New perspectives in cancer biology from a study of canonical and non-canonical functions of base excision repair proteins with a focus on early steps.

Alterations of DNA repair enzymes and consequential triggering of aberrant DNA damage response (DDR) pathways are thought to play a pivotal role in genomic instabilities associated with cancer development, and are further thought to be important predictive biomarkers for therapy using the synthetic lethality paradigm. However, novel unpredicted perspectives are emerging from the identification of several non-canonical roles of DNA repair enzymes, particularly in gene expression regulation, by different molecular mechanisms, such as (i) non-coding RNA regulation of tumour suppressors, (ii) epigenetic and transcriptional regulation of genes involved in genotoxic responses and (iii) paracrine effects of secreted DNA repair enzymes triggering the cell senescence phenotype. The base excision repair (BER) pathway, canonically involved in the repair of non-distorting DNA lesions generated by oxidative stress, ionising radiation, alkylation damage and spontaneous or enzymatic deamination of nucleotide bases, represents a paradigm for the multifaceted roles of complex DDR in human cells. This review will focus on what is known about the canonical and non-canonical functions of BER enzymes related to cancer development, highlighting novel opportunities to understand the biology of cancer and representing future perspectives for designing new anticancer strategies. We will specifically focus on APE1 as an example of a pleiotropic and multifunctional BER protein.

Quantitative PCR evaluation of deletions/duplications identified by array CGH.

Genomic deletions/duplications detected by array comparative genomic hybridization (aCGH) should be confirmed by an independent technology. This approach allows also to test, at low cost, inheritance of the imbalance. In the present study we explored the use of quantitative PCR (qPCR) to confirm aCGH-detected potentially clinically relevant imbalances. Only samples with DLRS <0.2 were tested for confirmation. aCGH results were confirmed in 102/118 cases (86.5%). A major element for non-confirmation was the dimension (and the probe coverage) of the putative aberration. Imbalances detected by 10 or less probes in aCGH assay were not confirmed in 11 out of 41 cases (26.8%), while those ones detected by 20 or more probes were always confirmed (46 cases). Among not confirmed imbalances, no statistical difference was found between deletions and duplication. Our data indicate that validation should be required for imbalances detected by less than 10 probes in aCGH assays.

Serum AP-endonuclease 1 (sAPE1) as novel biomarker for hepatocellular carcinoma.

Late diagnosis for Hepatocellular Carcinoma (HCC) remains one of the leading causes for the high mortality rate. The apurinic/apyrimidinic endonuclease 1 (APE1), an essential member of the base excision DNA repair (BER) pathway, contributes to cell response to oxidative stress and has other non-repair activities. In this study, we evaluate the role of serum APE1 (sAPE1) as a new diagnostic biomarker and we investigate the biological role for extracellular APE1 in HCC. sAPE1 level was quantified in 99 HCC patients, 50 non-HCC cirrhotic and 100 healthy controls. The expression level was significantly high in HCC (75.8 [67.3-87.9] pg/mL) compared to cirrhosis (29.8 [18.3-36.5] pg/mL] and controls (10.8 [7.5-13.2] pg/mL) (p < 0.001). The sAPE1 level corresponded with its protein expression in HCC tissue. sAPE1 had high diagnostic accuracy to differentiate HCC from cirrhotic (AUC = 0.87, sensitivity 88%, specificity 71%, cut-off of 36.3 pg/mL) and healthy subjects (AUC 0.98, sensibility 98% and specificity 83%, cut-off of 19.0 pg/mL). Recombinant APE1, exogenously added to JHH6 cells, significantly promotes IL-6 and IL-8 expression, suggesting a role of sAPE1 as a paracrine pro-inflammatory molecule, which may modulate the inflammatory status in cancer microenvironment. We described herein, for the first time to our knowledge, that sAPE1 might be considered as a promising diagnostic biomarker for HCC.

Human AP-endonuclease (Ape1) activity on telomeric G4 structures is modulated by acetylatable lysine residues in the N-terminal sequence.

Loss of telomeres stability is a hallmark of cancer cells. Exposed telomeres are prone to aberrant end-joining reactions leading to chromosomal fusions and translocations. Human telomeres contain repeated TTAGGG elements, in which the 3' exposed strand may adopt a G-quadruplex (G4) structure. The guanine-rich regions of telomeres are hotspots for oxidation forming 8-oxoguanine, a lesion that is handled by the base excision repair (BER) pathway. One key player of this pathway is Ape1, the main human endonuclease processing abasic sites. Recent evidences showed an important role for Ape1 in telomeric physiology, but the molecular details regulating Ape1 enzymatic activities on G4-telomeric sequences are lacking. Through a combination of in vitro assays, we demonstrate that Ape1 can bind and process different G4 structures and that this interaction involves specific acetylatable lysine residues (i.e. K27/31/32/35) present in the unstructured N-terminal sequence of the protein. The cleavage of an abasic site located in a G4 structure by Ape1 depends on the DNA conformation or the position of the lesion and on electrostatic interactions between the protein and the nucleic acids. Moreover, Ape1 mutants mimicking the acetylated protein display increased cleavage activity for abasic sites. We found that nucleophosmin (NPM1), which binds the N-terminal sequence of Ape1, plays a role in modulating telomere length and Ape1 activity at abasic G4 structures. Thus, the Ape1 N-terminal sequence is an important relay site for regulating the enzyme's activity on G4-telomeric sequences, and specific acetylatable lysine residues constitute key regulatory sites of Ape1 enzymatic activity dynamics at telomeres.