α-SYN expression. We observed that selective deletion of p27 by CRISPR/Cas9 technology in neural cells resulted in increased levels of α-SYN. Knock-down of the member of the same family p21Cip1 (p21) also led to increased α-SYN levels, indicating that p27 and p21 collaborate in the repression of α-SYN transcription. We demonstrated that this repression is mediated by the transcription factor E2F4 and the member of the retinoblastoma protein family p130 and that it is dependent of Cdk activity. Chromatin immunoprecipitation analysis revealed specific binding sites for p27, p21 and E2F4 in the proximal α-SYN gene promoter. Finally, luciferase assays revealed a direct action of p27, p21 and E2F4 in α-SYN gene expression. Our findings reveal for the first time a negative regulatory mechanism of α-SYN expression, suggesting a putative role for cell cycle regulators in the etiology of synucleinopathies.

ChIP-Seq analysis identifies p27(Kip1)-target genes involved in cell adhesion and cell signalling in mouse embryonic fibroblasts.

The protein p27Kip1 (p27), a member of the Cip-Kip family of cyclin-dependent kinase inhibitors, is involved in tumorigenesis and a correlation between reduced levels of this protein in human tumours and a worse prognosis has been established. Recent reports revealed that p27 also behaves as a transcriptional regulator. Thus, it has been postulated that the development of tumours with low amounts of p27 could be propitiated by deregulation of transcriptional programs under the control of p27. However, these programs still remain mostly unknown. The aim of this study has been to define the transcriptional programs regulated by p27 by first identifying the p27-binding sites (p27-BSs) on the whole chromatin of quiescent mouse embryonic fibroblasts. The chromatin regions associated to p27 have been annotated to the most proximal genes and it has been considered that the expression of these genes could by regulated by p27. The identification of the chromatin p27-BSs has been performed by Chromatin Immunoprecipitation Sequencing (ChIP-seq). Results revealed that p27 associated with 1839 sites that were annotated to 1417 different genes being 852 of them protein coding genes. Interestingly, most of the p27-BSs were in distal intergenic regions and introns whereas, in contrast, its association with promoter regions was very low. Gene ontology analysis of the protein coding genes revealed a number of relevant transcriptional programs regulated by p27 as cell adhesion, intracellular signalling and neuron differentiation among others. We validated the interaction of p27 with different chromatin regions by ChIP followed by qPCR and demonstrated that the expressions of several genes belonging to these programs are actually regulated by p27. Finally, cell adhesion assays revealed that the adhesion of p27-/- cells to the plates was much higher that controls, revealing a role of p27 in the regulation of a transcriptional program involved in cell adhesion.

POLE and POLD1 screening in 155 patients with multiple polyps and early-onset colorectal cancer.

Germline mutations in POLE and POLD1 have been shown to cause predisposition to colorectal multiple polyposis and a wide range of neoplasms, early-onset colorectal cancer being the most prevalent. In order to find additional mutations affecting the proofreading activity of these polymerases, we sequenced its exonuclease domain in 155 patients with multiple polyps or an early-onset colorectal cancer phenotype without alterations in the known hereditary colorectal cancer genes. Interestingly, none of the previously reported mutations in POLE and POLD1 were found. On the other hand, among the genetic variants detected, only two of them stood out as putative pathogenic in the POLE gene, c.1359 + 46del71 and c.1420G > A (p.Val474Ile). The first variant, detected in two families, was not proven to alter correct RNA splicing. Contrarily, c.1420G > A (p.Val474Ile) was detected in one early-onset colorectal cancer patient and located right next to the exonuclease domain. The pathogenicity of this change was suggested by its rarity and bioinformatics predictions, and it was further indicated by functional assays in Schizosaccharomyces pombe. This is the first study to functionally analyze a POLE genetic variant outside the exonuclease domain and widens the spectrum of genetic changes in this DNA polymerase that could lead to colorectal cancer predisposition.

p27Kip1, PCAF and PAX5 cooperate in the transcriptional regulation of specific target genes.

