Human SLX4 is a Holliday junction resolvase subunit that binds multiple DNA repair/recombination endonucleases.

Structure-specific endonucleases resolve DNA secondary structures generated during DNA repair and recombination. The yeast 5' flap endonuclease Slx1-Slx4 has received particular attention with the finding that Slx4 has Slx1-independent key functions in genome maintenance. Although Slx1 is a highly conserved protein in eukaryotes, no orthologs of Slx4 were reported other than in fungi. Here we report the identification of Slx4 orthologs in metazoa, including fly MUS312, essential for meiotic recombination, and human BTBD12, an ATM/ATR checkpoint kinase substrate. Human SLX1-SLX4 displays robust Holliday junction resolvase activity in addition to 5' flap endonuclease activity. Depletion of SLX1 and SLX4 results in 53BP1 foci accumulation and H2AX phosphorylation as well as cellular hypersensitivity to MMS. Furthermore, we show that SLX4 binds the XPF(ERCC4) and MUS81 subunits of the XPF-ERCC1 and MUS81-EME1 endonucleases and is required for DNA interstrand crosslink repair. We propose that SLX4 acts as a docking platform for multiple structure-specific endonucleases.

Role of EMT in the DNA damage response, double-strand break repair pathway choice and its implications in cancer treatment.

Numerous epithelial-mesenchymal transition (EMT) characteristics have now been demonstrated to participate in tumor development. Indeed, EMT is involved in invasion, acquisition of stem cell properties, and therapy-associated resistance of cancer cells. Together, these mechanisms offer advantages in adapting to changes in the tumor microenvironment. However, recent findings have shown that EMT-associated transcription factors (EMT-TFs) may also be involved in DNA repair. A better understanding of the coordination between the DNA repair pathways and the role played by some EMT-TFs in the DNA damage response (DDR) should pave the way for new treatments targeting tumor-specific molecular vulnerabilities, which result in selective destruction of cancer cells. Here we review recent advances, providing novel insights into the role of EMT in the DDR and repair pathways, with a particular focus on the influence of EMT on cellular sensitivity to damage, as well as the implications of these relationships for improving the efficacy of cancer treatments.

Destabilization of the TWIST1/E12 complex dimerization following the R154P point-mutation of TWIST1: an in silico approach.

BACKGROUND: The bHLH transcription factor TWIST1 plays a key role in the embryonic development and in tumorigenesis. Some loss-of-function mutations of the TWIST1 gene have been shown to cause an autosomal dominant craniosynostosis, known as the Saethre-Chotzen syndrome (SCS). Although the functional impacts of many TWIST1 mutations have been experimentally reported, little is known on the molecular mechanisms underlying their loss-of-function. In a previous study, we highlighted the predictive value of in silico molecular dynamics (MD) simulations in deciphering the molecular function of TWIST1 residues. RESULTS: Here, since the substitution of the arginine 154 amino acid by a glycine residue (R154G) is responsible for the SCS phenotype and the substitution of arginine 154 by a proline experimentally decreases the dimerizing ability of TWIST1, we investigated the molecular impact of this point mutation using MD approaches. Consistently, MD simulations highlighted a clear decrease in the stability of the α-helix during the dimerization of the mutated R154P TWIST1/E12 dimer compared to the wild-type TE complex, which was further confirmed in vitro using immunoassays. CONCLUSIONS: Our study demonstrates that MD simulations provide a structural explanation for the loss-of-function associated with the SCS TWIST1 mutation and provides a proof of concept of the predictive value of these MD simulations. This in silico methodology could be used to determine reliable pharmacophore sites, leading to the application of docking approaches in order to identify specific inhibitors of TWIST1 complexes.

FF483-484 motif of human Polη mediates its interaction with the POLD2 subunit of Polδ and contributes to DNA damage tolerance.

Switching between replicative and translesion synthesis (TLS) DNA polymerases are crucial events for the completion of genomic DNA synthesis when the replication machinery encounters lesions in the DNA template. In eukaryotes, the translesional DNA polymerase η (Polη) plays a central role for accurate bypass of cyclobutane pyrimidine dimers, the predominant DNA lesions induced by ultraviolet irradiation. Polη deficiency is responsible for a variant form of the Xeroderma pigmentosum (XPV) syndrome, characterized by a predisposition to skin cancer. Here, we show that the FF483-484 amino acids in the human Polη (designated F1 motif) are necessary for the interaction of this TLS polymerase with POLD2, the B subunit of the replicative DNA polymerase δ, both in vitro and in vivo. Mutating this motif impairs Polη function in the bypass of both an N-2-acetylaminofluorene adduct and a TT-CPD lesion in cellular extracts. By complementing XPV cells with different forms of Polη, we show that the F1 motif contributes to the progression of DNA synthesis and to the cell survival after UV irradiation. We propose that the integrity of the F1 motif of Polη, necessary for the Polη/POLD2 interaction, is required for the establishment of an efficient TLS complex.

