Genomic and protein expression analysis reveals flap endonuclease 1 (FEN1) as a key biomarker in breast and ovarian cancer

FEN1 has key roles in Okazaki fragment maturation during replication, long patch base excision repair, rescue of stalled replication forks, maintenance of telomere stability and apoptosis. FEN1 may be dysregulated in breast and ovarian cancers and have clinicopathological significance in patients. We comprehensively investigated FEN1 mRNA expression in multiple cohorts of breast cancer [training set (128), test set (249), external validation (1952)]. FEN1 protein expression was evaluated in 568 oestrogen receptor (ER) negative breast cancers, 894 ER positive breast cancers and 156 ovarian epithelial cancers. FEN1 mRNA overexpression was highly significantly associated with high grade (p= 4.89 x 10 - 57) , high mitotic index (p= 5.25 x 10 - 28), pleomorphism (p= 6.31 x 10-19), ER negative (p= 9.02 x 10-35 ), PR negative (p= 9.24 x 10-24 ), triple negative phenotype (p= 6.67 x 10-21) , PAM50.Her2 (p=5.19 x 10-13 ), PAM50.Basal (p=2.7 x 10-41), PAM50.LumB (p=1.56 x 10-26), integrative molecular cluster 1 (intClust.1) ( p=7.47 x 10-12), intClust.5 (p=4.05 x 10-12) and intClust. 10 (p=7.59 x 10-38 ) breast cancers. FEN1 mRNA overexpression is associated with poor breast cancer specific survival in univariate (p=4.4 x 10-16) and multivariate analysis (p=9.19 x 10-7). At the protein level, in ER positive tumours , FEN1 overexpression remains significantly linked to high grade, high mitotic index and pleomorphism (ps< 0.01). In ER negative tumours, high FEN1 is significantly associated with pleomorphism, tumour type, lymphovascular invasion, triple negative phenotype, EGFR and HER2 expression (ps<0.05). In ER positive as well as in ER negative tumours, FEN1 protein over expression is associated with poor survival in univariate and multivariate analysis (ps<0.01). In ovarian epithelial cancers , similarly, FEN1 overexpression is associated with high grade, high stage and poor survival (ps<0.05). We conclude that FEN1 is a promising biomarker in breast and ovarian epithelial cancer

REF-1 assay.

<p>(<b>A</b>) HCT116 nuclear extract was incubated with ³²P-labeled consensus (CON) or mutant (MUT) AP-1 oligonucleotide substrates, and binding reactions were resolved on a non-denaturing polyacrylamide gel. Control reactions without nuclear extract (no extract) are shown. The arrow designates the position of the AP-1-specific consensus binding complex, not seen with the MUT double-stranded DNA. Higher molecular weight non-specific complexes are observed. (<b>B</b>) Reduced wild-type (WT) or variant APE1 protein was incubated with HCT116 nuclear extract in the presence of the ³²P-labeled AP-1 CON DNA substrate. Shown is the AP-1-specific complex in the absence (no protein) or presence of the indicated reduced APE1 protein after phosphorimager analysis. Plotted is the relative AP-1 DNA binding activity, in comparison with reduced WT protein. Values represent the average and standard deviation of 3 independent experimental points.</p

Intracellular localization of APE1 protein variants.

<p>(<b>A</b>) Representative microscopy images of the mCherry APE1 fusion proteins following plasmid transfection into HeLa cells. Shown are the DAPI nuclear staining, mCherry fusion protein fluorescence and the merged images. (<b>B</b>) Comparative cytoplasm to nuclear distribution for the different mCherry APE1 proteins. Using densitometry, the ratio of exogenous cytoplasmic mCherry-tagged wild-type (WT) APE1 protein to endogenous cytoplasmic protein was divided by the ratio of exogenous nuclear mCherry-tagged WT APE1 protein to endogenous nuclear protein, and this value was designated as 1. The identical ratio was then determined for each of the APE1 variant proteins, and plotted relative to the WT value. Shown is the average and standard deviation of results from 3 separate extract preparations and western blot experiments.</p

Reconstitution assay using purified BER proteins.