The cyclin-dependent kinase inhibitor p27Kip1 (p27) also behaves as a transcriptional repressor. Data showing that the p300/CBP-associated factor (PCAF) acetylates p27 inducing its degradation suggested that PCAF and p27 could collaborate in the regulation of transcription. However, this possibility remained to be explored. We analyzed here the transcriptional programs regulated by PCAF and p27 in the colon cancer cell line HCT116 by chromatin immunoprecipitation sequencing (ChIP-seq). We identified 269 protein-encoding genes that contain both p27 and PCAF binding sites being the majority of these sites different for PCAF and p27. PCAF or p27 knock down revealed that both regulate the expression of these genes, PCAF as an activator and p27 as a repressor. The double knock down of PCAF and p27 strongly reduced their expression indicating that the activating role of PCAF overrides the repressive effect of p27. We also observed that the transcription factor Pax5 interacts with both p27 and PCAF and that the knock down of Pax5 induces the expression of p27/PCAF target genes indicating that it also participates in the transcriptional regulation mediated by p27/PCAF. In summary, we report here a previously unknown mechanism of transcriptional regulation mediated by p27, Pax5 and PCAF.

p27Kip1 and p21Cip1 collaborate in the regulation of transcription by recruiting cyclin-Cdk complexes on the promoters of target genes.

Transcriptional repressor complexes containing p130 and E2F4 regulate the expression of genes involved in DNA replication. During the G1 phase of the cell cycle, sequential phosphorylation of p130 by cyclin-dependent kinases (Cdks) disrupts these complexes allowing gene expression. The Cdk inhibitor and tumor suppressor p27(Kip1) associates with p130 and E2F4 by its carboxyl domain on the promoters of target genes but its role in the regulation of transcription remains unclear. We report here that p27(Kip1) recruits cyclin D2/D3-Cdk4 complexes on the promoters by its amino terminal domain in early and mid G1. In cells lacking p27(Kip1), cyclin D2/D3-Cdk4 did not associate to the promoters and phosphorylation of p130 and transcription of target genes was increased. In late G1, these complexes were substituted by p21(Cip1)-cyclin D1-Cdk2. In p21(Cip1) null cells cyclin D1-Cdk2 were not found on the promoters and transcription was elevated. In p21/p27 double null cells transcription was higher than in control cells and single knock out cells. Thus, our results clarify the role of p27(Kip1) and p21(Cip1) in transcriptional regulation of genes repressed by p130/E2F4 complexes in which p27(Kip1) and p21(Cip1) play a sequential role by recruiting and regulating the activity of specific cyclin-Cdk complexes on the promoters.

The p38 MAPK-MK2 axis regulates E2F1 and FOXM1 expression after epirubicin treatment.

E2F1 is responsible for the regulation of FOXM1 expression, which plays a key role in epirubicin resistance. Here, we examined the role and regulation of E2F1 in response to epirubicin in cancer cells. We first showed that E2F1 plays a key role in promoting FOXM1 expression, cell survival, and epirubicin resistance as its depletion by siRNA attenuated FOXM1 induction and cell viability in response to epirubicin. We also found that the p38-MAPK activity mirrors the expression patterns of E2F1 and FOXM1 in both epirubicin-sensitive and -resistant MCF-7 breast cancer cells, suggesting that p38 has a role in regulating E2F1 expression and epirubicin resistance. Consistently, studies using pharmacologic inhibitors, siRNA knockdown, and knockout mouse embryonic fibroblasts (MEF) revealed that p38 mediates the E2F1 induction by epirubicin and that the induction of E2F1 by p38 is, in turn, mediated through its downstream kinase MK2 [mitogen-activated protein kinase (MAPK)-activated protein kinase 2; MAPKAPK2]. In agreement, in vitro phosphorylation assays showed that MK2 can directly phosphorylate E2F1 at Ser-364. Transfection assays also showed that E2F1 phosphorylation at Ser-364 participates in its induction by epirubicin but also suggests that other phosphorylation events are also involved. In addition, the p38-MK2 axis can also limit c-jun-NH(2)-kinase (JNK) induction by epirubicin and, notably, JNK represses FOXM1 expression. Collectively, these findings underscore the importance of p38-MK2 signaling in the control of E2F1 and FOXM1 expression as well as epirubicin sensitivity.