EMT inducers catalyze malignant transformation of mammary epithelial cells and drive tumorigenesis towards claudin-low tumors in transgenic mice.

The epithelial-mesenchymal transition (EMT) is an embryonic transdifferentiation process consisting of conversion of polarized epithelial cells to motile mesenchymal ones. EMT-inducing transcription factors are aberrantly expressed in multiple tumor types and are known to favor the metastatic dissemination process. Supporting oncogenic activity within primary lesions, the TWIST and ZEB proteins can prevent cells from undergoing oncogene-induced senescence and apoptosis by abolishing both p53- and RB-dependent pathways. Here we show that they also downregulate PP2A phosphatase activity and efficiently cooperate with an oncogenic version of H-RAS in malignant transformation of human mammary epithelial cells. Thus, by down-regulating crucial tumor suppressor functions, EMT inducers make cells particularly prone to malignant conversion. Importantly, by analyzing transformed cells generated in vitro and by characterizing novel transgenic mouse models, we further demonstrate that cooperation between an EMT inducer and an active form of RAS is sufficient to trigger transformation of mammary epithelial cells into malignant cells exhibiting all the characteristic features of claudin-low tumors, including low expression of tight and adherens junction genes, EMT traits, and stem cell-like characteristics. Claudin-low tumors are believed to be the most primitive breast malignancies, having arisen through transformation of an early epithelial precursor with inherent stemness properties and metaplastic features. Challenging this prevailing view, we propose that these aggressive tumors arise from cells committed to luminal differentiation, through a process driven by EMT inducers and combining malignant transformation and transdifferentiation.

Crosstalk between inflammasome sensors and DNA damage response pathways.

Eukaryotic cells encounter diverse threats jeopardizing their integrity, prompting the development of defense mechanisms against these stressors. Among these mechanisms, inflammasomes are well-known for their roles in coordinating the inflammatory response against infections. Extensive research has unveiled their multifaceted involvement in cellular processes beyond inflammation. Recent studies emphasize the intricate relationship between the inflammasome and the DNA damage response (DDR). They highlight how the DDR participates in inflammasome activation and the reciprocal impact of inflammasome on DDR and genome integrity preservation. Moreover, novel functions of inflammasome sensors in DDR pathways have emerged, broadening our understanding of their roles. Finally, this review delves into identifying common signals that drive the activation of inflammasome sensors alongside activation cues for the DNA damage response, offering potential insights into shared regulatory pathways between these critical cellular processes.

Inflammasome-independent NLRP3 function enforces ATM activity in response to genotoxic stress.

NLRP3 is a pattern recognition receptor with a well-documented role in inducing inflammasome assembly in response to cellular stress. Deregulation of its activity leads to many inflammatory disorders including gouty arthritis, Alzheimer disease, and cancer. Whereas its role in the context of cancer has been mostly explored in the immune compartment, whether NLRP3 exerts functions unrelated to immunity in cancer development remains unexplored. Here, we demonstrate that NLRP3 interacts with the ATM kinase to control the activation of the DNA damage response, independently of its inflammasome activity. NLRP3 down-regulation in both broncho- and mammary human epithelial cells significantly impairs ATM pathway activation, leading to lower p53 activation, and provides cells with the ability to resist apoptosis induced by acute genotoxic stress. Interestingly, NLRP3 expression is down-regulated in non-small cell lung cancers and breast cancers, and its expression positively correlates with patient overall survival. Our findings identify a novel non-immune function for NLRP3 in maintaining genome integrity and strengthen the concept of a functional link between innate immunity and DNA damage sensing pathways to maintain cell integrity.

Opposite Roles for ZEB1 and TMEJ in the Regulation of Breast Cancer Genome Stability.

Breast cancer cells frequently acquire mutations in faithful DNA repair genes, as exemplified by BRCA-deficiency. Moreover, overexpression of an inaccurate DNA repair pathway may also be at the origin of the genetic instability arising during the course of cancer progression. The specific gain in expression of POLQ, encoding the error-prone DNA polymerase Theta (POLθ) involved in theta-mediated end joining (TMEJ), is associated with a characteristic mutational signature. To gain insight into the mechanistic regulation of POLQ expression, this review briefly presents recent findings on the regulation of POLQ in the claudin-low breast tumor subtype, specifically expressing transcription factors involved in epithelial-to-mesenchymal transition (EMT) such as ZEB1 and displaying a paucity in genomic abnormality.

