Abdelilah Aboussekhra

Mammalian DNA nucleotide excision repair reconstituted with purified protein components

Nucleotide excision repair is the principal way by which human cells remove UV damage from DNA. Human cell extracts were fractionated to locate active components, including xeroderma pigmentosum (XP) and ERCC factors. The incision reaction was then reconstituted with the purified proteins RPA, XPA, TFIIH (containing XPB and XPD), XPC, UV-DDB, XPG, partially purified ERCC1/XPF complex, and a factor designated IF7. UV-DDB (related to XPE protein) stimulated repair but was not essential. ERCC1- and XPF-correcting activity copurified with an ERCC1-binding polypeptide of 110 kDa that was absent in XP-F cell extract. Complete repair synthesis was achieved by combining these factors with DNA polymerase epsilon, RFC, PCNA, and DNA ligase I. The reconstituted core reaction requires about 30 polypeptides.

Two human homologs of Rad23 are functionally interchangeable in complex formation and stimulation of XPC repair activity.

XPC-hHR23B protein complex is specifically involved in nucleotide excision repair (NER) of DNA lesions on transcriptionally inactive sequences as well as the nontranscribed strand of active genes. Here we demonstrate that not only highly purified recombinant hHR23B (rhHR23B) but also a second human homolog of the Saccharomyces cerevisiae Rad23 repair protein, hHR23A, stimulates the in vitro repair activity of recombinant human XPC (rhXPC), revealing functional redundancy between these human Rad23 homologs. Coprecipitation experiments with His-tagged rhHR23 as well as sedimentation velocity analysis showed that both rhHR23 proteins in vitro reconstitute a physical complex with rhXPC. Both complexes were more active than free rhXPC, indicating that complex assembly is required for the stimulation. rhHR23B was shown to stimulate an early stage of NER at or prior to incision. Furthermore, both rhHR23 proteins function in a defined NER system reconstituted with purified proteins, indicating direct involvement of hHR23 proteins in the DNA repair reaction via interaction with XPC.

Repair of UV-damaged DNA by mammalian cells and Saccharomyces cerevisiae

Cells use many strategies to repair genomic damage caused by environmental agents and arising from the natural instability of the polynucleotide structure. Nucleotide excision repair is the most versatile DNA repair pathway and is the main defense of mammalian cells against UV-induced DNA damage. Defects in proteins involved in this pathway can lead to inherited disorders (such as xeroderma pigmentosum, Cockaynes syndrome and trichothiodystrophy) that are associated with hypersensitivity to sunlight. Most of the proteins and genes involved in these syndromes have now been identified. Study of UV-sensitive yeast RAD mutants has greatly aided this process and has revealed strong conservation of the components of nucleotide excision repair in eukaryotes. It has recently become clear that some of the proteins involved in the DNA repair process have dual functions and also participate in basal transcription and DNA replication.

p16(INK4A) induces senescence and inhibits EMT through microRNA-141/microRNA-146b-5p-dependent repression of AUF1.

Senescence and epithelial‐to‐mesenchymal transition (EMT) processes are under the control of common tumor suppressor proteins, EMT transcription factors, and microRNAs. However, the molecular mechanisms that coordinate the functional link between senescence and EMT are still elusive. We have shown here that p16INK4A‐related induction of senescence is mediated through miR‐141 and miR‐146b‐5p. These two microRNAs are up‐regulated in aging human fibroblast and epithelial cells. Furthermore, miR‐141 and miR146b‐5p trigger cell cycle arrest at G1 phase and induce senescence in primary human fibroblasts and breast cancer cells in the presence and absence of p16INK4A. Like p16INK4A‐induced senescence, miR‐141/miR146b‐5p‐related senescence is not associated with secretory phenotype, and is mediated through the RNA binding protein AUF1. We have further demonstrated that p16INK4A and its downstream miRNA targets inhibit EMT through suppressing the EMT inducer ZEB1 in an AUF1‐dependent manner. Indeed, AUF1 binds the mRNA of this gene leading to increase in its level. These results indicate that p16INK4A controls both senescence and EMT through repressing EMT‐related transcription factor via miR‐141/miR146b‐5p and their target AUF1. This sheds more light on the molecular basis of the tumor suppressive functions of p16INK4A, which represses both the proliferative and the migratory/invasive capacities of cells.