ATM and p53 regulate FOXM1 expression via E2F in breast cancer epirubicin treatment and resistance.

In this report, we investigated the role and regulation of forkhead box M1 (FOXM1) in breast cancer and epirubicin resistance. We generated epirubicin-resistant MCF-7 breast carcinoma (MCF-7-EPI(R)) cells and found FOXM1 protein levels to be higher in MCF-7-EPI(R) than in MCF-7 cells and that FOXM1 expression is downregulated by epirubicin in MCF-7 but not in MCF-7-EPI(R) cells. We also established that there is a loss of p53 function in MCF-7-EPI(R) cells and that epirubicin represses FOXM1 expression at transcription and gene promoter levels through activation of p53 and repression of E2F activity in MCF-7 cells. Using p53(-/-) mouse embryo fibroblasts, we showed that p53 is important for epirubicin sensitivity. Moreover, transient promoter transfection assays showed that epirubicin and its cellular effectors p53 and E2F1 modulate FOXM1 transcription through an E2F-binding site located within the proximal promoter region. Chromatin immunoprecipitation analysis also revealed that epirubicin treatment increases pRB (retinoblastoma protein) and decreases E2F1 recruitment to the FOXM1 promoter region containing the E2F site. We also found ataxia-telangiectasia mutated (ATM) protein and mRNA to be overexpressed in the resistant MCF-7-EPI(R) cells compared with MCF-7 cells and that epirubicin could activate ATM to promote E2F activity and FOXM1 expression. Furthermore, inhibition of ATM in U2OS cells with caffeine or depletion of ATM in MCF-7-EPI(R) with short interfering RNAs can resensitize these resistant cells to epirubicin, resulting in downregulation of E2F1 and FOXM1 expression and cell death. In summary, our data show that ATM and p53 coordinately regulate FOXM1 via E2F to modulate epirubicin response and resistance in breast cancer.

Phytochemical profile, antioxidant and cytotoxic activities of the carob tree (Ceratonia siliqua L.) germ flour extracts.

This work aimed to evaluate the phytochemical content and to determine the antioxidant and cytotoxic activities of methanol extracts of the carob tree (Ceratonia siliqua L.) germ flour. The extracts were rich in phenolic compounds, had considerable antioxidant activity, and reduced the viability of cervical (HeLa) cancer cells. The chemical content and the biological activities of the extracts were significantly affected by gender and cultivar. Female cultivar Galhosa had the highest levels of phenolic compounds, and the highest antioxidant activity. Extracts from the hermaphrodite trees and from the female cultivars Galhosa and Costela/Canela exhibited the highest cytotoxic activity. The most abundant compound was theophylline. The phenolic content was correlated to both antioxidant and cytotoxic activities. Our findings provide new knowledge about the health implications of consuming food supplemented with carob germ flour.

Lamellarin D bioconjugates I: Synthesis and cellular internalization of PEG-derivatives.

Herein is reported the design and synthesis of poly(ethylene glycol) derivatives of Lamellarin D with the aim of modulating their physicochemical properties and improving the biological activity. Mono-, di-, and tri-PEG conjugates with improved solubility were obtained in 18-57% overall yields from the corresponding partially protected phenolic derivatives of Lamellarin D. Conjugates 1-9 were tested in a panel of three human tumor cell lines (MDA-MB-231 breast, A-549 lung, and HT-29 colon) to evaluate their cytotoxicity. Several compounds exhibited enhanced cellular internalization, and more than 85% of the derivatives showed a lower GI(50) than Lam-D. Furthermore, cell cycle arrest at G2 phase and apoptotic cell-death pathways were determined for Lamellarin D and these derivatives.