EMT Transcription Factor ZEB1 Represses the Mutagenic POLθ-Mediated End-Joining Pathway in Breast Cancers.

A characteristic of cancer development is the acquisition of genomic instability, which results from the inaccurate repair of DNA damage. Among double-strand break repair mechanisms induced by oncogenic stress, the highly mutagenic theta-mediated end-joining (TMEJ) pathway, which requires DNA polymerase theta (POLθ) encoded by the POLQ gene, has been shown to be overexpressed in several human cancers. However, little is known regarding the regulatory mechanisms of TMEJ and the consequence of its dysregulation. In this study, we combined a bioinformatics approach exploring both Molecular Taxonomy of Breast Cancer International Consortium and The Cancer Genome Atlas databases with CRISPR/Cas9-mediated depletion of the zinc finger E-box binding homeobox 1 (ZEB1) in claudin-low tumor cells or forced expression of ZEB1 in basal-like tumor cells, two triple-negative breast cancer (TNBC) subtypes, to demonstrate that ZEB1 represses POLQ expression. ZEB1, a master epithelial-to-mesenchymal transition-inducing transcription factor, interacted directly with the POLQ promoter. Moreover, downregulation of POLQ by ZEB1 fostered micronuclei formation in TNBC tumor cell lines. Consequently, ZEB1 expression prevented TMEJ activity, with a major impact on genome integrity. In conclusion, we showed that ZEB1 directly inhibits the expression of POLQ and, therefore, TMEJ activity, controlling both stability and integrity of breast cancer cell genomes. SIGNIFICANCE: These findings uncover an original mechanism of TMEJ regulation, highlighting ZEB1 as a key player in genome stability during cancer progression via its repression of POLQ.See related commentary by Carvajal-Maldonado and Wood, p. 1441.

Comprehensive characterization of claudin-low breast tumors reflects the impact of the cell-of-origin on cancer evolution.

Claudin-low breast cancers are aggressive tumors defined by the low expression of key components of cellular junctions, associated with mesenchymal and stemness features. Although they are generally considered as the most primitive breast malignancies, their histogenesis remains elusive. Here we show that this molecular subtype of breast cancers exhibits a significant diversity, comprising three main subgroups that emerge from unique evolutionary processes. Genetic, gene methylation and gene expression analyses reveal that two of the subgroups relate, respectively, to luminal breast cancers and basal-like breast cancers through the activation of an EMT process over the course of tumor progression. The third subgroup is closely related to normal human mammary stem cells. This unique subgroup of breast cancers shows a paucity of genomic aberrations and a low frequency of TP53 mutations, supporting the emerging notion that the intrinsic properties of the cell-of-origin constitute a major determinant of the genetic history of tumorigenesis.

PARP3, a new therapeutic target to alter Rictor/mTORC2 signaling and tumor progression in BRCA1-associated cancers.

PARP3 has been shown to be a key driver of TGFß-induced epithelial-to-mesenchymal transition (EMT) and stemness in breast cancer cells, emerging as an attractive therapeutic target. Nevertheless, the therapeutic value of PARP3 inhibition has not yet been assessed. Here we investigated the impact of the absence of PARP3 or its inhibition on the tumorigenicity of BRCA1-proficient versus BRCA1-deficient breast cancer cell lines, focusing on the triple-negative breast cancer subtype (TNBC). We show that PARP3 knockdown exacerbates centrosome amplification and genome instability and reduces survival of BRCA1-deficient TNBC cells. Furthermore, we engineered PARP3-/- BRCA1-deficient or BRCA1-proficient TNBC cell lines using the CRISPR/nCas9D10A gene editing technology and demonstrate that the absence of PARP3 selectively suppresses the growth, survival and in vivo tumorigenicity of BRCA1-deficient TNBC cells, mechanistically via effects associated with an altered Rictor/mTORC2 signaling complex resulting from enhanced ubiquitination of Rictor. Accordingly, PARP3 interacts with and ADP-ribosylates GSK3ß, a positive regulator of Rictor ubiquitination and degradation. Importantly, these phenotypes were rescued by re-expression of a wild-type PARP3 but not by a catalytic mutant, demonstrating the importance of PARP3's catalytic activity. Accordingly, reduced survival and compromised Rictor/mTORC2 signaling were also observed using a cell-permeable PARP3-specific inhibitor. We conclude that PARP3 and BRCA1 are synthetic lethal and that targeting PARP3's catalytic activity is a promising therapeutic strategy for BRCA1-associated cancers via the Rictor/mTORC2 signaling pathway.