<p>(<b>A</b>) Wild-type (WT) and variant APE1 proteins were incubated with UDG and POLβ with ³²P-labeled 34U DNA substrate (1 pmol), and the reactions were resolved on a urea-polyacrylamide denaturing sequencing gel. The non-incised substrate (S), AP site incision product (P1), and gap-filling extension product (P2) were visualized and quantified using standard phosphorimaging analysis. NE = no enzyme. (<b>B</b>) Relative AP site cleavage efficiency. Shown are the averages and standard deviations of 4 independent reactions. (<b>C</b>) Relative gap-filling activity. Shown are the average and standard deviation of 4 independent assays.</p

Variant proteins and thermodynamic stability of folding as determined by chemical denaturation.

<p>(<b>A</b>) Following purification, wild-type (WT) and variant APE1 proteins were quantified and analyzed (1 µg) by SDS-polyacrylamide gel electrophoresis and Coomassie blue staining. Shown is a representative gel image. Molecular mass standards are indicated to the left in kDa. (<b>B</b>) Profile of maximum wavelength emission for tryptophan fluorescence relative to GdnHCl concentration for the wild-type (WT) and variant APE1 proteins (top). The free energies of protein unfolding in the absence of denaturant (ΔG_uw) and the values reflecting the dependence of the free energy on denaturant concentration (m_eq) are shown (bottom).</p

APE1 protein variants and oligonucleotide substrates.

<p>(<b>A</b>) Linear schematic of the 318 residue APE1 protein, including several reported amino acid substitutions. NLS = nuclear localization sequence; REF-1 = redox regulatory portion of the protein; italics = polymorphic variants; * = unique disease-associated variants; red text = variants with reduced AP endonuclease activity. The repair nuclease domain and several functionally important amino acids (C65, E96, D210, D283 and H309) are indicated. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065922#pone-0065922-t001" target="_blank">Table 1</a> for additional details. (<b>B</b>) The 18F NMR and 18G NMR oligonucleotides were used to design the double-stranded AP endonuclease substrate. (<b>C</b>) The 15P or 17P oligonucleotide was annealed to the 34G oligonucleotide to generate a 3′-recessed exonuclease/repair substrate, whereas the 34U oligonucleotide was annealed to 34G to create the uracil-containing duplex for the reconstitution assay. Oligonucleotides are written 5′ to 3′, with the non-labeled strands written upside-down. F = the AP site analog, tetrahydrofuran.</p

APE1 variants and predicted impact of amino acid change.

<p>Sorting intolerant from tolerant (SIFT) uses sequence homology to predict effects on protein function (<a href="http://sift.jcvi.org/" target="_blank">http://sift.jcvi.org/</a>). Scores <0.05 are considered deleterious, whereas those that are >0.5 are considered to be tolerated. The program polymorphic phenotypes (PolyPhen) predicts impact based on a set of empirical rules that apply to the protein’s sequence, phylogenetic and structural information (<a href="http://genetics.bwh.harvard.edu/pph/" target="_blank">http://genetics.bwh.harvard.edu/pph/</a>). Cologne University protein stability analysis tool (CupSat) predicts protein stability (<a href="http://cupsat.tu-bs.de/" target="_blank">http://cupsat.tu-bs.de/</a>), and was employed using the PDB APE1 protein structure (1DE8).</p>*<p>Described previously in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065922#pone.0065922-Hadi1" target="_blank">[33]</a>. N/A = not available. Once = it was observed a single time.</p

Re-sequencing of <i>APE1</i> exons in the NCI-60 cancer cell line panel.

<p>Re-sequencing was performed as described in Materials and Methods. All sequences were analyzed for homology to transcript variant 3 of APE1 (NM_080649.1). Genotypes have been divided into wild-type (D/D), heterozygous (D/E) or homozygous variant (E/E) for residue position 148 of APE1. Note: *, D/D for position 148, but Q/H heterozygous for position 51. Cancer type and cell line name are designated. Additional information is available at: <a href="http://dtp.nci.nih.gov/docs/misc/common_files/cell_list.html" target="_blank">http://dtp.nci.nih.gov/docs/misc/common_files/cell_list.html</a>.</p