Lamellarin D bioconjugates II: Synthesis and cellular internalization of dendrimer and nuclear location signal derivatives.

The design and synthesis of Lamellarin D conjugates with a nuclear localization signal peptide and a poly(ethylene glycol)-based dendrimer are described. Conjugates 1-4 were obtained in 8-84% overall yields from the corresponding protected Lamellarin D. Conjugates 1 and 4 are 1.4- to 3.3-fold more cytotoxic than the parent compound against three human tumor cell lines (MDA-MB-231 breast, A-549 lung, and HT-29 colon). Besides, conjugates 3 and 4 showed a decrease in activity potency in BJ skin fibroblasts, a normal cell culture. Cellular internalization was analyzed, and a nuclear distribution pattern was observed for 4, which contains a nuclear localization signaling sequence.

Synthesis and antitumor activity of mechercharmycin A analogues.

Several analogues of the cytotoxic thiopeptide IB-01211 or mechercharmycin A (1) have been synthesized. The cytotoxicity of 1 and the synthesized analogues were evaluated against a panel of three human tumor cell lines. Thiopeptide 1 and the most active derivatives 2 and 3c were chosen for further studies on effects on cell cycle progression and induction of apoptosis. Interestingly, the inhibition of cell division and activation of a programmed cell death by apoptosis were detected.

Glycogen synthase kinase-3beta binds to E2F1 and regulates its transcriptional activity.

GSK3beta and E2F1 play an important role in the control of proliferation and apoptosis. Previous work has demonstrated that GSK3beta indirectly regulates E2F activity through modulation of cyclin D1 levels. In this work we show that GSK3beta phosphorylates human E2F1 in vitro at serine 403 and threonine 433, both residues localized at its transactivation domain. This phosphorylation was not detected in vivo. However, co-immunoprecipitation experiments do reveal in vivo binding of these proteins. Moreover, uninhibitable and catalitycally inactive GSK3beta forms inhibit the transcriptional activity of a fusion protein containing E2F1 transactivation domain. Both forms of GSK3beta inhibit E2F1 with similar efficiency. Interestingly the effect was independent of the mutation of serine 403 and threonine 433 to alanine. This suggests that this transcriptional modulation is independent of GSK3beta kinase activity and phosphorylation state of serine 403 and threonine 433. The re-targeting of these GSK3beta forms to the nucleus results in a higher capacity to regulate E2F1 transcriptional activity. Depletion of the levels of GSK3beta protein using siRNA activates E2F1 transcriptional activity. The data presented in this study offer a new mechanism of regulation of E2F1 by direct binding of GSK3beta to its transactivation domain.

The structural plasticity of the C terminus of p21Cip1 is a determinant for target protein recognition.

The cyclin-dependent kinase inhibitory protein p21(Cip1) might play multiple roles in cell-cycle regulation through interaction of its C-terminal domain with a defined set of cellular proteins such as proliferating cell nuclear antigen (PCNA), calmodulin (CaM), and the oncoprotein SET. p21(Cip1) could be described as an intrinsically unstructured protein in solution although the C-terminal domain adopts a well-defined extended conformation when bound to PCNA. However, the molecular mechanism of the interaction with CaM and the oncoprotein SET is not well understood, partly because of the lack of structural information. In this work, a peptide derived from the C-terminal domain of p21(Cip1) that covers the binding domain of the three above-mentioned proteins was used to demonstrate that the C-terminal domain of p21 recognizes multiple ligands through its ability to adopt multiple conformations. The conformation is dictated by tertiary contacts rather than by the primary sequence of the protein. Our results suggest that the C-terminal domain of p21(Cip1) adopts an extended structure when bound to PCNA and probably when bound to the oncoprotein SET, but an alpha helix when bound to CaM.