Eugenol potentiates cisplatin anti-cancer activity through inhibition of ALDH-positive breast cancer stem cells and the NF-κB signaling pathway†

Triple‐negative breast tumors are very aggressive and contain relatively high proportion of cancer stem cells, and are resistant to chemotherapeutic drugs including cisplatin. To overcome these limitations, we combined eugenol, a natural polyphenolic molecule, with cisplatin to normalize cisplatin mediated toxicity and potential drug resistance. Interestingly, the combination treatment provided significantly greater cytotoxic and pro‐apoptotic effects as compared to treatment with eugenol or cisplatin alone on several triple‐negative breast cancer cells both in vitro and in vivo. Furthermore, adding eugenol to cisplatin potentiated the inhibition of breast cancer stem cells by inhibiting ALDH enzyme activity and ALDH‐positive tumor initiating cells. We provide also clear evidence that eugenol potentiates cisplatin inhibition of the NF‐κB signaling pathway. Indeed, the binding of NF‐κB to its cognate binding sites present in the promoters of IL‐6 and IL‐8 was dramatically reduced, which led to potent down‐regulation of the IL‐6 and IL‐8 cytokines upon combination treatment relative to the single agents. Similar effects were observed on proliferation, inhibition of epithelial‐to‐mesenchymal transition and stemness markers in tumor xenografts. These results provide strong preclinical justification for combining cisplatin with eugenol as therapeutic approach for triple‐negative breast cancers through targeting the resistant ALDH‐positive cells and inhibiting the NF‐κB pathway.

p16INK4A enhances the transcriptional and the apoptotic functions of p53 through DNA-dependent interaction

p16INK4A and p53 are two important tumor suppressor proteins that play essential roles during cell proliferation and aging through regulating the expression of several genes. Here, we report that p16INK4A and p53 co‐regulate a plethora of transcripts. Furthermore, both proteins colocalize in the nucleus of human primary skin fibroblasts and breast luminal cells, and form a heteromer whose level increases in response to genotoxic stress as well as aging of human fibroblasts and various mouse organs. CDK4 is also present in this heteromeric complex, which is formed only in the presence of DNA both in vitro using pure recombinant proteins and in vivo. We have also shown that p16INK4A enhances the binding efficiency of p53 to its cognate sequence presents in the CDKN1A promoter in vitro, and both proteins are present at the promoters of CDKN1A and BAX in vivo. Importantly, the fourth ankyrin repeat of p16INK4A and the C‐terminal domain of p53 were necessary for the physical association between these two proteins. The physiologic importance of this association was revealed by the inability of cancer‐associated p16INK4A mutants to interact with p53 and to transactivate the expression of its major targets CDKN1A and BAX in the p16‐defective U2OS cells expressing either wild‐type or mutated p16INK4A. Furthermore, the association between p16INK4A and p53 was capital for their nuclear colocalization, the X‐ray‐dependent induction of p21 and Bax proteins as well as the induction of apoptosis in various types of cells. Together, these results show DNA‐dependent physical interaction between p16INK4A and p53.

The DNA methyl-transferase protein DNMT1 enhances tumor-promoting properties of breast stromal fibroblasts

The activation of breast stromal fibroblasts is a crucial step toward tumor growth and spread. Therefore, it is extremely important to understand the molecular basis of this activation and determine the molecules and the mechanisms responsible for its sustainability. In the present report we have shown that the DNA methyl-transferase protein DNMT1 is critical for the activation of breast stromal fibroblasts as well as the persistence of their active status. Indeed, we have first revealed DNMT1 up-regulation in most cancer-associated fibroblasts relative to their corresponding adjacent normal fibroblasts. This effect resulted from HuR-dependent stabilization of the DNMT1 mRNA. Furthermore, ectopic expression of DNMT1 activated primary normal breast fibroblasts and promoted their pro-carcinogenic effects, both in vitro and in orthotopic tumor xenografts. By contrast, specific DNMT1 knockdown normalized breast myofibroblasts and repressed their cancer-promoting properties. These effects were sustained through inhibition of the IL-6/STAT3/NF-κB epigenetic cancer/inflammation positive feedback loop. Furthermore, we have shown that DNMT1-related activation of breast fibroblasts is mediated through upregulation of the RNA binding protein AUF1, which is also part of the loop. The present data demonstrate the critical function of DNMT1 in breast cancer-related sustained activation of breast stromal fibroblasts.

p16 Controls p53 Protein Expression Through miR-dependent Destabilization of MDM2

p16INK4A and p53 are two major tumor suppressor proteins that are both upregulated in response to various cellular stresses and during senescence and aging. p53 is a well-characterized transcription factor, while p16INK4A a cyclin-dependent kinase inhibitor encoded by the CDKN2A gene, and controls the expression of several genes through protein–protein interactions and also via miRNAs. This report demonstrates a p16INK4A-dependent positive regulation of p53 expression, at the protein level, in various human cells as well as in mouse embryonic fibroblasts. p16 suppresses p53 turnover through inhibition of its MDM2-related ubiquitination. This effect occurs through p16-related promotion of the MDM2 mRNA turnover via the p16INK4A downstream effectors miR-141 and miR-146b-5p, which bind specific sites at the 3′ untranslated region of the MDM2 mRNA. Implications: The current findings show p16INK4A-dependent stabilization of p53 through miR-141/miR-146b-5p–related posttranscriptional repression of MDM2, thus providing new insights into the complex functional link between p16INK4A and p53. Mol Cancer Res; 16(8); 1299–308. ©2018 AACR.