The cell-of-origin dictates the genomic landscape of breast cancers.

Aberrant cell proliferation induced by activated oncogenes triggers oxidative stress and uncontrolled DNA replication, promoting genomic instability. We recently reported that human mammary stem cells exhibit the unique capacity to withstand an oncogenic activation by dint of an anti-oxidant program driven by the ZEB1 transcription factor. This pre-emptive program prevents the onset of chromosomal instability, leading to the development of tumors with unique pathological features.

A stemness-related ZEB1-MSRB3 axis governs cellular pliancy and breast cancer genome stability.

Chromosomal instability (CIN), a feature of most adult neoplasms from their early stages onward, is a driver of tumorigenesis. However, several malignancy subtypes, including some triple-negative breast cancers, display a paucity of genomic aberrations, thus suggesting that tumor development may occur in the absence of CIN. Here we show that the differentiation status of normal human mammary epithelial cells dictates cell behavior after an oncogenic event and predetermines the genetic routes toward malignancy. Whereas oncogene induction in differentiated cells induces massive DNA damage, mammary stem cells are resistant, owing to a preemptive program driven by the transcription factor ZEB1 and the methionine sulfoxide reductase MSRB3. The prevention of oncogene-induced DNA damage precludes induction of the oncosuppressive p53-dependent DNA-damage response, thereby increasing stem cells' intrinsic susceptibility to malignant transformation. In accord with this model, a subclass of breast neoplasms exhibit unique pathological features, including high ZEB1 expression, a low frequency of TP53 mutations and low CIN.

The Heterodimeric TWIST1-E12 Complex Drives the Oncogenic Potential of TWIST1 in Human Mammary Epithelial Cells.

The TWIST1 embryonic transcription factor displays biphasic functions during the course of carcinogenesis. It facilitates the escape of cells from oncogene-induced fail-safe programs (senescence, apoptosis) and their consequent neoplastic transformation. Additionally, it promotes the epithelial-to-mesenchymal transition and the initiation of the metastatic spread of cancer cells. Interestingly, cancer cells recurrently remain dependent on TWIST1 for their survival and/or proliferation, making TWIST1 their Achilles' heel. TWIST1 has been reported to form either homodimeric or heterodimeric complexes mainly in association with the E bHLH class I proteins. These complexes display distinct, sometimes even antagonistic, functions during development and unequal prometastatic functions in prostate cancer cells. Using a tethered dimer strategy, we successively assessed the ability of TWIST1 dimers to cooperate with an activated version of RAS in human mammary epithelial cell transformation, to provide mice with the ability to spontaneously develop breast tumors, and lastly to maintain a senescence program at a latent state in several breast cancer cell lines. We demonstrate that the TWIST1-E12 complex, unlike the homodimer, is an oncogenic form of TWIST1 in mammary epithelial cells and that efficient binding of both partners is a prerequisite for its activity. The detection of the heterodimer in human premalignant lesions by a proximity ligation assay, at a stage preceding the initiation of the metastatic cascade, is coherent with such an oncogenic function. TWIST1-E protein heterodimeric complexes may thus constitute the main active forms of TWIST1 with regard to senescence inhibition over the time course of breast tumorigenesis.

PARP3 controls TGFß and ROS driven epithelial-to-mesenchymal transition and stemness by stimulating a TG2-Snail-E-cadherin axis.

Several members of the Poly(ADP-ribose) polymerase (PARP) family are essential regulators of genome integrity, actively prospected as drug targets for cancer therapy. Among them, PARP3 is well characterized for its functions in double-strand break repair and mitotis. Here we report that PARP3 also plays an integral role in TGFß and reactive oxygen species (ROS) dependent epithelial-to-mesenchymal transition (EMT) and stem-like cell properties in human mammary epithelial and breast cancer cells. PARP3 expression is higher in breast cancer cells of the mesenchymal phenotype and correlates with the expression of the mesenchymal marker Vimentin while being in inverse correlation with the epithelial marker E-cadherin. Furthermore, PARP3 expression is significantly upregulated during TGFß-induced EMT in various human epithelial cells. In line with this observation, PARP3 depletion alters TGFß-dependent EMT of mammary epithelial cells by preventing the induction of the Snail-E-cadherin axis, the dissolution of cell junctions, the acquisition of cell motility and chemoresistance. PARP3 responds to TGFß-induced ROS to promote a TG2-Snail-E-cadherin axis during EMT. Considering the link between EMT and cancer stem cells, we show that PARP3 promotes stem-like cell properties in mammary epithelial and breast cancer cells by inducing the expression of the stem cell markers SOX2 and OCT4, by increasing the proportion of tumor initiating CD44high/CD24low population and the formation of tumor spheroid bodies, and by promoting stem cell self-renewal. These findings point to a novel role of PARP3 in the control of TGFß-induced EMT and acquisition of stem-like cell features and further motivate efforts to identify PARP3 specific inhibitors.

Co-localization in replication foci and interaction of human Y-family members, DNA polymerase pol eta and REVl protein.

The progress of replicative DNA polymerases along the replication fork may be impeded by the presence of lesions in the genome. One way to circumvent such hurdles involves the recruitment of specialized DNA polymerases that perform limited incorporation of nucleotides in the vicinity of the damaged site. This process entails DNA polymerase switch between replicative and specialized DNA polymerases. Five eukaryotic proteins can carry out translesion synthesis (TLS) of damaged DNA in vitro, DNA polymerases zeta, eta, iota, and kappa, and REV1. To identify novel proteins that interact with hpol eta, we performed a yeast two-hybrid screen. In this paper, we show that hREV1 interacts with hpol eta as well as with hpol kappa and poorly with hpol iota. Furthermore, cellular localization analysis demonstrates that hREV1 is present, with hpol eta in replication factories at stalled replication forks and is tightly associated with nuclear structures. This hREV1 nuclear localization occurs independently of the presence of hpol eta. Taken together, our data suggest a central role for hREV1 as a scaffold that recruits DNA polymerases involved in TLS.

poliota-dependent lesion bypass in vitro.

Based upon phylogenetic relationships, the broad Y-family of DNA polymerases can be divided into various subfamilies consisting of UmuC (polV)-like; DinB (polIV/polkappa)-like; Rev1-like, Rad30A (poleta)-like and Rad30B (poliota)-like polymerases. The polIV/polkappa-like polymerases are most ubiquitous, having been identified in bacteria, archaea and eukaryotes. In contrast, the polV-like polymerases appear restricted to bacteria (both Gram positive and Gram negative). Rev1 and poleta-like polymerases are found exclusively in eukaryotes, and to date, poliota-like polymerases have only been identified in higher eukaryotes. In general, the in vitro properties of polymerases characterized within each sub-family are quite similar. An exception to this rule occurs with the poliota-like polymerases, where the enzymatic properties of Drosophila melanogaster poliota are more similar to that of Saccharomyces cerevisiae and human poleta than to the related human poliota. For example, like poleta, Drosophila poliota can bypass a cis-syn thymine-thymine dimer both accurately and efficiently, while human poliota bypasses the same lesion inefficiently and with low-fidelity. Even in cases where human poliota can efficiently insert a base opposite a lesion (such as a synthetic abasic site, the 3'T of a 6-4-thymine-thymine pyrimidine-pyrimidone photoproduct or opposite benzo[a]pyrene diol epoxide deoxyadenosine adducts), further extension is often limited. Thus, although poliota most likely arose from a genetic duplication of poleta millions of years ago as eukaryotes evolved, it would appear that poliota from humans (and possibly all mammals) has been further subjected to evolutionary pressures that have "tailored" its enzymatic properties away from lesion bypass and towards other function(s) specific for higher eukaryotes. The identification of such functions and the role that mammalian poliota plays in lesion bypass in vivo, should hopefully be forthcoming with the construction of human cell lines deleted for poliota and the identification of mice deficient in poliota.

Crosstalk between replicative and translesional DNA polymerases: PDIP38 interacts directly with Poleta.

Replicative DNA polymerases duplicate genomes in a very efficient and accurate mode. However their progression can be blocked by DNA lesions since they are unable to accommodate bulky damaged bases in their active site. In response to replication blockage, monoubiquitination of PCNA promotes the switch between replicative and specialized polymerases proficient to overcome the obstacle. In this study, we characterize novel connections between proteins involved in replication and TransLesion Synthesis (TLS). We demonstrate that PDIP38 (Poldelta interacting protein of 38kDa) directly interacts with the TLS polymerase Poleta. Interestingly, the region of Poleta interacting with PDIP38 is found to be located within the ubiquitin-binding zinc finger domain (UBZ) of Poleta. We show that the depletion of PDIP38 increases the number of cells with Poleta foci in the absence of DNA damage and diminishes cell survival after UV irradiation. In addition, PDIP38 is able to interact directly not only with Poleta but also with the specialized polymerases Rev1 and Polzeta (via Rev7). We thus suggest that PDIP38 serves as a mediator protein helping TLS Pols to transiently replace replicative polymerases at damaged sites.